This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Orgnization
    Geneva, 1986

         The International Programme on Chemical Safety (IPCS) is a
    joint venture of the United Nations Environment Programme, the
    International Labour Organisation, and the World Health
    Organization. The main objective of the IPCS is to carry out and
    disseminate evaluations of the effects of chemicals on human health
    and the quality of the environment. Supporting activities include
    the development of epidemiological, experimental laboratory, and
    risk-assessment methods that could produce internationally
    comparable results, and the development of manpower in the field of
    toxicology. Other activities carried out by the IPCS include the
    development of know-how for coping with chemical accidents,
    coordination of laboratory testing and epidemiological studies, and
    promotion of research on the mechanisms of the biological action of

        ISBN 92 4 154257 8  

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     2.1. General considerations
     2.2. Techniques
          2.2.1. Methods of isolation
          2.2.2. Methods of purification, identification,
                 and quantification
          2.2.3. Methods for assay quality assurance
     2.3. Specificity of analytical techniques
     2.4. Data evaluation
          2.4.1. Assay accuracy and precision
          2.4.2. Assay dynamic range


     3.1. General introduction
          3.1.1. Simple diffusion
          3.1.2. Filitration
          3.1.3. Specialized transport systems
     3.2. Gastrointestinal absorption
          3.2.1. General considerations
          3.2.2.  In vivo methods
         The site of gastrointestinal absorption
          3.2.3.  In vitro methods
         Isolated loops
         Isolated cells and vesicles
     3.3. Pulmonary absorption
          3.3.1. General considerations
          3.3.2.  In vivo methods
         Methods for the pulmonary exposure
                          of intact animals
         Methods for the pulmonary exposure
                          of anaesthetized animals
          3.3.3.  In vitro methods
         Perfused lungs
         Fluid-filled lung lobes
         Isolated cells
     3.4. Dermal absorption
          3.4.1. General considerations
          3.4.2.  In vivo methods
         Methods for dermal exposure
          3.4.3.  In vitro methods
         Isolated skin preparations
         Different cell populations

     3.5. Other routes of exposure
          3.5.1. General considerations
          3.5.2. The intravenous (iv) route
          3.5.3. Intraperitoneal (ip) absorption
          3.5.4. Intramuscular (im) administration
          3.5.5. Subcutaneous (sc) administration


     4.1. General considerations
     4.2. Invasive methods
          4.2.1. Qualitative methods
         Autoradiographic methods
          4.2.2. Quantitative methods
         Radiometric methods
         Chemical methods
     4.3. Non-invasive methods


     5.1. General considerations
     5.2. Methods for assessing reversible binding
          5.2.1. Extracellular sites
          5.2.2. Intracellular sites
     5.3. Methods for assessing irreversible binding


     6.1. General considerations
     6.2. Important enzymatic pathways in xenobiotic
          6.2.1. Phase I reactions
         Oxidation reactions
                  Cytochrome P-450 monooxy-
                                     genase system (EC
                  Microsomal flavin-containing
                                     monooxygenase (EC
                  Cooxidation by prosta-
                                     glandin H synthase
                  Miscellaneous peroxidative
                  Alcohol dehydrogenase (EC
                            and aldehyde 
                                     dehydrogenase (EC
                  Monoamine oxidase (EC
         Reduction reactions
                  Cytochrome P-450-
                                     dependent reactions
                  Flavoprotein-dependent reactions
                  Carbonyl reductases
         Hydrolysis reactions
                  Epoxide hydrolase (EC

          6.2.2. Phase II reactions
         UDP-glucuronosyltransferase (EC
         Mercapturic acid biosynthesis
                  Glutathione  S-transfer-
                                     ases (EC
                  Cysteine conjugate betalyase/
         Amino acid  N-acyltransferases
          N-acetyltransferases (EC
          N- and  O-methyltransferases
     6.3. Modulation of important metabolic pathways
          6.3.1. Physiological factors
         Genetic factors
         Sex hormones
                  Sex-linked differences
         Thyroid hormones
         Corticoid hormones
         Pituitary hormones
         Immune system
          6.3.2. Environmental factors
         Enzyme induction
          6.3.3. Pathological factors
         Liver disease
                  Acute viral hepatitis
                 Chronic hepatitis and cirrhosis
                 Obstructive jaundice
                                     and cholestasis
         Kidney disease
     6.4. Sampling procedures for parent compounds and
          metabolites  in vivo
          6.4.1. Non-invasive procedures
          6.4.2. Invasive procedures
     6.5. Experimental systems
          6.5.1. Systems with intact cellular structure
         Intact animals
         Isolated organs
         Freshly isolated cells
         Organs and cells in culture
          6.5.2. Cell-free systems
         Subcellular fractions of tissue
         Purified enzymes and/or reconstituted
                          enzyme systems
          6.5.3. Intestinal microflora
     6.6. Methods for assessing chemically reactive
          metabolites  in vitro


     7.1. General considerations
     7.2. Important excretory mechanisms
          7.2.1. Diffusion and filtration
     7.3. Sites of excretion
          7.3.1. Kidney
         Glomerular filtration
         Tubular secretion
         Tubular reabsorption
          7.3.2. Liver-biliary excretion
         Enterohepatic circulation
          7.3.3. Other excretory sites
     7.4. Modulation by physiological, environmental, and
          pathological factors
          7.4.1. Urinary excretion of xenobiotics
         pH and urine volume
         Inhibition and stimulation by
         Age differences
         Species differences
         Renal dysfunction
          7.4.2. Biliary excretion
         Species and age differences
         Effects of physiological compounds
         Effects of xenobiotics
         Hepatic disease and regeneration
     7.5. Methods for assessing excretion
          7.5.1. Whole animals
          7.5.2.  In vitro preparations
         Isolated organs
         Intestinal preparations
         Slices of renal cortex
         Other kidney preparations
         Purified membrane preparations


     8.1. General considerations
     8.2. Dose-independent kinetics
          8.2.1. One-compartment model
         Single dose
         Repeated dosing
          8.2.2. Two-compartment model
         Single dose
         Repeated dosing
     8.3. Kinetics of metabolites in the presence of
          parent compound
     8.4. Non-linear kinetics
     8.5. Physiological kinetic models
     8.6. Modulation of kinetics


     9.1. General considerations
     9.2. Analysis of parent compounds or metabolites
          9.2.1. Toxicokinetics and sampling strategy
          9.2.2. Dermal absorption
          9.2.3. Specimens in use
     9.3. Effect monitoring
     9.4. Monitoring of exposure to carcinogens
          9.4.1. Urinary mutagenicity
          9.4.2. Alkylation or arylation of proteins, 
                 peptides, amino acids, and nucleic acids
          9.4.3. Chromosomal damage
     9.5. Preanalytical error
          9.5.1. Physiological and environmental sources
                 of variation
          9.5.2. Variation associated with speciment 
                 collection and storage


     10.1. General considerations
     10.2. Analytical data
     10.3. Absorption data
     10.4. Distribution data
     10.5. Reversible binding data
     10.6. Metabolism data
     10.7. Excretion data
     10.8. Kinetic model data
     10.9. Human data



    Every effort has been made to present information in the 
criteria documents as accurately as possible without unduly 
delaying their publication.  In the interest of all users of the 
environmental health criteria documents, readers are kindly 
requested to communicate any errors that may have occurred to the 
Manager of the International Programme on Chemical Safety, World 
Health Organization, Geneva, Switzerland, in order that they may be 
included in corrigenda, which will appear in subsequent volumes. 


a,c    Professor A. Aitio, Institute of Occupational Health,
          Helsinki, Finland

a,c    Professor E.A. Bababunmi, Department of Biochemistry,
          University of Ibadan, College of Medicine, Ibadan,
          Nigeria  (Vice-Chairman)

a,b    Dr J.R. Bend, National Institute of Environmental Health
c,e       Sciences, Research Triangle Park, North Carolina,
          USA  (Rapporteur)

b      Professor H. Bräunlich, Institute of Pharmacology and
          Toxicology, Friedrich Schiller University of Jena,
          Jena, German Democratic Republic

c      Professor I. Darmansjah, Department of Pharmacology,
          University of Indonesia, Medical School, Jakarta,

a,b,c  Dr E. Dybing, Department of Toxicology, National
          Institute of Public Health, Oslo, Norway  (Chairman)

b      Professor H. Hoffmann, Academy of Sciences, Central
          Institute of of Microbiology and Experimental
          Therapy Jena, Jena, German Democratic Republic

a,b    Professor W. Klinger, Institute of Pharmacology and

c,e    Toxicology, University of Jena, Jena, German
          Democratic Republic

b,d,e  Dr D. Müller, Institute of Pharmacology and
          Toxicology, University of Jena, Jena, German
          Democratic Republic

a,c,e  Professor S.D. Nelson, Department of Medicinal Chemistry, 
          University of Washington, Seattle, Washington, USA

a,c,e  Professor O.G. Nilsen, Department of Pharmacology and
          Toxicology, University of Trondheim, Trondheim,

e      Dr Y. Ohno, Division of Medical Chemistry, National
          Institute of Hygienic Sciences, Tokyo, Japan

e      Dr A. Takahashi, Division of Medical Chemistry,
          National Institute of Hygienic Sciences, Tokyo, Japan

a,b    Dr A. Tanaka, Division of Medical Chemistry, National
c,e       Institute of Hygienic Sciences, Tokyo, Japan


Dr J. Parizek, International Programme on Chemical Safety,
       World Health Organization, Geneva, Switzerland  (Secretary)

a Planning meeting, Oslo, 15-18 August 1984.
b Preparatory meeting, Jena, 3-7 March 1985.
c Task Group meeting, Oslo, 8-13 September 1985.
d Representing the Institute of Pharmacology and Toxicology,
  Jena, at the Task Group meeting, Oslo, 8-13 September 1985.
e Contributed in drafting sections of the document.                 


    Since the publication of EHC 6:  Principles and Methods for 
Evaluating the Toxicity of Chemicals, Part I which included a 
section dealing with chemobiokinetics, substantial progress has 
been made in studies on toxicokinetics and recognition of their 
role in evaluating and elucidating the toxicity of chemicals.  For 
this reason, it was felt that a special monograph should be 
prepared on the subject to assist both those conducting the 
relevant studies and those using them in evaluations.  This 
includes the readers who use the IPCS documents for the evaluation 
of the effects of specific chemicals. 

    The outline of the document and the strategies used for its 
development were agreed on at a planning meeting convened for the 
International Programme on Chemical Safety by the National 
Institute of Public Health, Oslo, Norway, 15-18 August 1984.  
Dr E. Dybing was elected Chairman of the meeting, 
Professor E.A. Bababunmi, Vice-Chairman, and Dr J.R. Bend, 
Rapporteur.  DR DYBING agreed to guide the group of experts 
throughout the whole development of the monograph. 

    A second preparatory meeting was hosted for the International 
Programme on Chemical Safety by the Institute of Pharmacology and 
Toxicology of the Friedrich Schiller University of Jena, German 
Democratic Republic, 3-7 March 1985. 

    The text of the whole document was finalized at the Task Group 
meeting hosted by the National Institute of Public Health, Oslo, 
Norway, 8-13 September 1985. 

    The International Programme on Chemical Safety wishes to 
acknowledge the work of the Chairman (DR E. DYBING) and members of 
the Task Group, and of all who contributed to the preparation of 
the document including:  Professor S.D. Nelson who drafted section 2 
(which was later reviewed by Dr W.R. Porter, Abbott Laboratories, 
North Chicago, Illinois, USA); Professor W. Klinger who drafted 
sections 3, 4, and 5; Dr R. Bend who drafted section 6; 
Dr D. Müller who drafted section 6.3; Dr A. Tanaka who drafted 
section 7, and whose co-workers Dr A. Takahashi and Dr Y. Ohno 
drafted section 7.4 and 7.5.2 respectively; Professor O.G. Nilsen 
who drafted section 8 (which was later reviewed by Professor K. S. 
Pang, Faculty of Pharmacy, University of Toronto, Toronto, Canada); 
and Professor A. Aitio who drafted section 9.  The members of the 
Task Group drafted section 10. 

    The financial support is gratefully acknowledged of:  the 
National Institute of Public Health, Oslo, Norway, for the planning 
meeting and the Task Group meeting in Oslo; and the Ministry of 
Public Health of the German Democratic Republic, Berlin, and the 
Institute of Pharmacology and Toxicology of the Friedrich Schiller 
University of Jena for the preparatory meeting in Jena. 


2.1.  General Considerations

    The primary hypothesis underlying studies of the metabolism 
and toxicokinetics of a chemical is that the adverse biological 
effects of the substance are correlated with its concentration in 
the tissues of the organism (microorganisms, plants, or animals, 
including human beings) in which the toxic effect is observed.  
Consequently, it is always necessary to both identify and quantify 
the toxic chemicals that might be present in a sample.  The methods 
that have been used to accomplish these tasks cover the whole range 
of modern analytical techniques.  Some generally useful methods 
will be discussed in this section, but the primary emphasis will 
be on the criteria used to judge the acceptability of proposed 
methods and the mathematical techniques used to evaluate the data 

    It is nearly always possible to devise several acceptable 
methods to identify and quantify chemicals of interest.  Indeed, it 
is highly desirable to use more than one method, at times.  If two 
or more methods yield essentially the same results, confidence in 
each method is increased.  Several possible analytical methods 
should always be considered before beginning a new study.  The 
criteria that should be used for the final selection of a method 
are assay specificity, sensitivity, speed, simplicity, and cost. 

    In toxicokinetic studies, chemicals will nearly always be 
present in biological samples in very low concentrations.  After 
all, only chemicals of relatively low toxicity would be 
administered to test animals at the g/kg body weight level. 
Toxicological studies are more concerned with the chemicals that 
are toxic at the µg/kg body weight level.  Indeed, certain 
chemicals produce health hazards at even lower dose levels, 
especially after long-term exposure.  These extremely toxic 
substances pose an analytical challenge of the greatest magnitude, 
and individuals engaged in research on such extremely toxic 
substances must devote much of their time to developing analytical 
skills at the forefront of technology. 

    Because of the extremely low concentration of chemicals of 
toxicological interest in samples of biological origin, the 
analytical process usually consists of three stages (Smith & 
Stewart, 1981).  In the first stage, the toxic chemical is 
separated from the bulk of the biological material present in the 
sample (i.e., the sample matrix) and concentrated for further 
processing.  This sample-processing step is similar for most 
toxicological investigations, since the primary problems 
encountered are related to the nature of the biological materials 
to be removed (proteins, lipids, salts, etc.) and not to the nature 
of the toxic chemical.  Once the toxic chemical has been isolated 
from the sample matrix, additional separation steps are usually 
required in order to obtain material of sufficient purity to permit 
identification and quantification.  These steps are usually 
different for various toxic chemicals, as the methods used depend 

heavily on the chemical and physical properties of the chemical 
being investigated, as well as on the requirements of the specific 
instruments or techniques used for identification and 
quantification.  Finally, selected biological, chemical, or 
physical methods must be used to identify the toxic chemical 
unambiguously and to determine the exact amount present in the 
sample.  Obviously, this complex process may be subject to many 
errors that must be guarded against.  General problems associated 
with each of these stages will be discussed in section 2.2.  The 
most serious error that might arise is lack of specificity in the 
assay method; the analytical result may not be due to the test 
chemical in the sample, but may result from interfering sustances 
in the sample or from substances inadvertently introduced during 
the complex stages of sample preparation.  This problem will be 
addressed in section 2.3.  Whichever analytical methods may be 
employed to isolate, identify, and quantify the toxic chemical, 
certain general methods of assuring assay validity and 
statistically correct treatment of the data obtained can be used.  
These will be discussed in sections 2.2 and 2.4. 

    When a new chemical (not previously or not sufficiently studied 
for toxicological effects) is being tested, several analytical 
methods may be required during the course of a study.  These 
methods can be classified according to their intended sensitivity 
and specificity.  For example, early in the course of the study of 
a new chemical, relatively general approaches can be tried in order 
to develop a satisfactory method for preliminary studies without 
investing large amounts of time or money.  As the study progresses, 
it may become evident that these methods are not specific enough 
for the measurements that are required, or that the methods are not 
capable of providing all of the types of information desired.  More 
specialized methods may then have to be developed, once it is 
apparent that investing more time and money in the project is 
appropriate.  Ultimately, the importance of the chemical may 
dictate that extensive environmental monitoring and detailed 
studies of the mechanism(s) of toxicity are required.  These 
studies will only be possible if highly specialized analytical 
methods are used.  Reliability, ruggedness, and cost effectiveness 
during use may be major considerations in the selection of such 
methods, which are usually costly and time-consuming to develop. 

    A combination of analytical methods may be necessary for other 
reasons.  In any toxicokinetic study, the identity and purity of 
the chemical used in the test must be assured.  Analytical methods 
capable of detecting undesirable impurities will be required, as 
well as methods to assure that the chemical is of uniform potency 
from batch to batch.  Additional methods will be required to 
monitor the stability and uniformity of the form in which the test 
substance is administered to the organisms used in the 
toxicokinetic studies.  Finally, methods suitable to identify and 
quantify the test substance in toxicokinetic studies must be 
employed.  It is unlikely that a single analytical method will be 
of use for all of these purposes, though sometimes only minor 
modifications of a procedure may be required to accomplish most of 

the tests.  The necessity of using several different analytical 
approaches in such studies is frequently overlooked by scientists 
trained in areas other than analytical chemistry. 

2.2.  Techniques

2.2.1.  Methods of isolation

    In order to identify and quantify a toxic chemical in a sample 
of biological origin, it is first necessary to separate the 
chemical from the bulk of the biological components of the sample.  
The traditional separation methods in analytical chemistry have all 
been used for this purpose in the past, including precipitation, 
distillation, centrifugation, liquid-liquid extraction, liquid-
solid extraction, adsorption, ultra-filtration, and complexation.  
These techniques have been developed extensively by scientists in 
the fields of biochemistry, pharmacognosy, clinical chemistry, and 
agricultural chemistry.  The literature in these fields is a rich 
source of information for the scientist interested in metabolism.  
Many of the techniques required for the preparation of samples for 
further processing are standard, and textbooks in pharmaceutical 
analysis, pesticide residue analysis, clinical chemistry, or 
forensic toxicology should be consulted for examples of useful 
techniques (Henry et al., 1974; Sunshine, 1975; Tietz, 1976; Bauer 
et al., 1978; Connors, 1982; Schirmer, 1982; Baselt, 1984; 
Kratochvil et al., 1984). 

    It is usually necessary to desorb the toxic chemical from the 
macromolecular components of the sample and to remove proteins, 
lipids, etc., that may complicate further analysis.  Careful thought 
should be given to the method of sample preparation; it must be 
sufficiently gentle to allow recovery of the toxic chemical without 
degradation, yet sufficiently powerful to ensure the recovery of 
all, or nearly all, of the toxic chemical in the sample.  Any 
method that is used must be evaluated to make certain that recovery 
of the test chemical is consistent and reasonably complete.  Such 
an evaluation presupposes that methods are available to identify 
and quantify the test chemical and to separate it from substances 
that might interfere with the measurement process.  Consequently, 
though sample preparation is the first step in a complete assay 
method, it is usually the last step developed when a new method is 

    Sample preparation methods are usually evaluated by "spiking" 
experiments.  A known quantity of the test chemical is deliberately 
added to a sample known not to contain the substance.  After 
careful and thorough mixing, the sample is processed by the 
proposed technique and the amount of test chemical recovered is 
measured.  Ideally, this should be identical to the amount added. 

    Recovery of more than the amount added is a strong indication 
of sample contamination or the unsuspected presence of the test 
chemical in the sample prior to the "spiking" experiment.  Another 
possibility could be lack of specificity.  If the "spiking" 
experiment is repeated with different amounts of added test 

chemical, it may be possible to estimate the amount of chemical 
apparently present in the sample originally.  However, it may be 
that the chemical was not actually present initially, but that the 
sample became contaminated during processing, or, that substances 
present in the sample interfered with the measuring process.  In 
these cases, the process requires further investigation. 

    "Spiking" experiments may also reveal significant losses of the 
test chemical during processing.  If this is the case, the 
processing method will have to be modified in such a way as to 
reduce the losses. 

    The "spiking" experiments may show good recovery of the test 
chemical, but this could be the result of the fortuitous balancing 
of sample losses with interfering substances.  Measurement of the 
test chemical recovered in these experiments by two or more 
methods should yield consistent results.  Failure to do so 
indicates that the processing method requires further evaluation. 

    When a sample processing technique seems to be functioning 
adequately, it is still necessary to verify, by repeated 
experiments, that the results of the method are reproducible within 
the limits of precision required.  A frequently used method to 
improve precision is the thorough mixing into the original sample 
of a known amount of a standard substance that can be isolated 
along with the test chemical.  This internal standard is then used 
to obtain a relative measure of the amount of test chemical through 
appropriate calibration experiments.  Such internal standards 
should not be present in the original sample, should show similar 
recovery to the test chemical, and should be detectable by the same 
analytical technique. 

    From the foregoing remarks, it can be seen that validation of 
the sample processing stage may require elaborate experimentation.  
Despite the fact that most sample processing techniques are 
standard, the conditions used in actual experimentation require 
careful control.  Furthermore, validation of the processing 
technique for one type of sample matrix (e.g., plasma) does not 
guarantee that the method will work with a different sample matrix 
(e.g., urine or tissue homogenate).  It will be necessary to 
revalidate the sample preparation technique for each type of sample 
matrix encountered. 

    Sometimes results that seem anomalous will occur during the 
practical application of an analytical method.  It may be that 
unexpected interference with the method has occurred through 
contamination of the sample by residues extracted from glassware, 
pipettes, sample vial caps, etc., or through a failure of the 
sample processing technique to remove certain substances.  Such 
anomalous results merit investigation.  The specific steps that 
must be taken to remedy defects in the sample processing stage will 
depend on both the nature of the chemical under study and the 
nature of the processing procedure. 

    In recent years, techniques of sample preparation based on the 
column chromatography process of adsorption, partition, 
ultrafiltration, or complexation have become popular.  A relatively 
short column is employed, and elution solvents are selected to 
elute the compound of interest as a class with other compounds of 
similar chemical and physical properties.  Thus, it may be possible 
to recover the test compound and its degradation products or 
metabolites as a mixture, which can then be separated further.  
Such sample clean-up techniques are only useful for separating the 
test compound from substances with grossly different chemical and 
physical properties.  They may, in fact, also separate the test 
compound from metabolites or degradation products that are to be 
measured.  This potential problem illustrates the necessity of also 
validating sample processing techniques for suspected metabolites.  
This may not always be possible, unless authentic samples of the 
suspected metabolites are available for testing. 

    A variant of the "digital chromatography" technique, which is 
especially useful in preliminary studies, makes use of commercially 
available thin-layer chromatography plates that incorporate a 
"spotting zone" about 3 cm in height on a 20 cm high plate.  
Samples of up to 50 ml of plasma, urine, or 25% tissue homogenates 
can be applied to a 1 cm wide segment of the "spotting zone".  
After thorough drying, the toxic chemical can be eluted from the 
biological materials with a suitable solvent, such as methanol.  
The plate is developed only to the beginning of the conventional 
adsorbant layer, and this process is repeated several times.  As a 
result, the toxic chemical is concentrated in a narrow band at the 
beginning of the adsorbant layer, and conventional elution 
techniques can then be used to separate it from other materials 
that might also have been concentrated along with it.  This 
technique is especially easy to use if a radioisotope labelled form 
of the toxic chemical is available.  Excess unlabelled material can 
then be added to the sample to help dissociate the test substance 
from macromolecular binding sites.  The added unlabelled material 
also serves to reduce overall decomposition of the labelled test 
substance during analysis, by a dilution effect.  This technique 
can also be used for monitoring the uniformity and stability of the 
dosage, and may be applicable for determining the purity of the 
bulk test chemical as well.  Covalent binding of the labelled 
chemical to tissue components can readily be detected using this 
technique, since any bound material should remain in the "spotting 
zone."  These methods seem to be generally useful in the study of 
toxicokinetics of many substances, at least as a first approach.  
Of course, validation experiments must be performed with this 
method, as with any other. 

2.2.2.  Methods of purification, identification, and quantification

    Once a toxic chemical has been separated from the biological 
components of the sample matrix, it will normally be necessary to 
further purify it by additional separation procedures.  The degree 
of further purification required depends entirely on the 
selectivity and specificity of the measurement techniques to be 
used to identify and quantify the toxic chemical.  If a very 

general and nonselective measurement method is used, the burden of 
assuring adequate specificity, for the assay procedure as a whole, 
will fall on the purification stage.  On the other hand, if the 
toxic chemical possesses unique biological, chemical, or physical 
properties, these may be exploited to develop highly selective 
measurement methods for unambigously identifying and quantifying 
the substance.  If this is the case, very little, if any, 
purification may be required.  For this reason, purification 
procedures are normally developed simultaneously with the 
measurement procedures that will be employed.  Up-to-date reviews 
of the various methods, as applied to specific groups of chemicals, 
can be found in the Application Reviews of Analytical Chemistry. 

    The types of purification procedures that have been used in the 
past have varied widely.  Since the measurement of the metabolites 
or degradation products of the toxic chemical is usually required, 
methods that separate these products from the parent compound in 
such a way that they can be measured simultaneously are desirable.  
This goal is usually achieved by adopting a multiple, continous 
purification process, such as a chromatography system. 

    Most chromatography systems exploit differences in adsorption, 
partition, molecular size or shape, or complexation properties to 
effect a separation of chemicals that may otherwise have very 
similar chemical and physical properties.  The behaviour of a 
chemical in a given chromatographic system can usually be predicted 
adequately on the basis of knowledge of its chemical structure, 
thus allowing a reasonable choice of chromatographic technique 
(Lyman et al., 1981).  The fact that only small amounts of the test 
chemical are present in samples from toxicokinetic studies is an 
advantage when selecting chromatographic techniques, as a wider 
range of possible systems can be evaluated. 

    The mobile phase used in a chromatography system can be either 
a gas, liquid, or a supercritical fluid.  Because the hardware for 
gas chromatography (GC) has reached a higher state of technological 
improvement than the hardware for liquid or supercritical fluid 
chromatography, it is generally more reliable (Freeman, 1979; 
Jorgenson, 1984).  The separating power of gas chromatography 
equipment has been increased greatly through the use of capillary 
columns (Jennings, 1980).  GC systems are usually less costly to 
acquire, maintain, and operate than high performance liquid 
chromatography (HPLC) systems.  If a toxic chemical is volatile, or 
can be made volatile by simple chemical modifications, GC is 
usually preferred to HPLC.  GC equipment is also more readily 
interfaced to the mass spectrometer, a measuring device that can 
provide highly reliable identification of the chemicals being 
studied as well as quantification of the material identified.  
GC/MS systems provide a purification and measurement package that 
can be used in a wide variety of metabolic and toxicokinetic 
investigations (Watson, 1976; Millard, 1978; Message, 1984).  
However, highly-skilled operators are required to operate and 
maintain the equipment.  Despite the high initial cost of such 
systems, and despite the need for highly skilled personnel to 
operate them, it can be argued that such equipment is cost-
effective in the long run, if a wide variety of different toxic 
chemicals is to be studied. 

    Some toxic chemicals, or their metabolites, are not 
sufficiently volatile to be separated from contaminants by GC, and 
a liquid supercritical fluid mobile phase must be used instead.  In 
recent years, HPLC systems of increasing sophistication have become 
available.  Such systems are now often the first choice because of 
the flexibility in the choice of mobile and stationary phases for 
the chromatographic portion of the system as well as the wide 
variety of measurement devices that can be incorporated as 
detectors.  Detectors that measure ultraviolet, visible, or 
infrared absorption spectra, fluorescene, atomic absorption, 
refractive index, radioactivity, electrochemical properties, etc., 
have been found to be useful (Hadden et al., 1971; Tsuji & 
Morozowich, 1978; Snyder & Kirkland, 1979; Krstulovic & Brown, 
1982).  Interfacing an HPLC system to the mass spectrometer is not 
yet as well-established a technique as GC/MS, but progress in this 
area continues to be made (Cooks et al., 1983). 

    Despite the attractiveness of GC and HPLC techniques, thin-
layer chromatography (TLC) is still very useful and is especially 
powerful when combined with radioisotope tracer methods for 
establishing the identity and quantity of a toxic chemical and its 
metabolites.  Significant advantages of this combination are that 
nearly every toxic substance of interest can be studied using this 
technology, only one analytical instrument (a radiation counter) is 
required, labour and material costs are low, and the level of skill 
required to perform TLC/radiochemical assays is modest.  Mass 
balance experiments, an important requirement in validation 
studies, are easily performed.  The major drawbacks of this 
technique are the necessity of having a radioisotope labelled 
chemical, and that the procedures involved are slow and tedious 
compared to GC or HPLC methods.  However, for the scientist on a 
limited budget, working in a area with low labour costs and a 
shortage of personnel with advanced technical training, 
TLC/radioisotope methods are especially attractive.  The amount of 
radioactive material required is generally quite small, and it is 
used under experimental conditions that are unlikely to pose a 
health hazard.  Wastes can usually be disposed of by incineration 
without releasing significant amounts of radioactivity into the 
atmosphere.  However, it is essential that radioisotope use be 
supervised by individuals with adequate training in radiation 

    GC, HPLC, and TLC/radioisotope methods are commonly used for 
studying toxicokinetics of various chemicals.  Of these methods, 
TLC/radioisotope technology is the easiest method to implement, the 
method most likely to give useful results quickly, and the method 
least affected by the level of skill of the technicians employed.  
Although it rapidly becomes onorous if a large number of samples 
need to be analysed over long periods of time, the TLC/radioisotope 
method remains a good choice for early investigation of new 

    In some cases, isolation, purification, and quantification of a 
substance from a matrix can be carried out in only one stage.  One 
of the major developments of the past decade has been the 

application of immunochemistry to the measurement of hormones, 
drugs, toxins, and enzymes (Monroe, 1984).  Radioimmunoassay (RIA) 
is based on the existence of an equilibrium between an antigen (the 
component to be measured), an antibody, and a corresponding 
antigen-antibody complex in a system that includes trace amounts of 
radioactively labelled antigen.  A simple separation step is 
required in order to determine the amount of labelled antigen that 
is bound to the antibody or free in solution.  Newer enzyme 
immunoassay (EIA) techniques such as EMIT and ELISA do not require 
radioactivity and EMIT does not require a separation step.  These 
methods involve coupling an enzyme with an antibody or antigen.  
All of these techniques can be highly sensitive with the ability to 
measure concentrations of chemicals in the range of ng - µg/litre. 
Whether RIA or EIA techniques are used, the major disadvantage of 
the methods is the need for specific antibodies.  Development of 
monoclonal antibody techniques and the combining of liquid 
chromatographic separation with immunoassays will no doubt decrease 
problems associated with specificity.  As production techniques for 
antibodies are made more efficient, EIA techniques will be used 
more extensively because highly trained technicians and special 
instrumentation are not required. 

2.2.3.  Methods for assay quality assurance

    After the chemical under study and its metabolites have
been separated from the sample matrix and purified, they must
be identified and quantified.  Too often, in toxicokinetic
studies, it is not demonstrated that the methods of separation,
identification, and quantification used are adequate for the 
particular study.

    The method used to validate a particular purification and 
measurement process (i.e., to show that the process in fact 
measures what it purports to measure) depends, to some extent, on 
the techniques that are used.  Each scientist must become familiar 
with his instruments and techniques, and must learn the problems 
and pitfalls of these techniques and the way in which these can be 
overcome.  In addition to having the appropriate background 
knowledge, there are certain other general precautions that can be 
taken, and guidelines have been established by several 
organizations for proper assay quality control (APA, 1972; Federal 
Register, 1978; ASTM, 1979, 1981; ACS Committee on Environmental 
Improvement, 1980; Digne, 1984).  The following list summarizes the 
more important requirements: 

    (a)  Assure that the bulk chemical used in tests is of known 
         purity and free of undesirable contaminants.  A biological 
         assay of a highly purified sample of the bulk chemical 
         should demonstrate the same toxicological response as the 
         bulk chemical.

    (b)  Assure that dosage forms used in biological experiments 
         are of uniform content and are stable under conditions of 
         actual use.  A stability-indicating assay of the potency 
         of experimental dosage forms is required.  Such assays in 

         turn require validation (i.e., the assay must show a loss
         in potency in an artifically degraded sample consistent 
         with the amount of degradation induced).

    (c)  Assure that materials used as standards are of known 
         potency and purity.  Additional analytical tests may be 
         required, and it may be necessary to repeat these tests at 
         regular intervals.

    (d)  Assure that experimental work is conducted as planned, 
         that any modifications are documented, and that adequate 
         records of the experiments are maintained as they are 
         conducted.  Unusual observations should be noted and 

    (e)  Assure that adequate tests to challenge the ability of the 
         analytical procedures used to discriminate against 
         potentially interfering substances have been conducted.  
         These tests are discussed in section 2.3.

    (f)  Assure that uniform or controlled conditions have been, 
         or can be, maintained throughout the duration of the 
         experiment.  Verify experimentally that different analysts 
         or different instruments yield data of the same quality 
         with respect to assay sensitivity, precision, and accuracy.  
         These problems are discussed in section 2.4.

    (g)  Assure that the biological samples are stable during the 
         period prior to analysis, and that the analytical 
         procedure does not itself cause the substance being 
         analysed to degrade.  If it is impossible to avoid some 
         degradation, determine experimental conditions to assure 
         that such degradation will be consistent throughout the 
         period of experimentation.

    (h)  Assure that samples are analysed in a statistically valid, 
         randomized fashion.  Ideally, the analyst should not know 
         the identity of the samples until after the analysis has 
         been completed.  If it is necessary to assay samples in a 
         particular order, make certain that this information is 
         carefully documented.

    (i)  Assure that the right samples are being analysed.  Guard 
         against faulty labelling or record-keeping.

    (j)  Assure that instruments are properly maintained and in a 
         good working order.

    (k)  Assure that instruments are calibrated properly.  Verify 
         that calibration is performed at appropriate intervals.

    (l)  Assure that experimental samples and standards, and 
         "reagent blanks" or control samples are processed 
         identically.  Verify that any "reagent blank" samples are 
         appropriately selected.

    (m)  Assure that the results obtained are reproducible and 
         accurate.  There are many ways to achieve this aim, among 
         which are periodic reanalysis of experimental samples 
         previously assayed in the laboratory (internal control), 
         and the comparison of results obtained from the analysis 
         of samples that have been assayed in other laboratories 
         either by similar or different methods (external control).

If the preceding guidelines are adhered to, the quality of the data 
produced from toxicokinetic experiments should be controllable, 
with the result that interpretations made from such data are more 
likely to be grounded on fact. 

2.3.  Specificity of Analytical Techniques

    Assay specificity is perhaps the most serious problem 
encountered, since the chemicals being measured are present in such 
low concentrations in the samples that it is potentially possible 
for many other components in the sample to copurify with the 
chemicals of interest.  If this should occur, then these 
contaminants may interfere with either the identification or the 
quantification process or both.  Data obtained using measurement 
techniques of too general use should be regarded with suspicion.  
For example, most chemicals absorb light somewhere in the 
ultraviolet region of the spectrum, especially at low wavelengths.  
An assay method that depends on the measurement of the absorption 
of ultraviolet radiation at low wavelengths for quantification is 
unlikely to be very specific.  A purification technique of great 
power must therefore be used to minimize the potential for the 
occurrence of interference in the measuring process. 

    The use of "reagent blanks," i.e., samples that are supposedly 
free of the chemical being assayed, is helpful in demonstrating the 
lack of interfering substances.  Such "reagent blanks" must be 
selected with care, as they should be otherwise identical to the 
samples that will be analysed for the toxic chemical, except for 
the absence of the test chemical.  Because of the genetic diversity 
of biological test organisms, it is difficult to select an 
appropriate "reagent blank." 

    Although "blanks" provide some assurance that no instrument 
response will be obtained in the absence of the test chemical, a 
better approach is to select an instrument or bioassay that 
responds to some biological, chemical, or physical property of the 
test chemical that is not shared with many other substances.  If a 
bioassay is used, the organisms used in the assay should ideally 
respond only to the test chemical, or to a limited number of other 
chemicals that can be shown not to copurify with the test chemical.  
"Information rich" methods of analysis, such as mass spectrometry, 
infrared photometry, nuclear magnetic resonance spectrometry, or 
batteries of monoclonal antibodies provide the user with greater 
assurance that he or she is measuring the chemical under test, and 
not some interfering substance.  Unfortunately, at present, only 
mass spectrometry and specific immunoassays have adequate 
sensitivity to be used routinely in toxicokinetic studies. 

    It is advisable to use a second analytical technique to 
confirm, at least on a spot-check basis, that the usual method is 
producing correct results.  The second method should ideally be as 
different as possible, at both the purification stage and the 
measurement stage.  If both assay methods agree, there is less 
likelihood that interfering substances are present, since, if they 
were present, they would have to interfere, to the same extent, 
with both assays.  This is so unlikely that a confirming assay is 
often used as a check for assay specificity.  Of course, the 
confirming assay may be invalid if it is too similar to the 
original method. 

    Sometimes it is possible to increase the information content of 
the instrumental signal.  For example, if ultraviolet 
spectrophotometry is being used to monitor the effluent from a 
liquid chromatography column, measurements at a number of 
wavelengths could be obtained.  If an interfering substance is 
present in some samples, but not in others, or if an interfering 
substance is present in different amounts in each sample, the ratio 
of ultraviolet absorbances at two or more wavelengths will change 
(unless the interfering substance has an absorbance spectrum 
identical to that of the test chemical).  This extra data can be 
used for quality assurance purposes without complicating the normal 
data processing procedures. 

    "Spiking" experiments can sometimes be used to verify the 
absence of interfering substances.  A single sample is split into 
several subsamples, some of which are "spiked" with known, 
different amounts of authentic test chemical.  All of the 
subsamples are then analysed and the amounts found in the variously 
"spiked" samples are extrapolated to zero "spike" level.  The 
results should agree with the amounts actually found for the 
unspiked subsamples. 

    Finally, it is helpful to repeat the purification process on a 
large scale, using a representative sample.  Fractions 
corresponding to the chemical and its metabolites can then be 
collected and studied by a variety of other analytical techniques, 
in order to confirm their identities using all of the available 
methods for structure elucidation that can be employed.  If these 
procedures are followed, it will be reasonably certain that the 
assay is specific for the toxic chemical and its metabolites.  In 
practice, however, "suspicious" results will occasionally be 
observed, and, in this case, the sample material should be 
reassayed, preferably by a second method.  In this way, other 
sources of interference may be detected during the course of 

2.4.  Data Evaluation

2.4.1.  Assay accuracy and precision

    If the precautions noted previously are taken, the assay method 
should yield accurate results.  Nevertheless, however accurate the 
data may be, they may still not be sufficiently precise to be 

useful.  In the context of analytical methods, "accuracy" refers to 
how closely the average value reported for the assay of a sample 
agrees with the actual amount of substance being assayed in the 
sample, whereas "precision" refers to the amount of scatter in the 
measured values around the average result.  If the average assay 
result does not agree with the actual amount in the sample, the 
assay is said to be "biased", i.e., lacks specificity; bias can 
also be due to low recovery (section 2.3).  Assuring accuracy 
requires all of the elaborate experimentation discussed previously, 
while assuring precision requires repeating the experiment a number 
of times and then statistically analysing the averaged results.  
However, this improvement is achieved at great cost, as every 
halving of the degree of scatter in the average requires 
quadrupling the number of measurements.  High precision can be 
better achieved through improved experimental designs (Cochran & 
Cox, 1957; Aarons, 1981; Mitchell & Garden, 1982). 

    Before proceeding further, it is useful to discuss the level of 
precision that is really needed in toxicokinetic experiments.  The 
overall level of precision that can be achieved will always be 
limited by the intrinsic genetic variability of the organisms used 
in the experiments.  This can be reduced, to a certain extent, by 
clever experimental design, but it nevertheless remains a major 
limitation.  In the usual situation, statistical theory can be used 
to demonstrate that, if other sources of variation total less than 
about 30% of the biological component of variation, their overall 
effect will be small.  Thus, if the biological variation expected 
in the experiment will cause, on average, about a 20% relative 
scatter in the data, it is only necessary to reduce the relative 
scatter of the analytical procedures to less than 6% to achieve 
adequate precision.  This is well within the capabilities of GC, 
HPLC, TLC, or immunoassay methods used in conjunction with 
appropriate detectors and conventional laboratory sample handling 

    It can also be achieved by bioassay methods if sufficient 
replications of an appropriately designed assay are conducted.  
Bioassays require careful experimental design guided by appropriate 
statistical considerations in order to achieve high precision 
(Finney, 1978).  It is almost always necessary to compare each 
unknown sample to an authentic standard, using a procedure in which 
several dilutions of both sample and standard are tested 
simultaneously, in randomly selected test organisms, under 
identical conditions.  A relative potency is then calculated by 
plotting the responses obtained for the various dilutions of both 
the standard and unknown sample and calculating an average ratio of 
sample to standard potency using properly weighted nonlinear 
regression analysis.  With the advent of low-cost digital 
computers, there is no reason for failing to use the correct 
computational procedures.  Once a computer program has been 
obtained to perform potency ratio computations, it can be used for 
all future bioassay work.  The use of a calibration curve, which is 
standard practice for chemical or physical analytical data, is 
almost never permissible in bioassay work. 

    Data obtained from assays in which the final measurements are 
based on chemical reactivity or observation of a change in a 
physical property are usually analysed by means of a calibration 
curve.  Calibration curves must be monotonic (an increase in the 
concentration of the test chemical must always either increase or 
decrease the measured property or process).  Although computation 
is simplified if calibration curves are straight lines, this is by 
no means essential (Hubaux & Voss, 1970; Garden et al., 1980; 
Kurtz, 1983; Moler et al., 1983; Schwartz, 1983).  On the other 
hand, excessive curvature is undesirable, because the assay 
sensitivity is proportional to the rate of change in measured 
response to the rate of change in test chemical concentration, and 
this should be reasonably constant for the assay method to be 
useful.  Usable calibration curves can nearly always be well-
approximated by a cubic polynomial function.  Statistical theory 
can be used to select the best arrangement of standards to maximize 
assay precision.  Assay precision using data interpolated from a 
calibration curve is affected by the precision of the measurements 
of the unknown samples and the measurements of the standards (which 
ought to be the same), and the precision with which the parameters 
of the calibration curve can be determined.  The last depends on 
the design of the calibration experiments as well as on the 
measurement precision.  The concentrations for the standards should 
cover a wider range than that of the unknown samples.  If the 
calibration curve is a straight line, then the average 
concentration of the standards should be close to the anticipated 
average concentration of the unknowns.  The total number of 
standards analysed should be greater than, or equal to, the number 
of replicate measurements on a single unknown, but the best 
precision will be obtained when the number of standards assayed is 
equal to the number of replicate determinations for each unknown 
sample.  Replicating the measurements on unknown samples is usually 
the best way to improve precision, since it is likely that the 
number of standards used will greatly exceed the number of 
replicate measurements on unknown samples. 

    Whatever type of assay may be performed, it is essential that 
both the assay design and the data evaluation be conducted 
according to correct statistical principles.  If unsure how to 
proceed, a statistican should be consulted before any experimental 
work is begun.  Salient references that should be consulted include 
Youden & Steiner (1975), Montag (1982), Caulcutt & Boddy (1983), 
Bolton (1984), and Delaney (1984). 

2.4.2.  Assay dynamic range

    Although quality assurance and good experimental design will 
help to improve assay accuracy and precision, it is also necessary 
that the assay method be usable over a sufficiently wide range of 
concentrations for the toxic chemical and its metabolites. 

    The lower limit of usefulness for an analytical method has been 
perceived in different ways; frequently, the term "sensitivity" has 
been used to indicate the ability of an analytical method to 
measure small amounts of a substance accurately and with requisite 

precision.  This, however, is a misnomer; sensitivity correctly 
refers to the slope of the calibration curve.  The lower limit at 
which the test chemical can be distinguished from a "reagent blank" 
has been called the limit of detection for the analytical method.  
This is the lowest level at which the chemical being analysed can 
be identified.  It is more useful to define a lower limit at which 
adequate precision can be obtained (Long & Winefordner, 1983; 
Oppenheimer et al., 1983); this may be called the limit of 
quantification.  Typically, the limit of quantification occurs at a 
concentration fifteen to twenty times higher than the limit of 

    These concepts have limited use in practice; in toxicokinetic 
studies, it is the range of concentrations over which a reasonably 
uniform level of precision can be obtained that is important.  This 
range of concentrations may be called the dynamic range of the 
analytical method.  Typical assays useful for toxicokinetic studies 
should be able to measure the test chemical over a 100-fold range 
of concentrations.  This is not often easy to achieve without 
sacrificing some precision.  If the dynamic range is not wide 
enough, many invesitigators dilute and reassay samples that are too 
concentrated, but if this is done, it is necessary to demonstrate 
that the dilution process does not introduce additional error. 


3.1.  General Introduction

    Absorption is the process(es) by which an administered 
substance enters the body (OECD, 1981).  For the purposes of this 
document, absorption will be equated with the appearance of the 
chemical in the circulation.  The rate and extent of absorption of 
the administered substance can be estimated by various methods, 
with and without reference groups (i.e., a test group in which the 
substance is administered via another route that ensures complete 
availability of the dose).  These methods include: 

    (a)  determination of the amount of test substance and/or
         metabolites in urine, bile, faeces, and exhaled air,
         and that remaining in the carcass;

    (b)  comparison of a biological response (e.g., acute
         toxicity studies) between test and control and/or
         reference groups;

    (c)  comparison of the amount of dose excreted renally in
         test and reference groups; or

    (d)  determination of the area under the plasma steady
         state curve of the test substance and/or metabolites
         and comparison with data from a reference group
         (OECD, 1981).

    The rate of absorption can be determined from the plasma 
concentration time-curve of the test substance (sections, 

    The skin is the main barrier that separates mammals including 
man from environmental chemicals.  However, chemicals are absorbed 
via the skin and may produce damage.  The major routes by which 
toxicants enter the body are via the lungs, the gastrointestinal 
tract, and the skin.  Once the chemical has entered the blood-
stream, it may exert its toxic action directly in the blood or in 
any target tissue or organ to which the circulatory system 
transports the chemical. 

    Often, a chemical must pass through many "barriers" before 
reaching its target.  These barriers include the many membranes of 
the cells of the skin, the layers in the lung and gastrointestinal 
tract, the capillary cell, the cells of the tissue and organs where 
the chemical exerts its damaging effect, and the cells of the 
organs that eliminate the chemicals, mainly the liver and the 

    All cell membranes are similar:  they consist of a bimolecular 
layer of lipid molecules coated on each side with a protein layer, 
branches of which penetrate the lipid bilayer or even extend right 
through it.  At physiological temperatures, the lipids of the 
membranes (mainly phosphatidylcholine, cholesterol) have a 

quasifluid character, determined by the structure and relative 
proportion of unsaturated fatty acids.  The higher the 
concentration of unsaturated fatty acids, the higher is the 
fluidity.  Most foreign chemicals cross body membranes by simple 
diffusion.  The rate and extent of this diffusion or absorption is 
influenced by many factors, summarized in Table 1. 

Table 1.  Factors influencing the rate and extent of absorption of
a chemicala
Properties of the   Morphology and dimension of the absorbing
organism            body surface, perfusion of the absorbing
                    area, distribution and elimination processes,
                    general factors (e.g., nutritional status, 
                    age, disease)

Characteristics of  Relative molecular mass
the chemical        Physical state
                    - conformation
                    - aggregation
                    - dispersion
                    - acid or base characteristics
                    Solubility in various solvents

Characteristics of  Dose/concentration, duration of contact with
exposure            the absorbing surface

Exogenous factors   Formulation
                    - vehicle
                    - additives
                    Interaction with other chemicals
                    Physical conditions (e.g., temperature, 
a Modified from:  Scheler (1980).

3.1.1.  Simple diffusion

    Most organic molecules possess a certain degree of 
lipophilicity and cross membranes by diffusion through the lipid 
moiety.  The rate of transfer depends on the lipid solubility, 
which can be characterized by the lipid/water partition coefficient 
(most frequently determined are the oil/water or octanol/water 
partiton coefficents), and the concentration gradient across the 

    Chemicals exist in solution in ionized and/or non-ionized 
forms.  The charged (ionized) form is generally less able to 
penetrate cell membranes and, thus, diffusion is dependent on the 
lipid-soluble non-ionized form of the substance.  The dissociation 
constant or the negative logarithm of the dissociation constant 
(pKa) and the pH of the medium determine the degree of 

dissociation.  When the pKa of the chemical and the pH of the 
medium are equal, 50% of the chemical exists in the ionized form.  
 Note:  the pKa alone does not indicate whether a compound is an 
acid or a base, because a basic chemical can have a pKa greater 
than 7 and an acidic chemical a pKa lower than 7.  The degree of 
dissociation can be calculated according to the Henderson-
Hasselbach equation: 

                    (anion-) x (H+)
    for acids:  K = ------------------
                    (non-ionized acid)

                    (cation+) x (OH-)
    for bases:  K = ------------------
                    (non-ionized base)

These equations can be transformed to:

    for acids:  pKa - pH = log -----------

    for bases:  pKa - pH = log -----------

Consequently, organic acids are more likely to cross membranes by 
diffusion when they are in an acidic medium, and organic bases, 
when in an alkaline medium. 

3.1.2.  Filtration

    Very small hydrophilic compounds can pass through aqueous 
channels or pores.  This passage is called filtration, because it 
involves the bulk flow of water due to hydrostatic or osmotic 
forces.  The size and number of these channels or pores differ 
considerably in various membranes from 4 to 40 A (kidney 
glomerulus).  Such pores permit chemicals with a relative molecular 
mass ranging from 100 - 200 to up to 60 000 to pass. 

3.1.3.  Specialized transport systems

    These systems are important for nutrients and endogenous 
substances (sugars, amino acids, amines, etc.), but less important 
for most xenobiotics; they are relevant only for xenobiotics, such 
as amines or organic anions, that are very similar to endogenous 
substrates.  Active transport is characterized by:  a) the 
requirement of energy, or energy-producing metabolism; b) a certain 
selectivity with respect to the structure of the chemicals 
transported; c) a limited capacity so that the transport system can 
be saturated and a transport maximum is exhibited; and d) transport 
of a chemical proceeding against electrochemical or concentration 
gradients.  Active transport is important for the pulmonary uptake 
of, e.g., paraquat, a herbicide structurally similar to endogenous 
diamines such as putrescine and cadaverine, and for the organic 

anions phenol red and chromoglycate, as well as for the elimination 
of the chemicals by the kidneys (tubular secretion of weak acids 
and bases by acidic and basic carriers) and by the liver (biliary 
secretion of weak acids and bases and neutral compounds).  The 
central nervous system also has two transport systems for the 
active transport at the chorioid plexus, one for organic acids and 
one for organic bases. 

    Facilitated diffusion (without energy requirement and without 
movement of the chemical against a gradient) can account for the 
uptake of acidic chemicals into liver parenchymal cells (Müller & 
Klinger, 1975; Klaassen, 1980; Scheler, 1980; Klaassen & Watkins, 

    Phagocytosis and pinocytosis play an important role in the 
uptake of particulate matter, e.g., in the lungs (alveolar 
macrophages), in the subcutis (leukocytes, histiocytes), in the 
liver (Kupffer cells), and in the gastrointestinal tract 
(epithelial cells). 

3.2.  Gastrointestinal Absorption

3.2.1.  General considerations

    The gastrointestinal tract is a major site of absorption.  In 
principle, the contents of the gastrointestinal tract must be 
considered exterior to the body and absorption can take place along 
its full length, including the mouth and rectum.  In general, 
gastric juice is acidic and the intestinal contents, almost 
neutral.  Thus, a chemical will be absorbed predominantly in the 
part of the gastrointestinal tract where it exists in the most 
lipid-soluble form.  Even if only a small percentage is present in 
the non-ionized, diffusible form, e.g., weak acids in the 
intestine, the very large surface area (villi, microvilli), long 
contact time, and high concentration gradient (quick removal of 
absorbed material due to a high perfusion rate) generally provide 
high absorption rates, as equilibrium is always obtained.  The 
mechanisms by which some lipid-insoluble compounds are absorbed are 
not clear.  Some metals are absorbed by specialized transport 
systems (e.g., thallium, cobalt, and manganese by the iron-system, 
lead by the calcium-system).  Special attention must be paid to the 
stability of chemicals in the acidic stomach (e.g., esters can be 
hydrolysed), to the enzymes in the stomach and intestine (peptides 
will be split), and to the microbial flora, which have high 
hydrolytic and reductive capacity.  Moreover synthetic reactions 
can take place, e.g., nitrosamine formation from secondary amines 
in the stomach.  Absorption from the gastrointestinal tract can be 
modified by many factors such as nutrients (e.g., fat, milk), 
fibres in the diet, by starvation, by alcohol, and by drugs that 
alter stomach emptying time and/or motility of the gastrointestinal 

3.2.2.   In vivo methods  Measurement

    Measurement of the chemical and its metabolites in the blood 
stream, as well as in the urine, gives insight regarding the site, 
extent, and rate of absorption.  Bioavailability is the percentage 
of a given dose that appears after absorption and distribution with 
the portal blood through the liver in the circulating blood stream.  
It comprises absorption rate (the percentage of a given dose that 
disappears from the lumen of the gut) and the first pass-effect 
(the percentage of the absorbed chemical that is filtered out from 
the portal blood by accumulation and/or biotransformation in the 
liver).  Chemicals may be administered into the mouth cavity 
(without swallowing of the material), into the stomach via a 
gastric tube, or directly into the duodenum via a catheter.  If a 
chemical is absorbed from the mouth cavity or from the lower part 
of the rectum it does not pass the liver.  If it is administered 
directly into the duodenum it is not exposed to the acidic 
environment in the stomach.  Bioavailability may be determined by 
investigating the parent compound and its metabolites, mainly in 
the urine, or in the urine plus faeces.  The rate of uptake into 
the circulating blood stream can be determined by investigating the 
time-course of the concentration of the chemical in the blood 
(section  The site of gastrointestinal absorption

    The site of gastrointestinal tract absorption can also be 
determined in animals with sections of the gut separated by 
ligatures yet retaining the vascular and nervous connections with 
the body (suitable predominantly for small animals) or with 
intestinal loops prepared from various segments of the 
gastrointestinal tract with two external orifices (suitable only 
for such animals as the cat, rabbit, dog, pig) (Giraldez et al., 
1984).  Whereas  in situ sections can be prepared in anaesthetized 
animals only for a duration of a few hours, with all the possible 
influences of the anaesthesia on circulation, peristalsis etc., the 
so-called chronic intestinal loops are not filled with the 
physiological intestinal contents, the microflora may be altered 
and irritation and inflammation may influence absorption.  
Absorption can be investigated by determining the blood levels and 
excretion of the chemical and of its metabolites; luminal and 
vascular perfusion techniques, including lymph collection, can also 
be applied (Csąky, 1984; Windmueller & Spaeth, 1984). 

3.2.3.   In vitro methods  Isolated loops

    Various methods with isolated loops [uneverted, everted loops 
(sac preparations), loops consisting mainly of mucosa, intestinal 
sheets and isolated villi] have been developed (Csąky, 1984; 
Windmueller & Spaeth, 1984).  Most investigations have been 
carried out with various segments of the gastrointestinal tract and 

the rate of the diffusion process can be determined under optimal 
oxygenation and nutrition conditions in both directions (outside 
in, inside out).  Thus, simple diffusion can be determined under 
conditions in which the influence of perfusion and the liver are 
excluded.  However, the whole wall of the gastrointestinal tract 
is taken as the diffusion barrier in this model system and this is 
not true  in vivo.  Isolated cells and vesicles

    The absorption of chemicals can also be determined in isolated 
cells from the liver and gut and in isolated membrane vesicles 
(Csąky, 1984; Murer & Hildmann, 1984).  In these cases, only the 
cell membrane is a barrier to diffusion.  Time courses of uptake, 
possible facilitated and directed diffusion (the latter determined 
comparing both types of membrane vesicles), storage within the 
cells, etc., can be studied.  As the survival time of the cells and 
vesicles is limited and various functions decline at different 
rates, the use of freshly isolated cells and vesicles is 
restricted.  Cultured cells can also be used, but changes in cell 
characteristics (differentiation, special functions) during 
cultivation must be taken into account. 

3.3.  Pulmonary Absorption

3.3.1.  General considerations

    Chemicals that are absorbed by the lungs are usually gases 
(e.g., carbon monoxide, nitrogen oxides, ozone, sulfur oxides), 
vapours of volatile liquids (e.g., benzene, halogenated alkanes 
such as carbon tetrachloride), or aerosols (such as silica and 
asbestos).  The site of deposition of an aerosol depends on the 
size (diameter) of the particles and their charge.  Deposition in 
the nasopharyngeal segment is followed by absorption through the 
epithelium of this region and, after swallowing of the secreta, 
through that of the gastrointestinal tract.  Particles that are 
deposited in the tracheal, bronchial, and bronchiolar regions are 
cleared by the upward ciliary movement of the mucous layer.  This 
movement, normally rapid and efficient, is affected by smoking, 
coughing, and sneezing.  The mucous may be swallowed and the 
contents absorbed in the gastrointestinal tract.  Gases and 
vapours, as well as very small particles, reach the alveolar zone, 
where absorption takes place rapidly due in part to a very large 
surface area (80 - 100 m2 in adults) and a very thin diffusion 
barrier (4 µm).  Particles with an aerodynamic diameter smaller 
than 2 µm may not be deposited but exhaled.  Depending on polarity, 
gases and vapours (e.g., aldehydes, ammonia) may also be absorbed 
through the mucous membranes of the upper respiratory tract, and 
they may exert a considerable effect at this site of absorption. 

    Equilibrium is established rapidly between gases in the 
alveolar air and their concentration in the blood, which depends on 
the solubility of the gas or vapour in the blood; this varies 
widely.  The more soluble a chemical is in blood, the more must be 
dissolved to reach equilibrium and the more time is required to 

reach an equilibrium with body water.  In the case of a gas with 
low solubility, only a small percentage of the total gas in the 
lung is removed by the blood during respiration and the blood 
becomes saturated.  Increasing respiration rate does not change the 
rate of absorption, whereas increasing cardiac output markedly 
enhances this parameter.  However, it should be realized that 
respiration rate and cardiac output generally change concomitantly.  
For a gas with high solubility, almost all of the gas is 
transferred to the blood during each respiration, and saturation 
may not be reached.  Increasing the rate of respiration will 
increase the absorption rate considerably, whereas cardiac output 
is of minor importance.  The absorption of low-solubility gases is 
thus perfusion limited and that of high-solubility gases is 
respiration limited.  Thus, the absorption rate of gases and 
vapours with intermediate solubility is influenced by both the 
respiration and cardiac output rates.  These rules also hold true 
for liquid aerosols.  Particles are removed from the alveolar 
surface through phagocytosis by the aid of macrophages.  The 
macrophages migrate to the distal end of the bronchiolar system and 
are then removed by ciliary movement.  Unphagocytozed particles are 
also removed by ciliary movement.  In addition, both free and 
phagocytozed particles reach the lymphatic system, where they can 
remain for long periods (Witschi & Brain, 1985). 

3.3.2.  In vivo methods  Methods for the pulmonary exposure of intact animals

    Methods have been developed for the short-term and especially 
the long-term exposure of intact animals to gases, vapours, or 
defined aerosols.  In principle, open or closed circuit systems 
(the latter are much more complicated) can be used for whole-body 
exposures.  In such systems, the animal in the exposure chamber can 
move freely for periods of hours, days, weeks, or months, with or 
without exposure-free intervals. 

    Nose-only exposure, which avoids dermal and oral uptake and 
reduces contamination, requires restriction or even anaesthesia of 
the animal and can be performed for only short periods (several 
hours).  These exposure periods can then be repeated.  Blood (and 
tissue at the end of the study), urine, and faeces sampling can be 
carried out at intervals (Ther, 1965; Covert & Frank, 1980; Kennedy 
& Trochimowicz, 1983).  Methods for the pulmonary exposure of anaesthetized

    Pulmonary exposure of anaesthetized animals can be performed by 
cannulation of the trachea, with or without artifical respiration, 
in an open or a closed system.  Absorption can be measured by 
monitoring blood levels, and elimination by the sampling of urine 
via a catheter in the urinary bladder.  The generation and 
administration of aerosols are complicated (Covert & Frank, 1980; 
Kennedy & Trochimowicz, 1983), whereas exposure to distinct 
concentrations of gases or vapours can be performed in an open 

system without major technical problems (Ther, 1965).  However, it 
should be realized that such open systems involve exposure hazards.  
As an alternative, any unabsorbed chemical remaining in the lungs 
can be assayed after injection of small volumes of the chemical in 
a vehicle 1 - 2 mm above the bifurcation of the trachea of the rat, 
after killing the animal at the end of the absorption period 
(Schanker, 1978).  In all cases, no differentiation can be made 
between absorption through the tracheobronchial tract and/or the 
alveolar epithelium. 

3.3.3.   In vitro methods  Perfused lungs

    Isolated, ventilated, and perfused lungs have been used to 
investigate the tissue uptake of chemicals from the circulation 
(blood, plasma, or plasma substitutes) (Schanker, 1978).  Fluid-filled lung lobes

    Isolated, fluid-filled lung lobes perfused with blood, plasma, 
or plasma substitutes can also be used to assess the permeability 
of the alveolar epithelium and the capillary wall.  The estimation 
of permeability coefficients and of apparent membrane pore radii is 
possible, but may differ considerably from those in normal, 
ventilated organs (Schanker, 1978).  Isolated cells

    Freshly isolated lung cells (Bend et al., 1985) can be used to 
study of the kinetics of uptake.  As with isolated liver, 
intestinal, or kidney cells, the survival time is limited and 
various functions decline at different rates.  Up to the present, 
these isolated lung cells have been used predominantly for 
metabolism studies (Bend et al., 1985). 

3.4.  Dermal Absorption

3.4.1.  General considerations

    The skin is a relatively good barrier for lipid- and water-
soluble substances.  Nevertheless, many substances can be absorbed 
in sufficient quantities to produce acute or chronic systemic 
effects (Klaassen, 1980; Scheler, 1980).  For example, poisons have 
been developed for use in chemical warfare and these are readily 
absorbed through intact skin.  Most chemicals pass through the 
epidermal cells, which constitute the major part of the skin 
surface.  The cells of the sweat glands, the sebaceous glands, and 
the hair follicles seem to be of much less importance.  A chemical 
must cross a large number of cells (outer layer of horny, 
keratinized epidermal cells, spiny and germinal cells, corium) and 
many membranes to reach the systemic circulation.  This diffusion 
barrier is rate limiting for overall absorption.  The rate of 
diffusion of nonpolar chemicals is related to their lipid 
solubility and inversely related to their relative molecular mass; 

polar substances may diffuse through the protein filaments.  The 
structure, function, and also the permeability of the skin vary 
from one region of the body to another, and hydration, abrasion, or 
removal of the stratum corneum distinctly enhance permeability, 
sometimes by a factor of 10 or more.  Damage to skin by acids, 
bases, and skin irritants followed by inflammation drastically 
increases skin permeability for a wide range of chemicals.  
Moreover, many solvents increase permeability.  Dimethyl sulfoxide 
(DMSO) is one of the best known of these solvents. 

3.4.2.   In vivo methods  Methods for dermal exposure

    Methods for dermal exposure must take into account species 
differences and microlesions that appear after shaving in 
furbearing species.  If a definite area of skin or even the whole 
animal is exposed to a chemical, the solvent used, water uptake by 
skin, etc., can influence the absorption rate, much more than the 
physical and chemical properties of the substance under 
consideration.  After various lengths of exposure, blood samples 
can be assayed or the appearance of the chemical and of its 
metabolites can be monitored in urine.  The skin can also be 
analysed after washing and cleaning.  Precautions should be taken 
to avoid inhalation exposure. 

3.4.3.   In vitro methods  Isolated skin preparations

    Isolated skin preparations of adult animals, with or without 
fur (e.g., nude mice, pigs), as well as of newborn animals (e.g., 
rats and mice without fur), can be used as a diaphragm in a 
diffusion chamber.  The rate of diffusion can be determined without 
the influence of perfusion.  Again, the influence of water and 
solvents may play the most important role.  Different cell populations

    Different cell populations can also be obtained from skin and 
can be used for the study of absorption (i.e., uptake processes).  
In the epidermis, cell to cell connections play a much greater role 
than those in organs such as liver and kidney.  Thus, possible 
artefacts must be taken into account. 

3.5.  Other Routes of Exposure

3.5.1.  General considerations

    In general, chemicals enter the interior of the body from the 
exterior by crossing the epithelial barriers of the skin, lung, or 
gastrointestinal tract.  Penetration via a lesion (e.g., wound) is 
the exception.  A parenteral route is often chosen to study the 
action of a chemical in order to control dose, concentration, etc., 
and to avoid uncertainties of bioavailability (Scheler, 1980). 

3.5.2.  The intravenous (iv) route

    The intravenous (iv) route introduces the chemical in solution 
directly into the blood stream, avoiding the process of absorption. 

3.5.3.  Intraperitoneal (ip) administration

    In general, after intraperitoneal (ip) administration of a 
chemical, absorption is facilitated by the large surface of the 
peritoneal cavity.  The chemical mainly enters the liver by the 
portal circulation; thus, first pass effects must be considered. 

3.5.4.  Intramuscular (im) administration

    In general, the chemical is readily absorbed after 
intramuscular (im) administration, because of the good perfusion of 
muscular tissue, but it must also pass several membranes. 

3.5.5.  Subcutaneous (sc) administration

    After subcutaneous (sc) administration, absorption is 
relatively slow.  Changes in the perfusion by vasoactive compounds 
(e.g., vasoconstriction by sympathomimetics, vasodilatation by 
local anaesthetics) as well as peculiarities in the formulation 
(solvent, microcrystals, etc.) can strongly influence the rate of 


4.1.  General Considerations

    Distribution is the process(es) by which an absorbed substance 
and/or its metabolites circulate and partition within the body.  
Two approaches can be used for the analysis of distribution 

    (a)  the qualitative approach using information obtained
         by whole-body autoradiographic techniques; and

    (b)  the quantitative approach using information obtained
         by sacrificing animals at different times after
         exposure and determining the concentration, and
         amount of, the test substance and/or metabolites in
         tissues and organs (Klaassen, 1980; OECD, 1981).

    The distribution of a chemical in the blood usually occurs 
rapidly.  Distribution to the different tissues and organs is 
determined by the blood flow through the capillary walls, and 
interstitial and cell barriers, by the concentration gradient of 
the free, unbound chemical, and by the affinity to binding sites in 
the tissues and organs.  Thus, all considerations on diffusion 
given in section 3, are also valid for distribution. 

    The concentration of a chemical in blood (blood level) is 
dependent on the dose, the rate of elimination, and the volume of 
distribution (section  The greater the volume of 
distribution, the lower the blood level.  But, as the chemical may 
be found in various tissues and organs in different concentrations 
according to its hydro- and lipophilicity, this distribution volume 
is a fictitious space.  There are very few chemicals that are 
distributed in plasma (e.g., trypan blue, indocyanine green), 
plasma plus interstitial = extracellular water (chloride, inulin, 
thiosulfate), or in extracellular plus intracellular = total body 
water (antipyrine).  Chemicals with high lipophilicity occur in 
much higher concentrations in fat and many are accumulated in the 
liver and kidney.  The apparent volume of distribution is an 
estimation of the magnitude of the distribution volume, based on 
the blood level, if the chemical were distributed equally in the 
body after one single exposure.  Only the circulatory system is a 
distinct, closed "compartment" where chemicals are distributed 
rapidly.  Distribution to the various tissues and organs is usually 
markedly delayed; this second type of distribution volume is termed 
a "peripheral compartment" and there is an equilibrium of the free 
chemical between the so-called rapid, or central, and the slow or 
peripheral compartment.  As the free chemical is eliminated (Fig. 
1), the chemical from the peripheral compartment is slowly released 
into the circulation (rapid or central compartment). 


    In contrast to the fictitious volumes of distribution and 
compartments, some organs and tissues are morphologically defined 
compartments due to special diffusion barriers or to storage 
capacities.  Many chemicals do not enter the central nervous system 
(CNS) as readily as other organs and tissues, because there are 
special diffusion barriers:  the capillary endothelial cells of the 
CNS have very tight junctions with very few, if any, pores, and the 
capillaries are surrounded by specialized glial cells termed 
astrocytes.  This so-called blood-brain barrier is not absolute, 
but it does exclude many chemicals.  The characteristics of the 
blood-brain barrier vary in different brain areas.  Chemicals with 
high lipid solubility readily enter the CNS, but hydrophilic 
compounds (e.g., ions) are almost totally excluded.  Similarly, an 
efficient blood-testicular barrier exists.  In pregnancy, the 
embryo and fetus are readily reached by most chemicals, which pass 
the placenta by simple diffusion.  The number of layers that 
represent a diffusion barrier in the placenta varies with the 
species and within one species with the state of gestation.  
Usually the more lipid-soluble chemicals will cross the placenta 
more readily and an equilibrium between the concentrations of the 
free chemical in maternal and fetal compartments (e.g., maternal 
and fetal plasma water) will be reached sooner or later.  In 
general, the placental-fetal unit belongs to the peripheral 
compartment of the mother. 

4.2.  Invasive Methods

4.2.1.  Qualitative methods  Autoradiographic methods

    Autoradiographic methods have been developed to study the 
distribution of chemicals after labelling with radioactive isotopes 
(3H, 14C, 35S, etc.) with alpha-, beta-, and gamma-radiation 
(Amlacher, 1974; Rogers, 1979, 1985; Nagata, 1984), in the whole 
body of experimental animals.  By killing the animals at different 
times after administration or by taking biopsies, the time curves 
of distribution in the various tissues and organs as well as the 
distribution patterns at equilibrium can be obtained.  
Autoradiographic methods can also be used for distribution studies 
at the tissue and cell levels. 

4.2.2.  Quantitative methods  Radiometric methods

    After administration of a radiolabelled chemical, blood and 
tissue samples are taken at intervals, either after killing the 
animals or by obtaining biopsies, and the activities determined by 
different techniques, depending on the radiolabel and radiation 
type.  In this way, time distribution curves as well as 
distribution patterns can be obtained.  Chemical methods

    If sensitive and specific chemical detection methods are 
available (section 2), distribution-time curves and patterns can be 
studied after killing the animals or by taking biopsies at 
different intervals after administration. 

4.3.  Non-Invasive Methods

    Non-invasive methods are desirable in studying the distribution 
of chemicals in expensive non-rodents, and especially in man.  
Limited information can be obtained through the detection of 
chemicals and their metabolites in saliva, breath, and urine (using 
radiometric, chemical, or stable isotope methods), as well as 
through whole-body scanning, after administration of radiolabelled 
substances (Breimer & Danhof, 1980; Posti, 1982; Walther et al., 
1983; Zylber-Katz et al., 1984; Kretschko & Berg, 1985; Krumbiegel 
et al., 1985a,b).  However, developments in this field, such as 
positron imaging, X-ray fluorescence, neutron activation, and 
magnetic pneumography, deserve special attention.  They include 
positron imaging, X-ray fluorescence, neutron activation, and 
magnetic pneumography. 


5.1.  General Considerations

    Many proteins including albumin, glycoproteins, lipoproteins, 
and specialized proteins for metals, bind chemicals (Davison, 1971; 
Vallner, 1977).  Plasma albumin, which  binds the greatest variety 
of foreign chemicals, i.e., acidic, basic, and neutral substances, 
has been thoroughly investigated.  The main binding force is 
hydrophobic bonding, but other reversible bonds such as hydrogen, 
van der Waal's, and ionic bonds are also involved.  As hydrophobic 
bonding plays the most important role, the extent of binding is 
directly correlated with lipophilicity.  The high relative 
molecular mass of albumin (about 68 000) prevents the bound 
chemical from crossing the capillary walls, and this fraction is 
not directly available for extravascular distribution.  But, as 
unbound (free) compound leaves the vessels by diffusion, bound 
compound dissociates from the protein and the relative degree of 
dissociation remains constant.  This process continues until the 
free chemical in the extravascular water equilibrates with the 
free chemical in the plasma-water.  Plasma-albumin binding of 
chemicals does not influence active processes such as carrier-
mediated transport in the tubular cell in the kidney. 

    Depending on the structure and character of a chemical (acidic, 
basic, neutral), different parts of the albumin molecule serve as 
the binding sites; the binding capacity (number of binding sites) 
is limited and therefore saturable.  Consequently, competition 
between chemicals for the same binding site(s) occur.  The 
competing chemicals are generally similar in structure, but not 
always, and competition phenomena depend on the concentrations and 
the affinities of the particular compounds.  Competitive release 
has been observed with certain drugs, the sudden high concentration 
of the free drug leading to potent pharmacological or toxic 

    Protein binding of chemicals is also observed in tissues or 
organs.  The liver and the kidneys have a high capacity to bind 
certain chemicals.  In both organs, substances are bound to so-
called carriers, responsible for active transport, and also to 
specific binding proteins.  Special binding proteins for organic 
anions (glutathione transferases) and for metals (metallothioneins) 
have been described.  Different types of binding are observed in 
bone with toxic metals (lead, strontium), halogens (fluoride) and 
other compounds (e.g., tetracyclines).  Certain metals can replace 
calcium cations and fluoride and tetracyclines can replace 
hydroxylanions in the hydroxyapatite lattice structure of bone by 
an exchange adsorption reaction, a typical surface phenomenon.  The 
chemicals diffuse into the hydration shell of the hydroxyapatite 
crystals, which have a large surface area.  Fluoride and 
radioactive metals may cause damage at the site of storage, others 
are not toxic to the bone, but may serve as a depot.  Release by 
ionic exchange, pH changes, or osteoclastic activity can lead to an 
increased concentration in plasma resulting in toxic reactions, if 
enough chemical is mobilized. 

5.2.  Methods for Assessing Reversible Binding

5.2.1.  Extracellular sites

    The binding of chemicals to serum, plasma, or albumin (crude or 
purified fractions) is estimated by various techniques including 
ultrafiltration techniques, (Sephadex)-gel filtration, or 
equilibrium dialysis (Davison, 1971; Vallner, 1977).  Assay of the 
chemicals can be accomplished by various analytical methods 
(section 2); radiometric methods are used when radiolabelled 
chemicals are studied. 

    The following physical methods for studying chemical-protein 
binding can be carried out only in very special cases: 

    (a)  ultraviolet and visible absorption spectroscopy of
         free and bound chemical;

    (b)  fluorescence spectroscopy;

    (c)  optical rotatory dispersion and dichroism; or

    (d)  nuclear magnetic resonance.

5.2.2.  Intracellular sites

    The same methods used for extracellular sites, especially 
ultrafiltration techniques, can be used in the study of reversible 
binding to cell fractions (nuclei, nuclear membranes, mitochondria, 
smooth and rough endoplasmic reticulum, lysosomes, cytosol, special 
membranes) and others such as synaptosomes. 

5.3.  Methods for Assessing Irreversible Binding

    Irreversible binding of chemicals and especially of reactive 
metabolites to macromolecules (DNA, RNA, proteins, and lipids) 
plays an important role and is studied predominantly using 
radiolabelled chemicals (Pohl & Branchflower, 1981).  The term 
"irreversible binding" (or "covalent binding") is used when the 
bound proportion does not change after isolation, dilution, and 
washing procedures; frequently this binding is also stable during 
denaturation.  Only recently have other techniques, such as mass 
spectroscopy, X-ray crystallography, and immunochemical analysis, 
been applied to assess the nature of the irreversible binding 
(Berlin et al., 1984a). 


6.1.  General Considerations

    In this section, metabolism refers to the process or processes 
by which an administered xenobiotic chemical is structurally 
altered in the body by either enzymatic or nonenzymatic reactions.  
In this context, the terms biotransformation and metabolic 
transformation are used interchangeably with metabolism.  
Xenobiotic denotes a relatively small (relative molecular mass < 
1000), non-nutrient chemical that is foreign to the species in 
which biotransformation is being studied, though certain compounds 
biosynthesized by some species (e.g., alkaloids, glycosides) are 
xenobiotics in others. 

    The major role of biotransformation is to convert poorly 
excretable lipophilic compounds to more polar entities that can be 
readily excreted in the urine and/or the bile.  In the absence of 
metabolism, such xenobiotics accumulate in the mammalian body, 
increasing the potential for a toxic response.  Examples of such 
compounds are certain polychlorinated biphenyl (PCB) and 
polychlorinated dibenzofuran (PCDF) congeners (Masuda et al., 
1985).  On the other hand, biotransformation is less likely in 
xenobiotics that have high water/oil partition ratios (hydrophilic 
compounds), which are rapidly excreted in urine. 

    Two or more sequential enzymatic reactions are routinely 
required to convert lipophilic xenobiotics to metabolites that are 
efficiently excreted.  Williams (1959) classified the pathways 
involved into phase I and phase II reactions.  Oxidation, 
reduction, and hydrolysis are termed phase I reactions, whereas 
conjugation and synthesis are phase II reactions.  Normally, one or 
more phase I reactions precede phase II metabolism.  Initially, 
xenobiotic metabolism was associated with detoxication.  However, 
it is now known that both phase I and phase II reactions function 
in metabolic activation processes as well.  Many different types of 
compounds are converted to their ultimate toxic chemical species 
during metabolism; a few of the best studied examples include 
acetaminophen, 2-acetylaminofluorene, aflatoxin B1, benzo( a)pyrene, 
carbon tetrachloride, diethylnitrosamine, dimethylnitrosamine, and 

    Historically, most  in vitro studies of xenobiotic metabolism 
were conducted on mammalian liver, because this organ generally 
contains a high concentration of biotransformation enzymes, is 
relatively large, and consists of few cell types.  Compared with 
the liver, extrahepatic tissues do not normally play a major 
quantitative role in the biotransformation of foreign compounds, 
though there are exceptions.  Interest in the xenobiotic-
metabolizing enzymes of extrahepatic tissues has increased 
markedly during the last decade, largely in an attempt to 
understand the relationships between metabolism and target organ or 
cell toxicity (Gram, 1980; Rydström et al., 1983; Bend & Serabjit-
Singh, 1984).  It is now known that, for certain compounds, the 
relative ability of a tissue or cell type to metabolically activate 

a chemical versus the ability of the same tissue or cell type to 
detoxify the reactive/toxic metabolite(s) can determine the 
severity and location of the lesion.  The best example of this 
phenomenon is the lung-specific toxin 4-ipomeanol, which is 
biotransformed to a metabolite by the cytochrome P-450 (P-450) 
monooxygenase (EC system.  The metabolite formed causes 
highly selective necrosis of the nonciliated bronchiolar 
epithelial (Clara) cells of several species (Boyd, 1980).  
Endoplasmic reticulum and P-450-dependent enzyme activity are also 
concentrated in Clara cells (Devereux et al., 1981, 1982) compared 
with other lung cells such as the alveolar type II cell and the 
alveolar macrophage.  Thus, the susceptibility of Clara cells to 
4-ipomeanol is related to their ability to rapidly oxidize it to a 
toxic metabolite.  It may also be related to their inability to 
maintain intracellular glutathione (GSH) at a concentration 
sufficient to detoxify the electrophilic metabolite formed.  For a 
recent review of the metabolic activation of xenobiotics to toxic 
metabolites, see Guengerich & Liebler (1985). 

    The contribution of the intestinal flora to the  in vivo 
metabolism and toxicity of xenobiotics should not be forgotten 
(Scheline, 1980; Goldman, 1982), and can be especially important 
for chemicals that undergo enterohepatic circulation. 

    The remainder of this section will deal with the enzymes known 
to be important in xenobiotic metabolism, their modulation by 
physiological, environmental, and pathological factors, non-
invasive methods for studying xenobiotic metabolism  in vivo, and 
 in vitro preparations that are used to study organ-specific and 
cell-specific biotransformation. 

6.2.  Important Enzymatic Pathways in Xenobiotic Metabolism

6.2.1.  Phase I reactions  Oxidation reactions

    Oxidation is the first step in the metabolism of most 
xenobiotics, and there are several different enzyme systems that 
oxidize chemicals in mammals.  The most important pathways are 
discussed here.  Cytochrome P-450 monooxygenase system (EC

    This is the most important enzyme system involved in the phase 
I metabolism of xenobiotics, primarily because of its great 
versatility (for reviews, see Sato & Omura, 1978; Johnson, 1979; 
Coon & Persson, 1980; Lu & West, 1980; Wislocki et al., 1980; Wolf, 
1982; Estabrook, 1984; Johnson et al., 1985; Levin et al., 1985).  
In addition to most xenobiotics, it metabolizes several classes of 
endogenous compounds, including fatty acids, prostaglandins, 
steroids, and vitamins.  The P-450 system is membrane bound and has 
two major components, cytochrome P-450, a haemoprotein, and NADPH-
cytochrome P-450 reductase, a flavoprotein that contains both FMN 
and FAD prosthetic groups.  This enzyme also reduces cytochrome c 

and is known as NADPH cytochrome c reductase; its role in 
xenobiotic metabolism has been reviewed by Masters (1980).  A major 
reason for the wide variety of substrates that are oxidized by this 
system is that there are many forms or isozymes of P-450.  These 
differ in substrate specificity, as well as in their response to 
the administration of enzyme inducers, such as phenobarbital (PB) 
or 3-methylcholanthrene (3-MC).  Detailed protein purification and 
characterization studies have shown that there are at least 10 
distinct P-450 proteins in rat liver (Guengerich et al., 1982; 
Levin et al., 1985).  This is not the only reason for substrate 
diversity, however, for single purified P-450 isozymes in 
reconstituted monooxygenase systems catalyse many different 

    The overall oxidation of a substrate, RH, can be summarized by 
the following equation (1), where NADPH, one reduced form of 
nicotinamide adenine dinucleotide, is shown as the required 

    RH + O2 + NADP + H+ + ----> ROH + H2O + NADP+              (1)

    The sequence of events that occurs during a monooxygenase 
reaction is now well understood (Estabrook, 1984).  P-450 contains 
one molecule of iron-protoporphyrin IX as its prosthetic group.  
Normally, in microsomal preparations, this iron is in the ferric 
(Fe+3) state.  Oxidized P-450 first reacts with a molecule of 
substrate to form an enzyme-substrate complex.  Next, one electron 
is donated to this complex from NADPH via NADPH-cytochrome P-450 
reductase, which converts RH-P-450(Fe+3) to RH-P-450(Fe+2).  The 
P-450(Fe+2)-substrate complex then reacts with molecular oxygen to 
form an oxycytochrome P-450 ternary complex.  This complex may then 
accept one additional electron from NADPH via NADPH-P-450 
reductase, or from NADH by cytochrome b5 reductase (EC to 
form the equivalent of a two-electron reduced complex of 
haemoprotein, oxygen, and substrate, which dissociates to yield 
oxidized substrate, P-450(Fe+3) and water.  The reaction is termed a 
monooxygenation because one atom of atmospheric oxygen is 
transferred to the substrate; the other is incorporated into water.  
A simplified reaction scheme is given in Fig. 2. 

    Some of the reactions catalysed by the P-450 monooxygenase 
system include aliphatic hydroxylation, epoxidation, aromatic 
hydroxylation, heteroatom ( N-,  O-,  S-) dealkylation, oxidative 
deamination, nitrogen oxidation, oxidative desulfuration, oxidative 
dehalogenation, and oxidative denitrification (Wislocki et al., 

    There are 2 types of P-450-dependent monooxygenase systems in 
mammals.  The most important one for xenobiotic metabolism, and the 
one described above, is associated with the endoplasmic reticulum 
(microsomal fraction) of the liver and many extrahepatic tissues 
(lung, kidney, placenta, small intestine, skin, adrenal, testis, 
ovary, eye, pancreas, mammary gland, aorta walls, brain, nasal 
epithelial membranes, colon, salivary glands, prostate, heart, 
lymph nodes, spleen, thymus, and thyroid) (Gram, 1980; Bend & 

Serabjit-Singh, 1984).  The other P-450 monooxygenase system is 
associated with the mitochondria of steroid-metabolizing tissues 
(adrenal, ovary, testis) and contains an FAD-flavoprotein and an 
iron sulfur protein that facilitate electron transfer from NADPH to 
P-450 (Estabrook et al., 1973).  The mitochondrial P-450 system is 
normally involved with the metabolism of endogenous compounds 
including cholesterol, cholecalciferol, and deoxycorticosterone, 
and has a much higher degree of substrate specificity than the 
microsomal system (Estabrook, 1984). 


    Although the P-450 system is concentrated in the endoplasmic 
reticulum of hepatocytes, it is also present at lower 
concentrations in the nuclear membrane, in the Golgi apparatus, and 
in the plasma membrane (Stasiecki & Oesch, 1980).  Presumably, this 
is also true for extrahepatic tissues. 

    There are several parameters associated with the microsomal 
P-450 system that can be readily assayed.  The total content of 
microsomal P-450, and of the related microsomal haemoprotein 
cytochrome b5, can be measured by the method of Omura & Sato 
(1964a,b).  P-450 is quantified from the difference spectrum 
generated from carbon monoxide-saturated and reduced versus reduced 
microsomal preparations and b5 from the difference spectrum between 
oxidized and reduced microsomes.  However, it must be remembered 
that, because of the diversity of P-450 isozymes present in 
microsomes, changes in total P-450 content seldom correlate with 
changes in monooxygenase activity. 

    Microsomal NADPH-P-450 reductase activity can also be measured 
(Gigon et al., 1969), though it is much easier to quantify the 
enzyme activity as NADPH-cytochrome c reductase activity (Masters, 

    Studies of the biotransformation of a xenobiotic, and the rate 
of this process, using the P-450 system in microsomal or 10 000 x g 
supernatant preparations of liver and/or a variety of extrahepatic 
tissues (Burke & Orrenius, 1979), can be an important step in 
evaluating the toxicity of a chemical.  In general, it is best to 
perform initial studies with hepatic microsomes (or 10 000 x g 
supernatant preparations), in which the P-450 is present in the 
highest concentration.  However, since the different isozymes of 
P-450 are not distributed equally in different tissues and cells, 
the ratio of oxidative detoxication and toxication reactions can 
vary from tissue to tissue.  Thus, in some cases, it will be more 
appropriate to study a toxication pathway in microsomes from an 
extrahepatic tissue.  In studies of this type, it is necessary to 
buffer (to approximately pH 7.4) the microsomal preparation 
(105 000 x g pellet) and to add NADPH or an NADPH-generating system 
(e.g., NADP, glucose-6-phosphate, and glucose-6-phosphate 
dehydrogenase), as cofactor, prior to incubation.  Since an 
individual xenobiotic is frequently oxidized by several different 
pathways, it may be necessary to separate and to chemically 
identify the various metabolites formed in order to understand the 
nature of the toxication pathway(s) (section 2).  Microsomal flavin-containing monooxygenase (EC

    There is also a P-450-independent monooxygenase localized in 
the endoplasmic reticulum of mammalian liver and extra-hepatic 
tissues, which can be detected in virtually all nucleated cells.  
This flavin-containing monooxygenase (FMO) was originally isolated 
from pig liver (Ziegler & Mitchell, 1972) and has recently been 
purified from rat, mouse, and rabbit liver (Kimura et al., 1983; 
Tynes et al., 1985) and mouse and rabbit lung (Williams et al., 
1984b; Tynes et al., 1985).  The enzyme contains the coenzyme 
FAD and requires NADPH as a cofactor; its purification, 
characterization, kinetic mechanism, and catalytic properties have 
been reviewed by Ziegler (1980, 1984) and Poulsen (1981).  The 
physiological substrate for the enzyme is believed to be 
cysteamine, which is oxidized to cystamine. 

    Although the FMO does not catalyse oxidation reactions at 
carbon as does the P-450 system, it can oxidize certain nitrogen-
containing (Ziegler, 1984), sulfur- and selenium-containing 
(Poulsen, 1981; Ziegler, 1984), and phosphorus-containing 
xenobiotics (Smyser & Hodgson, 1985).  Tertiary amines (e.g., 
dimethylaniline, chlorpromazine, imipramine) are oxidized to 
fairly stable amine oxides by this enzyme and almost all 
 N,N-disubstituted alkylamines and arylamines that do not have 
negatively charged functional groups are substrates (equation 2). 

    R3N -----> R3N+ -O-                                        (2)

    Secondary amines, such as  N-benzylamphetamine and 
desimipramine, are rapidly  N-oxygenated through this enzyme.  The 
initial oxidation product is the corresponding hydroxylamine, which 
is further oxidized to a nitrone (equation 3).                     

                FMO                   FMO
    RCH2NHCH2R ----->  RCH2N(OH)CH2R ----->  RCH=N+ (O-)CH2R  (3)

    Aliphatic nitrones readily decompose in aqueous solution to 
produce aldehydes and primary hydroxylamines. 

    Only primary amines that readily form imine tautomers, such as 
2-naphthylamine, are substrates for the FMO.  Consequently, the 
 N-oxygenation of most primary xenobiotic aromatic amines is 
catalysed by the P-450 system. 

    Other nitrogen-containing substrates for the FMO are 
1,2-disubstituted hydrazines and  N-substituted aziridines (Prough, 

    Since there is a large number of drugs and environmental 
pollutants that contain sulfur, it is of considerable interest that 
FMO preferentially catalyses the oxidation of sulfur atoms in 
compounds that contain both sulfur and nitrogen (Poulsen, 1981).  
FMO is apparently a more universal sulfur oxidase than P-450.  
Thus, it catalyses the oxidation of:  alkyl and aryl thiols, free of 
anionic groups initially, to the disulfide; alkyl and aryl 
disulfides to sulfinic acids; cyclic disulfides to sulfoxides; 
thiocarbamides or thioureas to formamidine sulfonic acids, through 
intermediate sulfenic and sulfinic acids; and thioether containing 
organophosphates or carbamates to their corresponding sulfoxides 
(Poulsen, 1981; Ziegler, 1984; Tynes & Hodgson, 1985).  The 
oxidation of thiocyanates, carbodithoic acids, and dithiocarbamates 
is also catalysed by this enzyme. 

    It is apparent that FMO is an important enzyme for the 
metabolism of several classes of xenobiotics.  However, the 
relative importance of FMO in toxication/detoxication is still 
being evaluated.  Because some compounds are susbstrates for both 
the FMO and the P-450 monooxygenases, it may be necessary to 
determine which one of these pathways (or both) is responsible for 
the oxidative metabolism of a given toxicant.  Fortunately, 
several methods are now available to distinguish between the two 
systems.  For example, only the P-450 system is induced following 
the administration of compounds such as PB or 3-MC, P-450-dependent 
activity can be selectively inhibited by carbon monoxide, by 
lipophilic primary alkylamines, or by specific antibodies to 
NADPH-P-450 reductase (Ziegler, 1984; Tynes & Hodgson, 1985).  
Trypsin proteolysis of mouse liver microsomes, under appropriate 
conditions, also removes NADPH-P-450 reductase activity leaving FMO 
activity intact (Tynes & Hodgson, 1985). 

    Not much is known about the number of different forms or 
isozymes of FMO and their endogenous regulation.  However, recent 
reports have demonstrated the presence of the enzyme in the lung of 
rabbit and mouse in forms that are immuno-chemically and 
catalytically different from those in the liver (Williams et al., 
1984b; Tynes et al., 1985).  Moreover, the rabbit pulmonary isozyme 
is induced during pregnancy, whereas that in liver is not (Williams 
et al., 1984b). 

    It is worth reemphasizing that P-450 isozymes catalyse several 
reactions not catalysed by FMO.  These include aliphatic 
hydroxylation, aromatic hydroxylation, epoxide formation, and 
 N-,  O-, and  S-dealkylation reactions that occur via rearrangement 
of initial alpha-carbon hydroxylation products (i.e., FMO does not 
catalyse oxidation at carbon).  Cooxidation by prostaglandin H synthase (EC

    Marnett et al. (1975) originally demonstrated, in preparations 
of sheep seminal vesicles, that several organic chemicals, 
including benzo( a)pyrene, are oxidized during the prostaglandin H 
synthase-catalysed conversion of arachidonic acid to 
prostaglandins.  Over the last 5 years, many  in vitro studies have 
focused on this metabolic pathway because of its potential 
toxicological importance.  This reaction converts certain 
xenobiotics to electrophilic metabolites (Marnett, 1981, Marnett & 
Eling, 1983; Krauss & Eling, 1984).  Prostaglandin H synthase 
catalyses 2 distinct enzymatic reactions; its cyclooxygenase 
activity converts arachidonic acid to prostaglandin (PG)G2, a 
hydroperoxyendoperoxide, and its peroxidase activity reduces (PG)G2 
to (PG)H2, a hydroxyendoperoxide.  Xenobiotic oxidation is 
catalysed by the hydroperoxidase of prostaglandin H synthase, and 
the reaction is called cooxidation (Marnett, 1981; Marnett & Eling, 
1983).  The reduction of (PG)G2 by prostaglandin H synthase 
requires the donation of single electrons and these can come from 
the substrate that is cooxidized, though this is not always the 
case (Marnett & Eling, 1983).  Many xenobiotics that mediate toxic 
responses, including acetaminophen, 2-aminofluorene, 2-amino-4-(5-
nitrofuryl)thiazole, diethylstilbestrol (DES), benzo( a)pyrene 
7,8-dihydrodiol, 7,8-dihydrobenzo( a)pyrene, and 4-phenetidine, are 
known substrates for this reaction, and 2-aminofluorene and benzo
( a)pyrene 7,8-dihydrodiol are converted to potent mutagens by 
it (Krauss & Eling, 1984). 

    Prostaglandin H synthase activity is high at several 
extrahepatic sites that are low in P-450 monooxygenase activity. 
These include skin, kidney medulla, lung of certain species, 
platelets, and the endothelial cells lining blood vessels.  Thus, 
it is possible that prostaglandin H synthase complements and/or 
serves as an alternative to, the P-450 monooxygenases for the 
metabolic activation of certain carcinogens; the relative 
importance of these 2 pathways for the formation of ultimate 
carcinogens  in vivo is still not known.  However, this one electron 
oxidation pathway cannot be ignored when reactions for the 
formation of toxic xenobiotics are being considered. 

    Ram seminal vesicles are very rich in prostaglandin H synthase 
and buffered incubation mixtures containing substrate, arachidonic 
acid, and ram seminal vesicle microsomes are excellent for testing 
for prostaglandin H synthase-dependent cooxidation (Marnett & 
Eling, 1983; Krauss & Eling, 1984).  Miscellaneous peroxidative pathways

    Phenols and arylamines are excellent substrates for peroxidase-
catalysed one electron oxidation, and complex product mixtures 
often result from the interactions of the free radicals produced.  
A few examples of the role of peroxidases (EC in toxicity 
are described below. 

    Certain arylamines cause methaemoglobinaemia, which is 
currently believed to be due to their oxidation by oxyhaemoglobin 
in the erythrocyte (Eyer, 1983).  Haemoglobin in erythrocytes has 
also been shown to activate styrene to metabolites that cause 
sister chromatid exchanges (Norppa et al., 1983).  Phenol, 
benzidine, and methylaminoazobenzene are irreversibly bound to 
nuclear DNA of polymorphonuclear leukocytes, and this reaction is 
catalysed by myeloperoxidase-endogenous hydrogen peroxide in these 
cells (O'Brien, 1985).  DES is converted to  Z,Z-dienestrol by PGHS 
and estrogen-inducible peroxidases (Metzler, 1984), and this 
peroxidative pathway is the only oxidative route for DES metabolism 
that has been demonstrated in the fetal mouse reproductive tract, 
the site for transplacental carcinogenesis.  This implies, but does 
not prove, that peroxidases play a role in DES tumourigenesis. 

    These few examples have been given only to emphasize that H2O2-
dependent peroxidases can activate certain classes of xenobiotics 
to reactive chemical species, which in turn may mediate a toxic 
response.  Consequently, the potential of peroxidases to contribute 
to xenobiotic activation must be considered.  Alcohol dehydrogenase (EC and aldehyde
dehydrogenase (EC

    An important metabolic pathway for alcohols and aldehydes is 
oxidation to aldehydes and ketones, and to carboxylic acids, 
respectively.  Mammalian liver alcohol dehydrogenase is a zinc-
containing, cytosolic NAD+-dependent enzyme that occurs as a family 
of isozymes, and which catalyses the oxidation of primary and 
secondary aliphatic, arylalkyl, and cyclic alcohols (Bosron & Li, 
1980; McMahon, 1982).  Although the enzyme is widely distributed in 
mammalian tissues, it is found in highest concentrations in the 
liver.  As illustrated in equation (4), this enzyme also catalyses 
the reverse reaction, by which aldehydes are reduced to primary 
alcohols, in the presence of NADH+. 

    CH3CH2OH + NAD+ <----->  CH3CHO + H+ + NADH               (4)

    However, the  in vivo reduction of aldehydes by this enzyme is 
not normally a quantitatively important metabolic reaction. 

    Isozymes of aldehyde dehydrogenase are also widely distributed 
in mammalian tissues, but with the highest concentrations 
occurring in the liver.  The typical cytosolic aldehyde reductase 
requires NAD+ as a cofactor, and aliphatic and aromatic aldehydes 
are readily oxidized to carboxylic acids, as shown in equation (5). 

    CH3CHO + NAD+ <----->  CH3COOH + H+ + NADH                (5)

    For review articles, see Weiner (1980) and McMahon (1982). 

    Although this is a reversible reaction, the carboxylic acids 
formed are either converted rapidly to their ester glucuronide 
derivatives (catalysed by UDP-glucuronosyl-transferase) (section or they are polar enough to be excreted unchanged.  
Consequently, the reverse reaction is generally not of importance 
 in vivo. 

    The general concensus is that aldehyde dehydrogenase is the 
major enzyme functioning during the  in vivo oxidation of aldehydes 
(McMahon, 1982).  However, both aldehyde oxidase (Rajagopalan, 
1980; Weiner, 1980) and xanthine oxidase (Rajagopalan, 1980) also 
catalyse the oxidization of aldehydes  in vitro and may contribute 
to their oxidation  in vivo. Monoamine oxidase (EC

    The monoamine oxidases are localized in the outer membrane of 
the mitochondrion and are widely distributed in most mammalian 
tissues, exceptions being the erythrocyte and plasma.  This enzyme 
system catalyses the oxidative deamination of a wide variety of 
monoamines of both endogenous (e.g., neurotransmitter amines, 
amines formed by gastrointestinal microflora) and exogenous origin, 
as shown in equation (6). 

    RCH2NH2 + O2 + H2O  ----->  RCHO + NH3 + H2O2              (6)

    For reviews, see Tipton (1980) and Fowler & Ross (1984). 

    Primary aliphatic amines larger than methylamine are deaminated 
by this enzyme system; alpha-alkyl-substituted amines are not 
substrates, however.  Amines containing an aryl group are also 
oxidized to aldehydes, but there must be at least one unsubstituted 
methylene group between the aromatic and amine moieties.  Monoamine 
oxidases can also metabolize some secondary (e.g., isoamylamine; 
 N-methylbenzylamine) and tertiary amines, with allylic or methyl 

    These enzymes are flavoproteins that contain one molecule of 
FAD per molecule and consist of two subunits of nearly identical 
size.  There are 2 forms (A and B) of monoamine oxidase, and the 
relative proportions of the two enzymes vary in different species 
and in different tissues of the same species.  The A form of the 
enzyme is most active (lower Km) with amines generally believed to 
be neurotransmitters (e.g., serotonin, norepinephrine, 
epinephrine), whereas the B form is more active with xenobiotic 

amines (e.g., benzylamine, 2-phenethylamine).  However, this is an 
oversimplification and considerable substrate overlap is known 
(Fowler & Tipton, 1984); for example, both A and B forms of 
monoamine oxidase from rat liver oxidize dopamine and tyramine.  Reduction reactions

    Several functional groups, including nitro, azo, tertiary amine 
 N-oxide, aldehydes, ketones, sulfoxides, and alkyl polyhalides, 
are reduced by mammals  in vivo and tissue preparations  in vitro. 
The realization that free radicals are sometimes formed as labile 
intermediates during reductive metabolism, and contribute to 
toxicity, has markedly increased interest in this area of research 
(Mason, 1980; Hewick, 1982; Kalyanaraman, 1982; Anders, 1984; 
Ziegler, 1984).  Although some of the reactions occur under aerobic 
conditions  in vitro, anaerobic conditions facilitate formation of 
amines from nitrocompounds or for the cleavage of azo compounds.  
Consequently, it is recognized that the intestinal microflora are 
important for reductive metabolism  in vivo (Scheline, 1973, 1980; 
Goldman, 1982).  Cytochrome P-450-dependent reactions

    Several reduction reactions occur when hepatic microsomes are 
incubated with NADPH (or NADH), under anaerobic or aerobic 
conditions.  Such reactions can be catalysed by the P-450 
monooxygenase system or only by its flavoprotein component, NADPH-
P-450 reductase.  If the reaction is inhibited by carbon monoxide, 
P-450 is known to be involved.  Microsomal NADPH-dependent 
reactions not inhibited by carbon monoxide are generally due to the 
flavoproteins NADPH-P-450 reductase, and to a lesser extent, NADH-
cytochrome b5 reductase. 

    In addition to being oxidatively metabolized, many 
polyhalogenated alkanes are converted by a P-450-dependent, one-
electron reduction pathway to a free radical intermediate and 
inorganic halide (Anders, 1984; Anders & English, 1985).  The 
radical formed may, in turn, abstract a hydrogen atom from 
microsomal lipid (initiating lipid peroxidation) to form a reduced 
alkyl halide, undergo alpha- or beta-elimination to form a carbene 
or alkene, respectively, or undergo a second one-electron reduction 
reaction to form a carbanionic intermediate.  The latter forms a 
reduced alkyl halide on reaction with a proton. 

    The best studied example of this reaction is the reduction of 
carbon tetrachloride (CCl4) to chloroform (CHCl3), which occurs 
 in vitro under aerobic or anaerobic conditions and  in vivo.  The 
trichloromethyl radical formed (-CCl3) is believed to be a major 
contributor to CCl4-mediated hepatotoxicity.  Halothane, 
trichlorofluoromethane, hexachloroethane, pentachloroethane, and 
DDT are also dehalogenated by this P-450-dependent reductive 

    Several other classes of xenobiotics are also efficiently 
reduced by the P-450 monooxygenase system, under anaerobic 
conditions.  These include tertiary amine  N-oxides (converted to 
tertiary amines), hydroxylamines (primary amines), hydrazo 
derivatives (primary amines), and epoxides (unsaturated 
hydrocarbons).  Flavoprotein-dependent reactions

    The first step of the NADPH-dependent reduction of aromatic 
nitro and azo compounds by hepatic microsomes is not inhibited by 
carbon monoxide, indicating that NADPH-P-450 reductase (EC 
catalyses these reactions.  The reduction of aromatic nitro 
compounds to primary amines normally proceeds via intermediate 
nitroso and hydroxylamine derivatives, and that of aromatic azo 
compounds via hydrazo intermediates.  However, there is now 
convincing evidence that the initial step in these reactions is the 
formation of an anion radical (nitro anion free radical, RNO2-, and 
azo anion free radical, R- N-NR1, respectively) which is formed by 
a one-electron transfer from a reduced flavoprotein (Mason, 1980).  
In the presence of oxygen, the anion radicals are rapidly 
reoxidized to the parent aromatic nitro or azo compound, 
concomitant with the generation of the superoxide anion radical (O2-).  
This futile cycling explains the toxicity of compounds, such as 
nitrofurantoin and paraquat, which is due to superoxide generated 
under conditions in which little or no metabolism of the xenobiotic 
is apparent. 

    NADPH-P-450 reductase is widely distributed in mammals, and 
consequently, these potentially toxic reactions occur in different 
tissues and subcellular organelles.  For example, Moreno et al. 
(1984a,b) have documented the formation of nitro anion radicals 
(from nitrofurantoin and nitrofurtinox) and azo anion radicals 
(from arsenazo III) in the outer membrane of rat liver 

    Under anaerobic conditions, and with the appropriate cofactor, 
the cytosolic fraction of mammalian liver will also reduce many 
classes of xenobiotics (for a more detailed discussion, see Hewick 
(1982)).  These include aromatic nitro and azo compounds, amine 
 N-oxides, sulfoxides, nitrosamines, and hydroxamic acids.  Of 
these, the reduction of aromatic nitro and azo compounds and of 
nitrosamines are important toxication-detoxication reactions.  
Three flavoproteins present in cytosol are believed to function as 
nitro-reductases; two (xanthine oxidase and aldehyde oxidase) 
contain molybdenum and the third is DT-diaphorase (EC, 
NADH or NAD(P)H dehydrogenase, quinone).  Several exogenous nitro 
compounds are known to serve as electron acceptors for xanthine 
oxidase (EC as do certain  N-oxides.  Aldehyde oxidase 
(EC can also utilize a number of electron acceptors, which 
are reduced during the oxidation of aldehydes.  These include 
sulfoxides, nitrosamines, hydroxamic acids, azo dyes, and aromatic 
nitro-compounds (Kitamura & Tatsumi, 1984).  DT-diaphorase can 
utilize electrons equally well from NADPH or NADH and reduces 
quinones to hydroquinones and catechols, and nitro compounds to 
their hydroxylamine derivatives.  Carbonyl reductases

    As mentioned earlier (section, both alcohol and 
aldehyde dehydrogenases can function as reductases in the presence 
of NAD+.  In addition, there are a number of other carbonyl 
reductases that are NADP+-dependent.  These enzymes have been 
classified into two groups, aldehyde reductases (EC and 
carbonyl reductases (EC  Aldehyde reductases are 
localized in the cytosol, have a broad sub-strate specificity, a 
monomeric structure of low relative molecular mass and are widely 
distributed in extrahepatic tissues (von Wartburg & Wermuth, 1980).  
They reduce aromatic and aliphatic aldehydes as well as some 

    Carbonyl reductases share many properties with aldehyde 
reductase, for they prefer NADP+ as a cofactor, have low relative 
molecular massess, are localized in the cytosol and widely 
distributed in mammalian tissues (Felsted & Bachur, 1980).  
However, the carbonyl reductases differ from aldehyde reductases in 
substrate specificity and inhibitor selectivity.  In general, 
aldehyde reductases reduce only aldehydes whereas carbonyl 
reductases reduce both aldehydes and ketones.  Insensitivity to 
inhibition by barbiturates also differentiates carbonyl reductases 
from aldehyde reductases. 

    The reduction of carbonyl-containing xenobiotics is an 
important metabolic pathway  in vivo (McMahon, 1982), and it 
appears that the NADP+-dependent enzymes are primarily responsible 
for the catalysis of this reaction.  Hydrolysis reactions

    Certain xenobiotics, such as esters and amides, undergo 
hydrolysis, when administered to animals.  Hydrolysis reactions can 
also be important for the sequential metabolism of chemicals that 
are converted to epoxides by the P-450 system.  These reactions are 
classified as phase I because they release functional groups 
(RCOOH, RNH2, ROH) that are sites for conjugation (phase II) 
reactions.  Epoxide hydrolase (EC

    Epoxide hydrolases catalyse the hydration of epoxides to trans-
dihydrodiols and they are important enzymes in toxication-
detoxication processes (Hammock et al., 1980; Lu & Miwa, 1980; 
Oesch, 1980; Guengerich, 1982; Hernandez & Bend, 1982; Timms et 
al., 1984).  Unsaturated aliphatic and aromatic hydrocarbons are 
converted to epoxides (alkene and arene oxides, respectively) by 
P-450 monooxygenases.  Certain of these electrophilic epoxides 
react covalently with macro-molecules, including protein and DNA, 
and they can produce acute or chronic toxicity, including necrosis, 
mutagenesis, carcinogenesis, and teratogenesis (Daly et al., 1972).  
In most cases, the diols produced by epoxide hydrolase are less 
toxic than the substrate.  However, with some polycyclic aromatic 
hydrocarbons, the diols are precursors for potent carcinogenic and 

mutagenic products.  For example, benzo( a)pyrene 7,8-dihydrodiol 
is converted to highly toxic benzo( a)pyrene 7,8-dihydrodiol-9,10-
epoxides by the P-450 system (Jerina et al., 1976) or prostaglandin 
H synthase (Marnett & Eling, 1983). 

    There are two distinct types of epoxide hydrolases, both of 
which are widely distributed in mammalian tissues.  One type is 
localized primarily in the endoplasmic reticulum, although 
immunochemically related activity is also found in the nuclear 
membrane.  A second type of epoxide hydrolase is localized in the 
cytosol (Hammock et al., 1980).  The microsomal and cytosolic 
enzymes have different properties, including substrate 
specificities.  Thus, styrene 7,8-oxide is only a substrate for the 
microsomal hydrolases, whereas trans-beta-methylstyrene 7,8-oxide 
is hydrated primarily by the cytosolic enzyme(s).  Several inducers 
of xenobiotic metabolizing enzymes, including PB, 3-MC, Aroclor(R) 
1254 and trans-stilbene oxide induce microsomal, but not cytosolic, 
epoxide hydrolase activity (Timms et al., 1984).  There is also 
evidence of more than one form of microsomal epoxide hydrolase 
(Guengerich, 1982; Watabe et al., 1983; Timms et al., 1984).  Carboxylesterases/amidases

    Many xenobiotic esters and amides are hydrolysed  in vivo.  
These reactions are discussed together because highly purified 
carboxylesterases have been demonstrated to cleave carboxylesters, 
carboxyamides, and carboxythioesters, producing a carboxylic acid 
and an alcohol, amine, or mercaptan, respectively (Junge & Krisch, 
1975; Heymann, 1980, 1982).  Carboxylesterase refers to a wide 
variety of enzymes that have esterase (and amidase) activity.  
Esterases are divided into three groups on the basis of their 
substrate specificity, and the  in vitro effects of paraoxon and 
Hg2+, but this classification must not be regarded as absolute.  
A-esterases (EC; arylesterases) preferentially hydrolyse 
aromatic esters, hydrolyse the organophosphate paraoxon, and are 
inhibited by Hg2+; B-esterases (EC; carboxylesterases) 
preferentially hydrolyse aliphatic esters, are inhibited by 
paraoxon, and are not influenced by Hg2+; and C-esterases (EC; acetylesterases) preferentially hydrolyse esters of acetic 
acid, are activated by Hg2+ and are not influenced by paraoxon 
(Heymann, 1980).  The B-esterases are the most important group for 
the metabolism of xenobiotics.  This class includes enzymes with 
carboxylesterase/amidase, cholinesterase, and arylamidase 

    B-type esterases are present in almost all mammalian tissues, 
occur as multiple isozymes, and are concentrated in the liver.  
They are localized predominantly in the endoplasmic reticulum of 
the liver and other tissues.  The B-type esterase activity present 
in plasma is probably due to release of liver isozymes. 

    Hydrolysis of esters and amides can lead to either detoxication 
or metabolic activation.  For example, hydrolysis of hydroxamic 
acids has been implicated in the formation of proximate mutagens 

(Thorgeirsson et al., 1980).  The functional groups uncovered 
during hydrolysis normally undergo phase II metabolism, as 
discussed below. 

6.2.2.  Phase II reactions

    Most phase II reactions markedly increase the water solubility 
of xenobiotics.  Exceptions are acetylation and methylation 
reactions.  UDP-glucuronosyltransferase (EC

    Probably the most common conjugation reaction is the synthesis 
of glucuronic acid derivatives (glucuronides) of both endogenous 
and exogenous compounds.  Aliphatic alcohols, phenols, carboxylic 
acids, mercaptans, primary and secondary aliphatic amines, and 
carbamates are converted to their beta-glucuronide derivatives by 
UDP-glucuronosyltransferase (UDP-GT).  This enzyme also exists in 
the form of several isozymes, is widely distributed in mammalian 
tissues but is most concentrated in the liver.  UDP-GT activity is 
primarily localized in the endoplasmic reticulum (microsomal 
fraction of tissue homogenate) (Dutton, 1980; Kasper & Henton, 
1980; Burchell, 1981; Caldwell, 1982a; Mulder, 1982; Burchell et 
al., 1985). 

    UDP-GT catalyses the translocation of glucuronic acid to a 
substrate from the cosubstrate UDPGA (UDP-alpha-glucuronic acid) as 
shown in equation (7). 

    R-OH + UDPGA  ----->  R-O-Glucuronide + UDP                (7)

    During the reaction, inversion occurs resulting in the 
formation of beta-D-glucuronides.  Glucuronide conjugates excreted 
in the bile can be hydrolysed to their aglycone by beta-
glucuronidase in the intestinal microflora.  The released 
xenobiotic (i.e., aglycone) can then be reabsorbed, and the cycle 
repeated.  This process is called enterohepatic circulation and 
accounts for the prolonged excretion of some xenobiotics that are 
readily metabolized (section 

    Certain glucuronides are electrophilic in nature and glucuronic 
acid serves as a leaving group during chemical reaction (Stogniew & 
Fenselau, 1982).  Consequently, glucuronides may function in 
toxication processes.  Consistent with this hypothesis, van Breeman 
& Fenselau (1985) recently demonstrated the covalent binding of the 
aglycone portion of several carboxylic acid glucuronides to 
nucleophilic sites on serum albumin via transacylation reactions.  Sulfotransferases

    Another very common phase II reaction for phenols is the 
conjugation with sulfate to form sulfate monoesters.  Other 
xenobiotic substrates for this pathway include aliphatic alcohols, 
primary and secondary amines, hydroxylamines, and sulfhydryl 
compounds, such as thiophenols.  These reactions are catalysed by a 

family of cytosolic enzymes, the sulfotransferases, which require 
3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the cofactor (for 
reviews, see Jakoby et al. (1980, 1984a) and Mulder (1981, 1982, 
1984).  The reaction is shown in equation (8). 

R-OH + PAPS  ---->  R-O-SO3-  + Adenosine 3',5'-Bisphosphate   (8)

    The sulfotransferases have been divided into several groups as 
a result of substrate specificity determinations with purified 
enzymes (Jakoby et al., 1984a).  The aryl sulfotransferases (EC are active with phenols, hydroxylamines (e.g.,  N-hydroxy-
2-acetylaminofluorene), tyrosine esters, and catecholamines; the 
alcohol sulfotransferases (EC are active with primary 
and secondary steroid alcohols; and amine sulfotransferases 
(EC are active with arylamines (to form sulfamates), an 
activity not catalysed by either purified aryl or alcohol 

    Certain sulfate esters are chemically reactive and can alkylate 
nucleophilic sites on macromolecules (Mulder, 1981).  Thus, these 
phase II-metabolites may be involved in toxicity. 

    As mentioned previously, phenols are substrates for both
sulfotransferases and glucuronosyltransferases.  Generally, 
glucuronide metabolites will predominate after administration of a 
phenol or phenolic precursor to mammals, because sulfate formation 
is a high affinity, low capacity system, whereas glucuronidation is 
a lower affinity, high capacity system.  Sulfation is a low 
capacity system due to depletion of sulfate (Mulder, 1981).  Mercapturic acid biosynthesis

    A large variety of compounds, mostly xenobiotics, are excreted 
in urine as mercapturic acid derivatives, which are chemically 
 S-substituted- N-acetylcysteines (Fig. 3).  This is a very 
important reaction in detoxication because the xenobiotics that 
participate in the initial reaction, the formation of  S-substituted 
GSH derivatives, are electrophilic (Wood, 1970; Chasseaud, 1976, 
1979; Tate, 1980).  The first step in mercapturic acid formation, 
illustrated in Fig. 3, is catalysed by the GSH  S-transferases, and 
the endogenous tri-peptide GSH (L-gamma-glutamyl-L-cysteinyl-
glycine) is a required cosubstrate.  Subsequently, the glutamic 
acid residue is removed from the  S-substituted GSH conjugate by 
gamma-glutamyltranspeptidase, an enzyme with very high activity in 
the kidney, but which is also present in liver.  Next, the glycine 
moiety is removed by as yet unspecified dipeptidases, which have 
cysteinylglycinase activity.  The resulting  S-substituted cysteine 
is converted to the corresponding mercapturic acid by 

    Although xenobiotic mercapturic acids are normally the major 
thioether products present in urine, smaller amounts of the 
cysteine conjugates are also frequently excreted, and in at least 
one fish species, they are the major urinary metabolites (Yagen et 
al., 1984).  All 4 thioethers formed during mercapturic acid 
biosynthesis are routinely excreted in bile. 

FIGURE 3  Glutathione  S-transferases (EC

    The GSH  S-transferases are a family of dimeric proteins in the 
cytosolic fraction of mammalian liver and extrahepatic tissues 
(Jerina & Bend, 1977; Jakoby, 1978; Jakoby & Habig, 1980; Smith & 
Litwack, 1980; Mannervik, 1985).  However, some GSH  S-transferase 
isozymes are also localized in microsomes (Morgenstern & DePierre, 
1985) and within the mitochondrial matrix (Sies et al., 1980) of 
the liver.  A wide variety of functional groups is converted to 
 S-substituted GSH adducts by this family of enzymes, and the 
reactions fall into two general groups, namely the displacement of 
good leaving groups from carbon or heteroatoms by the nucleophilic 
attack of the GSH thiol group or its addition to activated double 
bonds. Leaving groups from saturated carbon centres include 
halogen, sulfate, sulfonate, phosphate and nitro groups, and these 
reactions are facilitated if the carbon atom is benzylic or 
allylic.  Halogens are readily displaced from aromatic compounds as 
long as they are activated by the presence of electron-withdrawing 
groups (e.g., nitro).  Strained rings such as alkene and arene 
oxides and four-membered lactones are readily cleaved by GSH 
 S-transferases.  There are also many examples of thiol addition to 
an "activated" double bond having a potent electron-withdrawing 
substituent (e.g., alpha,beta-unsaturated ketones).  The major 
factor in the transferase-catalysed reaction of these substrates 
with GSH is the electrophilicity of the carbon atom where the thiol 

    GSH  S-transferases also catalyse a number of reactions where an 
 S-substituted GSH adduct is not formed, or where this adduct is 
oxidized GSH.  Examples of these reactions include the release of 
nitrate from nitrate esters, the release of cyanide from 

thiocyanates, and the positional isomerism of double bonds in the 
conversion of DELTA5-3-ketosteroids to DELTA4-3-ketosteroids.  Some 
GSH  S-transferases also have peroxidase activity. 

    The role of the GSH  S-transferases in the metabolism of 
electrophilic carcinogens (Chasseaud, 1979) and of alkene and arene 
oxides (Jerina & Bend, 1977; Hernandez & Bend, 1982) has been 
reviewed as has the regulation of GSH content (Reed & Beatty, 
1980).  Although catalysis by GSH  S-transferases is almost always 
associated with detoxication, a few susbstrates (e.g., ethylene 
dihalides) are metabolized to toxic products (Anders, 1984).  Cysteine conjugate beta-lyase/thiomethylation

    In addition to being acetylated to mercapturic acids, some 
 S-substituted cysteine conjugates can also be hydrolysed.  The key 
enzyme in this reaction sequence is cysteine conjugate beta-lyase 
(EC (Tateishi & Shimizu, 1980; Jakoby et al., 1984b), 
which cleaves the cysteine adduct to a free thiol, ammonia, and 
pyruvate (equation 9). 

    RSCH2CH(NH2)COOH ---------->  RSH + NH3 + CH3COCOOH        (9)

    This enzyme is present in the cytosolic fraction of rat liver 
and kidney and also in the microflora of the gut (Rafter et al., 
1983).  The requirement for a good substrate with mammalian enzymes 
is the presence of a good leaving group on the beta-carbon of the 
alpha amino acid; such substrates include  S-4-bromophenyl-L-
cysteine,  S-2,4-dinitrophenyl-L-cysteine,  S-1,2-dichlorovinyl-L-
cysteine and  S-2-benzothiazolyl-L-cysteine (Jakoby et al., 1984b).  
Each of the substrates of beta-lyase inactivates the enzyme by a 
suicidal process, and this occurs once in about every 600 catalytic 

    Since thiols may be toxic and they are more lipophilic than 
their cysteine conjugate precursors, cysteine conjugate beta-lyase 
is generally a toxication pathway.  For example, trichloroethylene 
is metabolized to  S-1,2-dichlorovinyl-L-cysteine which is 
converted by beta-lyase activity to a sulfur-bearing electrophile 
that is implicated in the renal toxicity of both the parent 
compound and its cysteine conjugate. 

    The thiols formed by mammalian or bacterial beta-lyase  in vivo
are substrates for  S-methyltransferase (equation 10), an enzyme 
widely distributed in mammalian tissues. 

RSH +  S-Adenosyl-L-Methionine -->
                        RSCH3 +  S-Adenosyl-L-Homocysteine     (10)

    This pathway accounts for the thiomethyl metabolites formed 
from several classes of xenobiotics.  Thiomethyl metabolites can 
also be further oxidized, presumably by the microsomal flavin-
containing monooxygenase to the corresponding sulfoxide and sulfone 
derivatives (Rafter et al., 1983).  Amino acid  N-acyltransferases

    Several types of xenobiotic carboxylic acids (aromatic, 
heteroaromatic, arylacetic, cinnamic, and aryloxyacetic) are 
conjugated with a variety of endogenous amino acids including 
glycine, glutamine, and taurine, prior to excretion in mammals 
(Caldwell, 1982a).  An amide (peptide) bond is formed between the 
carboxylic acid group and the alpha-amino group of the amino acid 
during conjugation.  The reactions involved in the conversion of a 
carboxylic acid (e.g., benzoic acid) to its glycine derivative 
(hippuric acid) are illustrated in equations (11) to (13). 

    RCOOH + ATP ---->  RCO~AMP + PPi                          (11)

    RCO~AMP + CoA-SH ---->  RCO~S-CoA + AMP                   (12)

    RCO~S-CoA + H2NCH2COOH ---->  RCONHCH2COOH                (13)

    Conversion of the acid to its CoA ester derivative is the rate 
limiting step in this sequence.  The enzyme that catalyses the 
final reaction is acyl-CoA:  amino acid  N-acyltransferase(s), which 
is localized in the mitochondria of kidney and liver.  The amino 
acid substrate specificity, which can vary from species to species, 
resides in the specific  N-acyltransferase that catalyses this 
reaction (Killenberg & Webster, 1980).   N-acetyltransferases (EC

    Biotransformation of the primary amino group of xenobiotics by 
acetylation is a common metabolic pathway, whereas acetylation of 
xenobiotic hydroxyl and sulfhydryl groups is apparently unknown 
(Weber & Glowinski, 1980; Caldwell, 1982b).  Primary aliphatic and 
aromatic amines, sulfonamides, hydrazines, and hydrazides are 
readily acetylated  in vivo, and the reaction is catalysed by 
acetyl CoA:   N-acetyltransferases, as shown in equation (14). 

    RNH2 + CH3CO SCoA ---->  RNHCOCH3 + CoASH                 (14)

    This family of enzymes is cytosolic in nature and is widely 
distributed.  There are also enzymes that hydrolyse  N-substituted 
acetamides (section; and the extent to which a species 
excretes free versus acetylated amines depends on the relative 
rates of the acetylation and deacetylation reactions, on the 
physical and chemical properties of the two products, and whether 
or not the amine is metabolized by competing pathways.  Some 
acetylated hydroxamic acids are chemically reactive and have been 
implicated as ultimate carcinogens (King, 1974).   N- and  O-methyltransferases

    Since  S-adenosyl-L-methionine (Ad-Met)-dependent  S-methyl-
ation has already been discussed (section, it will not be 
discussed in this section.  Other functional groups that are 
methylated include aliphatic and aromatic amines,  N-heterocyclics, 
and mono- and polyphenols.  The most important enzymes for the 

catalysis of these methylation reactions with xenobiotics are 
catechol  O-methyltransferase (EC, histamine 
 N-methyltransferase (EC, and indolethylamine 
 N-methyltransferase (Borchardt, 1980).  These enzymes all catalyse 
the transfer of a methyl group from Ad-Met to phenolic or amine 
substrates ( O- and  N-methyltransferases, respectively).  
Methylation is generally not a quantitatively important metabolic 
pathway for xenobiotics, but it is an important pathway in the 
intermediary metabolism of both  N- and  O-containing catechols and 
amines (Borchardt, 1980; Caldwell, 1982b). 

6.3.  Modulation of Important Metabolic Pathways

    As described above, virtually all of the enzymes that catalyse 
xenobiotic metabolism exist as families containing a number of 
isozymes or forms, often with overlapping but different substrate 
specificity.  Different isozymes of the same enzyme are distributed 
unevenly in various tissues and cells, respond in different ways to 
the administration of enzyme inducers, and are expressed 
differentially as a function of age and sex.  Consequently, these 
and other factors can markedly alter the metabolism and toxicity of 
a xenobiotic. 

6.3.1.  Physiological factors  Age

    A detailed review of the effects of neonatal development on 
xenobiotic metabolism has been published by Klinger (1982).  Only 
low levels of P-450-dependent monooxygenation are detectable in the 
fetal liver of most species.  After birth, a marked increase is 
observed and maximum activities are reached at different times, 
depending on the species and the substrate.  In old age, activities 
decrease again.  Available studies indicate that extrahepatic aryl 
hydrocarbon hydroxylase (AHH) activity generally develops 
postnatally in all organs, in part, parallel to the development in 
the liver. P-450-dependent  N-dealkylations have been investigated 
in many species and with many substrates.  In rats, with all 
substrates, maximum hepatic activities were found from the 30th to 
the 60th day of life, and in mice, in the third week of life. 

    A reaction of toxicological significance is the P-450-dependent 
reduction of carbon tetrachloride (section  Newborn rats 
are resistant to the hepatotoxic action of this substance, because 
of deficient metabolism.  Alcohol dehydrogenase activity in the 
liver increases postnatally in all species that have been 
investigated.  Different isozymes have been detected in mouse and 
man including:  one in fetuses, two in newborn offspring, and four 
in adults.  Old rats and mice eliminate ethanol more slowly than 
young adults.  However, this is due to a decreased liver NAD+ 
concentration rather than to altered alcohol dehydrogenase activity 
(Klinger, 1982). 

    The age-dependence of UDP-GT activity changes concomitantly 
with the expression of different forms of this enzyme.  In rats, 
the late fetal cluster of UDP-GTs (with 4-nitrophenol, 
2-aminophenol, and 1-naphthol as substrates) appears in the last 
five fetal days and before birth reaches levels higher than those 
in the adult.  The activity of neonatal cluster of UDP-GTs (with 
phenolphthalein, testosterone, estradiol, and bilirubin as 
substrates) is low at birth and increases postnatally.  This holds 
true in the glucuronidation of many substances in other species. 

    Epoxide hydrolase, GSH  S-transferases, sulfotransferases, and 
 N-acyl and  N-acetyltransferases also show age-dependent changes 
in activity (Klinger, 1982).  Genetic factors

    Species differences in xenobiotic metabolism are very 
important; both quantitative and qualitative differences are known.  
While this aspect of metabolism is only mentioned here, it must be 
considered when selecting an animal model for studies of xenobiotic 

    Genetic factors are also responsible for important differences 
in xenobiotic metabolism between members of the same species, as in 
the genetic regulation of enzyme induction by the Ah locus (Nebert 
& Negishi, 1984) (section  Defective oxidation of 
debrisoquine occurs in up to 10% of Americans, Europeans, and 
Nigerian Africans, and is due to the absence of a specific isozyme 
of P-450 (Mbanefo et al., 1980; Breimer et al., 1984). 

    Genetic differences in the activity of alcohol dehydrogenase 
in human beings are also well known.  Five percent of Europeans and 
65% of Japanese rapidly convert ethanol to acetaldehyde.  
Similarly, there are marked ethnic differences due to genetic 
polymorphism in  N-acetylation (Weber & Glowinski, 1980), and 90% 
of Japanese, 45% of Europeans, and 18% of Egyptians are classified 
as rapid acetylators. 

    It is also worth noting that the activity of certain metabolic 
pathways can be diminished indirectly by genetic defects.  Thus, 
conjugation of electrophilic xenobiotics with GSH is lower in 
individuals deficient in glucose-6-phosphate dehydrogenases and GSH 
reductase activity (Wellhöner, 1982).  Sex hormones  Sex-linked differences

    Sex-linked differences in P-450-dependent xenobiotic metabolism 
are known in both rats and mice.  After sexual maturation, male 
rats exhibit markedly higher activity with some substrates than 
females; in mice the reverse is true (Klinger, 1982).  In the 
neonatal period, imprinting by androgens is responsible for the 
development of a male type of steroid and xenobiotic metabolism in 
rats (Skett & Gustafsson, 1979), and these increases in metabolic 

activity are associated with isozymes of P-450 that are neonatally 
imprinted by androgens in male rats and mice (Chung et al., 1981). 
Glucuro-nidation activity is also higher in male than in female 
rats and neonatal treatment with estrogens depresses the activity 
in males (Lamartiniere et al., 1979).  Similar sex differences in 
rats are also known for hepatic GSH  S-transferases (Baines et al., 

    In contrast to results found with rats and mice, no differences 
between the sexes are detectable in P-450 and b5 concentrations, 
NADPH-cytochrome c reductase, P-450-dependent AHH, aniline 
hydroxylase, benzphetamine  N-demethylase, 4-nitroanisole 
 O-demethylase and 7-ethoxycoumarin  O-deethylase activities, or 
epoxide hydrolase activity in human liver microsomes.  The pattern 
of the metabolites of benzo( a)pyrene formed is qualitatively 
similar in both sexes (Kremers et al., 1981).   In vivo, there is no 
difference between sexes in the rate of  N-demethylation of 
aminopyrine, measured by the breath test (Pirotte & El Allaf, 
1983).  The elimination of metamizol and caffeine, which are 
demethylated by different P-450-isozymes, is also the same in men 
and women (Simon et al., 1985).  Pregnancy

    In all the species investigated, hepatic drug metabolism is 
generally inhibited in pregnancy (for review, see Kato (1977)).  
Neale & Parke (1973) found a decrease in hepatic P-450 
concentration, and in biphenyl-4-hydroxylase and UDP-GT activities 
in pregnant rats, whereas the activity of biphenyl-2-hydroxylase 
was not inhibited.  In spite of lower activities per kg liver in 
pregnant rats, P-450-dependent biotransformation capacity can be 
the same or even higher because of a greater liver weight (Schlede 
& Borowski, 1974; Symons et al., 1982). 

    Hepatic monoamine oxidase and catechol  O-methyltransferase 
(Parvez & Parvez, 1975) and sulfotransferase (Pulkinen, 1966) 
activities are also inhibited in the rat during pregnancy, whereas 
alcohol and acetaldehyde dehydrogenase activities appear to be 

    However, pregnancy has also been reported to induce microsomal 
monooxygenase activity in a tissue- and isozyme-specific manner.  
Thus, a form of FMO (Williams et al., 1984b) and a specific P-450 
isozyme efficient in the catalysis of prostaglandin omega-
hydroxylase (Williams et al., 1984a) are induced in rabbit lung but 
not in liver during pregnancy. 

    The clearance of drugs that are metabolized is often decreased 
in pregnant women.  A typical example is the markedly delayed 
elimination of caffeine (Knutti et al., 1981).  Thyroid hormones

    Generally, administration of thyroid hormones inhibits some 
P-450-dependent biotransformation reactions and increases others, 
suggesting isozyme selective regulation (Kato, 1977).  The effects 
of T3, which inhibits ethylmorphine  N-demethylation and stimulates 
7-ethoxycoumarin  O-deethylase, were most marked in beta-
naphthoflavone (BNF)-induced rats (Müller et al., 1985).  In 
clinical studies, drug metabolism is generally impaired in 
hypothyroid patients, whereas in hyperthyroidosis both increases 
and decreases are possible.  Corticoid hormones

    Many investigations have been performed a adrenalectomized 
animals (Kato, 1977).  As results have been contradictory, it is 
not clear whether cortisol administration is able to normalize 
decreased activities in adrenalectomized animals.  The induction of 
the isozyme P-450PCN by dexamethasone and pregnenolone 16-alpha-
carbonitrile (PCN) is not a corticoid effect, because endogenous 
corticoids do not induce this form of P-450 (Heuman et al., 1982).  
Corticoid hormones may increase  N-acetylation, since, in patients 
with hyperadreno-corticoidism and in patients treated with 
cortisone, the rate of acetylation of 4-aminobenzoic acid  is 
higher, while it is decreased in Addison's disease 
(hypoadrenocorticoidism).  Pituitary hormones

    ACTH regulates P-450-dependent AHH activity in microsomes 
prepared from the adrenal cortex of rats, whereas 3-MC and 2,3,7,8-
tetrachlorodibenzo- p-dioxin (TCDD) are not effective as inducers 
(Guenthner et al., 1979).  In juvenile, but not in adult, rats, 
ACTH enhances adrenal P-450 concentration and AHH activity with 
DMBA as a substrate (Hallberg et al., 1983).  Immune system

    Treatment with monospecific immunomodulants is known to 
decrease hepatic P-450 concentrations and monooxygenase activity 
(Williams et al., 1981).  Interferon or inducers of interferon also 
have this effect (Singh et al., 1982).  These decreased rates have 
been associated with reduced acetaminophen hepatotoxicity 
following treatment with interferon (Renton & Dickson, 1984).  
However, destruction or removal of the thymus and splenectomy had 
no significant effect on P-450-dependent reactions in rats (Klinger 
et al., 1983). 

6.3.2.  Environmental factors  Enzyme induction

    It is well known that certain types of organic chemicals 
markedly increase the activity of various xenobiotic metabolizing 
enzymes, when administered to mammals.  Compounds that increase 

activity by enhancement of the synthesis of the enzymes involved 
are called enzyme inducers and the process is termed enzyme 
induction (Conney, 1967; Estabrook & Lindenlaub, 1979; Bresnick et 
al., 1984).  More than 300 chemicals are known to be inducers, and 
they include drugs, pesticides, industrial chemicals, and 
polycyclic aromatic hydrocarbons (PAHs).  The importance of 
induction in the study of chemical toxicity is at least two-fold; 
if metabolic pathways that lead to detoxication are induced, 
reduced toxicity is anticipated, but, if more of a compound is 
converted to a toxic metabolite, increased toxicity will normally 
be observed. 

    The chemicals that induce xenobiotic metabolizing enzymes are 
lipophilic in nature, often have a relatively long half-time in the 
animal, and are normally substrates for the P-450 monooxygenase 
system.  Formerly, enzyme inducers were crudely  classified into 
two main groups on the basis of their characteristics; "PB-like" 
and "PAH or 3-MC-like".  Whereas different compounds of the PAH 
class induce the same major isozymes of P-450, there are 
quantitative differences in the relative amounts of the isozymes 
induced.  Moreover, there are other compounds such as PCN, ethanol, 
isosafrole, isoniazid, and clofibrate that also are isozyme-
selective inducers of the P-450 system.  For these reasons, it is 
no longer accurate to simply classify enzymes inducers as "PB-like" 
and "3-MC-like". 

    PB inducers cause a profound proliferation of the endoplasmic 
reticulum in the liver of rats, induce multiple microsomal 
xenobiotic metabolizing enzymes including NADPH-P-450 reductase, 
epoxide hydrolase (Oesch, 1980) and specific isozymes of P-450 (Lu 
& West, 1980; Guengerich et al., 1982; Johnson et al., 1985; Levin 
et al., 1985), of UDP-GT (Burchell, 1981; Burchell et al., 1985) 
and carboxylesterase (Heymann, 1980).  Certain subunits of 
cytosolic GSH  S-transferase are also increased selectively in rat 
liver by PB administration (Jakoby & Habig, 1980; Mannervik, 1985). 
PB-like inducers also show some induction of renal and intestinal 
xenobiotic metabolizing enzymes, but this varies according to the 
enzyme, species, and the amount of inducer administered.  Many 
drugs and pesticides (e.g., DDT) are inducers of the PB class. 

    PAH inducers (e.g., BNF, 3-MC, TCDD, certain PCB, PCDF, and PBB 
congeners) do not cause a profound proliferation of the endoplasmic 
reticulum in the liver of rats and induce fewer microsomal enzymes.  
For example, NADPH-P-450 reductase is not induced and epoxide 
hydrolase may or may not be induced, depending on the inducer, the 
dose, and the age of the rats.  However, PAHs are potent inducers 
of specific isozymes of P-450 (Lu & West, 1980; Guengerich et al., 
1982; Johnson et al., 1985; Levin et al., 1985) and of UDP-GT 
(Burchell, 1981; Burchell et al., 1985), which are not induced by 
PB, and this induction occurs in both hepatic and extra-hepatic 
tissues.  In general, the administration of PAHs induces more 
isozymes of P-450 in the liver than in extra-hepatic tissues 
(Philpot et al., 1985).  The process of enzyme induction can be an 
important determinant of, or contributor to, chemical-mediated 

toxicity.  Induction of certain isozymes of P-450 by PAHs is known 
to be regulated genetically and this occurs at the Ah locus (Nebert 
& Negishi, 1984) (section 

    In contrast, both PAHs and PB induce subunit 1 of cytosolic GSH 
 S-transferases in rat liver (Mannervik, 1985) and neither 
increases sulfotransferase activity in rat hepatocytes (Moldéus et 
al., 1976; Burke & Orrenius, 1978). 

    From the above discussion, it should be obvious that enzyme 
inducers can markedly affect xenobiotic metabolism and toxicity, 
and, for this reason,  in vivo studies with control versus PB- 
and/or PAH-induced animals are common.  The effects of enzyme 
induction on toxicity with a given chemical provide preliminary 
information on the biochemical mechanisms involved and may also 
indicate the best  in vitro system (e.g., PB- or BNF-induced) to 
use for the biosynthesis of toxic metabolite(s).  Inhibition

    Inhibitors are known for all the enzymes that catalyse 
xenobiotic metabolism described in this document (relevant 
references are quoted in appropriate sections).  The P-450 
monooxygenase system has been best studied in this respect and a 
number of compounds are known that inhibit by being suicide 
substrates (Ortiz de Montellano, 1984), by being alternative 
substrates for monooxygenase activity ( in vivo and  in vitro), 
or by forming a spectral complex with P-450, after  in vivo 
administration (Hodgson & Philpot, 1974).  SKF 525-A and piperonyl 
butoxide are both inhibitors of the last type, and they can be used 
in both  in vivo and  in vitro studies to help determine whether or 
not the P-450 system is involved in the metabolic activation of a 

    Frequently, competing metabolic pathways are operative in the 
biotransformation of a xenobiotic or its phase I metabolites.  In 
this case, selective inhibitors can be used to help determine the 
relative importance of one particular pathway compared with another 
in toxication.  In  this context, lipophilic primary alkylamines 
and carbon monoxide selectively inhibit the P-450 monooxygenases 
compared with the FMO (Ziegler, 1984), 2,6-dichloro-4-nitrophenol 
and pentachlorophenol selectively inhibit sulfotransferases 
compared with UDP-GT (Mulder, 1984), cyclohexene oxide selectively 
inhibits epoxide hydrolase compared with the GSH  S-transferases. 
Harmaline and (±)-alpha-methyltryptamine are very selective 
inhibitors of the A form of MAO, whereas imipramine is a selective 
inhibitor of the B form of this enzyme (Benedetti & Dostert, 1985). 

6.3.3.  Pathological factors

    Some pathological influences on xenobiotic metabolism have been 
briefly mentioned above (section 6.3.1) and selected aspects of 
this topic are discussed below.  Pathological conditions, such as 
malnutrition and neoplasia, which exert a complicated or 
contradictory effect on xenobiotic metabolism, are not discussed 

further.  However, it must be realized that these conditions can 
compromise chemical metabolism.  Liver disease

    The liver is the major site of metabolism for most xenobiotics.  
Consequently, liver dysfunction is likely to have detrimental 
effects on this process (Kato, 1977).  Acute viral hepatitis

    In mild, acute viral hepatitis, the P-450 content and the rates 
of AHH and ethylmorphine  N-demethylation in the human liver are 
unchanged (Farrell et al., 1979).  The  in vivo pharmacokinetics of 
drugs in patients with acute hepatitis have been frequently 
determined.  For example, the half-times of hexobarbital (Breimer 
et al., 1975), aminopyrine (Windorfer et al., 1977), and diazepam 
(Klotz et al., 1975) are significantly increased in patients with 
hepatitis.  On the other hand, neither the rate of elimination of 
phenobarbital nor the pattern of excreted metabolites (hydroxylated 
and conjugated metabolites) is changed (Alvin et al., 1975).  
However, it must be remembered that the liver blood flow and the 
binding of drugs to plasma proteins can also change as a result of 
liver disease, and so the observed effects need not be on the 
xenobiotic metabolizing enzymes  per se.  Chronic hepatitis and cirrhosis

    In "active cirrhosis" in human beings, the hepatic content of 
total P-450 and its associated AHH and coumarin 7-hydroxylase 
activities are decreased (Kratz, 1976; Farrell et al., 1979; Brodie 
et al., 1981). 

    Many human  in vivo studies have been carried out.  Changes in 
pharmacokinetics seem to be caused by the diminution of the 
metabolic capacity of the liver rather than by changes in liver 
blood flow (Klotz et al., 1979).  The elimination of drugs 
metabolized by the P-450 monooxygenases is generally delayed, and 
caffeine is more slowly eliminated in patients with cirrhosis 
(Desmond et al., 1980).  An inhibition of glucuronidation is 
indicated  by the decreased excretion of acetaminophen glucuronides 
(Hammer & Prellwitz, 1966). 

    Hepatic acetylation and esterase activities are also lower in 
human beings with cirrhosis, though ethanol oxidation appears to be 
impaired only in advanced cirrhosis (Kato, 1977).  Obstructive jaundice and cholestasis

    In bile duct ligated rats, hepatic P-450 and b5 concentrations 
and NADPH cytochrome c reductase were diminished (Mackinnon & 
Fouts, 1975).  In rabbits with obstructive jaundice, the P-450-
dependent metabolism of various substrates was impaired, the extent 
depending on the substrate and the degree of disease (McLuen & 
Fouts, 1960).  In man, P-450-dependent  N-demethylation was not 

altered in cholestatic hyperbilirubinaemia, as indicated by a 
normal aminopyrine breath test, whereas this reaction was impaired 
in hepato-cellular disease (Hepner & Vessell, 1977).  However, the 
elimination of other drugs can be delayed in cholestasis (Kato, 
1977).  The contradictory results reported are probably because of 
differences in the extent of liver cell damage in the various 
studies as a result of cholestasis.  Kidney disease

    The pharmacokinetics of many drugs is changed in patients with 
renal diseases.  In general, this is due to a diminution in the 
excretion of the unchanged foreign compound and its metabolites 
rather than to inhibition of renal metabolism itself.  
Nevertheless, hepatic xenobiotic metabolism is inhibited in man 
with chronic renal failure and the elimination of many drugs that 
are oxidized, glucuronidated, acetylated, or hydrolysed is delayed 
(Kato, 1977).  In rats with chronic renal insufficiency, the 
hepatic microsomal P-450 concentration and rates of benzphetamine 
and aminopyrine  N-demethylation were lower (Black & Arias, 1975).  
Xenobiotic metabolism has also been investigated in obstructive 
hydronephrotic rabbit kidney; the P-450 concentration and rate of 
biphenyl-4-hydroxylation in renal microsomes were reduced, as was 
the induction of P-450 and acetanilide hydroxylation by 
administration of 3-MC (Zenser et al., 1984).  Diabetes

    Many papers have been published describing xenobiotic 
metabolism in alloxan diabetes.  In the liver of alloxan-treated 
rats, a P-450 isozyme that preferentially catalyses aniline 
hydroxylation was induced (Past & Cook, 1982), whereas the rate of 
 in vitro metabolism of many other substrates was decreased (Kato, 
1977).  In human beings with diabetes, the rate of elimination of 
drugs is diminished or unchanged.  Recent investigations have 
indicated that P-450 function depends on secondary alterations in 
the liver in diabetes.  In liver fibrosis and in "hepatitis", the 
P-450 concentration, and the activities of AHH and 7-ethoxycoumarin 
 O-deethylation are decreased, whereas in steatosis, only the total 
P-450 concentration is reduced (Salmela, 1984). 

    Earlier results concerning the effects of diabetes on UDP-GT 
were contradictory (Kato, 1977), but Price & Jollow (1982) 
demonstrated that the decreased hepatotoxicity of acetaminophen in 
alloxan-induced diabetes was due to increased rates of 
glucuronidation and sulfation and higher GSH levels. 

6.4.  Sampling Procedures for Parent Compounds and Metabolites 
 In Vivo

6.4.1.  Non-invasive procedures

    The terminal products of xenobiotic metabolism are excreted in 
the urine and/or bile.  The biliary metabolites are in turn emptied 
into the small intestine and subsequently excreted with the faeces, 

though it must be remembered that reduction and hydrolysis 
reactions are catalysed by the enzymes of the intestinal flora, 
that nonpolar xenobiotic metabolites can be absorbed through the 
intestinal walls to initiate enterohepatic circulation, and that 
the faecal metabolites result from the combined action of metabolic 
systems in the experimental animal and the intestinal microflora.  
Detailed methods have been described for the collection of both 
urine (Mulder et al., 1981) and faeces (Matthews, 1981).  It is 
advisable to employ a metabolism cage that efficiently separates 
the urine and faeces, otherwise there can be extraction of faecal 
metabolites into the urine (section 7). 

    Some xenobiotics and/or their metabolites are also volatile, 
and the lipid peroxidation of the endoplasmic reticulum, which 
follows the administration of carbon tetrachloride, results in the 
exhalation of alkanes.  If 14C-radiolabelled xenobiotics are 
available for study, 14C-carbon dioxide may also be expired.  Thus, 
under certain conditions, it is advisable to monitor exhaled air 
for the presence of the parent compound, metabolites, and/or 
indicators of toxicity.  Methods for the collection of exhaled 
hydrocarbons have recently been described (Wendel & Dumelin, 1981), 
as have methods for collecting and quantifying exhaled 14C-carbon 
dioxide (Bircher & Preisig, 1981) (section 7.5.1). 

    As described in section 7.3.4, chemicals and/or their 
metabolites are excreted in saliva, milk, tears, and sweat. 
Although the amounts excreted by these routes are minor compared 
with urinary and biliary excretion, the secretions can also be 
assessed for a xenobiotic and its metabolites, by non-invasive 

    Once collected, the fluids containing the chemicals of interest 
should be analysed according to the principles outlined in section 

6.4.2.  Invasive procedures

    The simplest invasive procedure for the sampling of 
xeno biotics/metabolites  in vivo is accomplished by the removal of 
blood with a hypodermic needle and syringe or by cannulation of a 
vein, to allow repeated sampling. 

    There are also several methods for collecting bile.  The 
simplest applies only to experimental animals that have a gall 
bladder, which excludes rats.  An animal can be anaesthesized and 
killed, at a fixed time after treatment with a xenobiotic, and the 
bile carefully removed from the gall bladder by needle and syringe.  
The simplest method for rats is to anaesthetize them, expose the 
bile duct by opening the abdominal cavity, and to cannulate the 
bile duct.  This preparation can be used for up to 8 h, if efforts 
are taken to prevent dehydration and hypothermia.  It is also 
possible to collect bile continuously for several days via a 
permanent biliary cannula fixed to the skull of the rat.  The 
surgical procedures required for bile duct cannulation have been 
described in detail by Mulder et al. (1981). 

    It is also possible to collect urine from the bladder of both 
male and female rats.  In males, the bladder is cannulated 
directly (under anaesthesia), whereas in females, the bladder is 
cannulated via the urethra.  These procedures have also been 
described in detail by Mulder et al. (1981) (section 7.5.1). 

6.5.  Experimental Systems

6.5.1.  Systems with intact cellular structure

    There are several aspects of xenobiotic metabolism and of the 
relationships between this metabolism and toxicity that often make 
studies in systems with intact cellular architecture a requirement 
for elucidating mechanisms of  in vivo metabolism and toxicity.  
First, the biotransformation of a chemical usually requires the 
sequential involvement of several enzymatic pathways, which 
frequently differ in subcellular localization; the interactions of 
the various pathways can best be studied prior to cell disruption. 
Second, individual cell types can serve as sensitive indices for 
chemical-mediated toxicity, and these indices can be pathological, 
biochemical, or physiological.  Finally, cells may contain 
metabolically important enzymatic pathways that are degraded during 
tissue disruption or which, in the absence of the appropriate 
cofactor or subcellular fraction, are not active  in vitro.  Intact animals

     In vivo studies offer the advantage of studying metabolism
under conditions in which the chemical exerts its biological,
possibly toxic, effect.  However, it must be remembered that
the hepatic contribution to xenobiotic metabolism  in vivo
usually predominates and that the metabolites excreted are
generally the products of phase II enzymes (i.e., conjugates).  Isolated organs

    The methods for the use of isolated perfused liver (Meijer et 
al., 1981), kidney (Newton & Hook, 1981), lung (Smith & Bend, 
1981), in situ small intestine (Windmueller & Spaeth, 1981), and 
testis (VanDemark & Ewing, 1963; Lee & Nagayama, 1980) preparations 
in studies of xenobiotic metabolism have been discussed in detail.  
Certain complicating factors inherent to  in vivo studies of 
chemical metabolism are eliminated in perfused organs.  These 
include the concentration of substrate reaching the tissue, 
particularly if it is an extra-hepatic organ, and redistribution to 
and from other tissues.  Other advantages of the use of perfused 
organ systems are that metabolism can be studied solely in the 
organ that shows a toxic response, and that cell-free perfusion 
media can be used allowing easy recovery of both the xenobiotic and 
its metabolites.  Disadvantages include that experience is 
required to successfully perfuse organs, that perfused organs are 
viable only for a limited period of time, and that comprehensive 
analysis of total (i.e., polar and nonpolar) metabolites from all 
compartments (i.e., perfusion medium and organ) in the system is 
necessary to guarantee accurate interpretation of experimental data 
(section  Freshly isolated cells

    Methods for the preparation of suspensions enriched in 
hepatocytes (Fry & Bridges, 1979; Moldéus et al., 1983), alveolar 
type II and Clara cells and alveolar macrophages (Devereux & Fouts, 
1981; Devereux, 1984), interstitial and spermatogenic cells of the 
testis (Mukhtar et al., 1978), tip (differentiated) and crypt 
(undifferentiated) cells from small intestine (Schiller & Lucier, 
1978; Pinkus, 1981), basal and differentiated keratinocytes of skin 
(Coomes et al., 1984; Pohl et al., 1984), and renal cells (Ormstad 
et al., 1981) from rat, rabbit, and/or mouse and for the use of 
these cell preparations in studies of xenobiotic 
metabolism/toxicity appear in the literature (Bend & Serabjit-
Singh, 1984).  Intact cells serve as an experimental system 
intermediate in complexity between the perfused organ and 
subcellular fractions or purified enzymes isolated from homogenized 
whole tissue.  Some advantages of cells are that they routinely 
contain a full complement of the enzymes and cofactors present in 
the intact tissue, that damage to the cells is a sensitive 
toxicological end-point, that all cells are exposed approximately 
equally to added chemicals, and that the isolation/enrichment of 
cell populations of a single type makes it possible to locate the 
quantitatively important activation/detoxication pathways to 
different cell types.  This is of particular interest in tissues 
such as lung and kidney, which consist of many cell types. 

    There are also some disadvantages associated with the use of 
isolated cell preparations for studies of xenobiotic metabolism.  
Mixed-cell populations are of limited use, especially if they are 
not characterized and identified morphologically.  Moreover, the 
enzymatic digestion that is normally used to release cells from the 
basement membrane may cause selective degradation of certain of the 
enzymes (e.g., NADPH-P-450 reductase) that are important in 
xenobiotic metabolism.  The procedures used for cell isolation and 
enrichment, particularly for extrahepatic tissues, are also 
expensive (labour intensive), and the yields of specific cell types 
are often too low for comprehensive studies to be made with cells 
from the same animal or group of animals.  Organs and cells in culture

    Conditions required for the successful culture of epithelial 
cells and whole organs have been developed over the last decade, 
and these preparations are used for the investigation of 
xenobiotic metabolism, particularly of carcinogens, in extrahepatic 
tissues.  The major problem with these systems is in terms of 
extrapolation to the  in vivo situation.  For example, there is 
usually rapid dedifferentiation of cells in culture and the P-450 
monooxygenase system is selectively affected.  Thus, although 
cultured cells and organs make excellent experimental systems for 
mechanistic studies of the relationships between metabolism and 
toxicity, the precise relationships between toxication and 
detoxication reactions are unlikely to reflect those that occur  in 
 vivo in the cells/organs from which cultures were derived.  A 
major advantage of this system is that cells of human origin can be 
studied (e.g., Autrup et al., 1979, 1982). 

6.5.2.  Cell-free systems  Subcellular fractions of tissue homogenate

    One of the easiest methods for studying the specific pathways 
involved in the metabolism of a xenobiotic is to use subcellular 
fractions prepared by differential centrifugation of whole tissue 
homogenate.  As mentioned in the section on enzymatic pathways 
(section 6.2), different enzymes are localized in different 
subcellular fractions; consequently, the concentration of enzymes 
can be increased by using the appropriate subcellular fraction as 
an enzyme source (e.g., microsomal pellet for P-450 monooxygenases, 
FMO, epoxide hydrolase, UDP-GT, and carboxylesterases/amidases).  
The reaction of interest can often be segregated for study from 
other competing or sequential pathways by the addition of a single 
cofactor.  For example, UDP-GT is inactive in microsomes unless 
UDP-glucuronic acid is added.  If a differential tissue 
distribution of the enzymes metabolizing a xenobiotic to a toxic 
metabolite is suspected, the appropriate sub-cellular fraction of 
various extrahepatic tissues can also be conveniently studied.  Purified enzymes and/or reconstituted enzyme systems

    Homogeneous isozymes of xenobiotic metabolizing enzymes are 
advantageous for certain types of experiments.  For example, the 
catalytic properties of individual forms of P-450 can be determined 
in reconstituted monooxygenase systems containing P-450 isozyme, 
NADPH-P-450 reductase, and phospholipid (Lu & West, 1980).  
Similar experiments can be carried out in systems containing 
isozymes of UDP-GT and phospholipid (Burchell, 1981).  With such 
studies, it is possible to assess position-specific metabolism and 
substrate specificity; however, rates of substrate turnover 
sometimes do not agree with those found in microsomes.  This 
information can be important in understanding the metabolic basis 
of toxication/detoxication, particularly if sequential pathways 
are required for metabolic activation. 

6.5.3.  Intestinal microflora

    Intestinal microflora contain enzymes that can metabolize 
xenobiotics by many pathways under the anaerobic conditions that 
are physiological for the intestinal tract (Scheline, 1973, 1980; 
Goldman, 1982; Rafter et al., 1983).  These include hydrolysis 
(beta-glucuronides, sulfates, esters of xenobiotic carboxylic 
acids, amides, and sulfamates),  C-decarboxylation and 
dehydroxylation reactions, dealkylation, dehalogenation, 
heterocyclic ring fission, reduction (nitro groups, azo groups, 
epoxides,  N-oxides, sulfoxides, aldehydes, ketones, and alkenes),
aromatization (cyclohexanecarboxylic acid derivatives), nitrosamine 
formation and degradation, and methylation.  Thus, microflora can 
contribute to the metabolism of many different types of xenobiotics 
 in vivo.  The fact that intestinal microflora are difficult to 
isolate and culture means that the number and diversity of 
organisms may be underestimated. 

    There are many different species of bacteria (up to 60 in human 
beings) (Donaldson, 1964) that can be isolated from the 
gastrointestinal tract.  Because of the acidic nature of the 
stomach contents, most bacteria entering the intestinal tract via 
the mouth are destroyed.  The lower the pH in the stomach (the 
rabbit has a very acidic stomach juice), the fewer organisms 
present.  The microflora in the proximal jejunum of the human and 
in proximal small intestine of the rabbit are sparse, whereas the 
proximal small intestine of mice, rats, and guinea-pigs contain 
greater numbers and varieties of bacteria.  Man and the common 
experimental animal species show marked similarities in the numbers 
and types of organisms in the distal small intestine, and the 
numbers of bacteria increase markedly in this transition zone 
between the small and large intestines.  Drasar et al. (1970) 
reported that faecal lactobacilli and streptococci are much more 
plentiful in mice and rats than in other species; in contrast, 
faeces from guinea-pigs and rabbits contain fewer enterobacteria. 
Thus, the contribution of intestinal microflora to xenobiotic 
metabolism  in vivo, and the portion of the alimentary tract 
involved, may vary from species to species. 

    An  in vivo approach can be used to study the contribution of 
intestinal microflora to xenobiotic metabolism and toxicity.  
Either antibiotics, alone or in combination (e.g., neomycin, 
bacitracin, and tetracycline) (Remmel et al., 1981), are 
administered to an animal to reduce gut flora or gnotobiotic 
(germ-free) animals (Savage, 1977; McLafferty & Goldman, 1981) are 
used.  Germ-free animals are derived by aseptic Cesarean section 
and are raised and kept under conditions that prevent infection 
with bacteria, fungi, parasites, or viruses.  Animals raised this 
way are said to be barrier-derived, and they are available from 
commercial breeders.  Needless to say, they must be kept in an 
aseptic environment, and the xenobiotics administered to them, and 
their food and water must be sterilized. 

    Studies of xenobiotic metabolism in germ-free animals appear to 
be preferable to those in antibiotic-treated animals, because the 
antibiotics used can interfere with distribution and metabolism of 
the xenobiotic (Remmel et al., 1981). 

6.6.  Methods for Assessing Chemically Reactive Metabolites  In Vitro

    It is now known that the cytotoxic, mutagenic, teratogenic, 
immunotoxic, and carcinogenic effects produced following the 
administration of inert or relatively non-toxic chemicals are 
generally related to the formation of reactive electrophiles during 
their metabolism.  These electrophilic products can react 
irreversibly (covalently) with nucleophilic sites on tissue 
macromolecules, such as protein, lipid, RNA, and DNA.  Sometimes 
these covalent interactions are related to the final toxicological 
event (e.g., interaction of ultimate carcinogenic or mutagenic 
metabolite with DNA), but, in other cases, they are not.  In any 
event, the electrophilic nature of many toxic metabolites provides 
a convenient  in vitro method for their detection.  For studies of 
this type, it is very important to use a xenobiotic that is at 

least 99% pure.  Because of the small amount of xenobiotic 
metabolite normally bound covalently to macromolecules, studies are 
greatly facilitated if radiolabelled xenobiotic is available; high 
specific activity is normally required.  Obviously, the radiolabel 
must be in a functional group or position that is both 
enzymatically and nonenzymatically stable (Pohl & Branchflower, 

    Radiolabelled substrate is incubated with purified enzymes, 
various subcellular fractions of tissue homogenate, isolated intact 
cells, or perfused organs, and the appropriate cofactors under 
conditions normally used for studying xenobiotic metabolism.  
Depending on the experimental system, it may be necessary to add 
exogenous protein, lipid, RNA, or DNA.  Precipitation/differential 
extraction or equilibrium dialysis procedures are then performed to 
separate the xenobiotic and its extractable metabolites from 
xenobiotic metabolite covalently bound to macromolecules (for 
experimental details, see Pohl & Branchflower, 1981). 

    To show unequivocally that covalent reaction of a xenobiotic 
metabolite with a macromolecule has occurred, it is necessary to 
hydrolyse the macromolecules and to separate and chemically 
identify the metabolite-modified amino acid, lipid, or nucleoside.  
In a few cases, such products have been rigorously characterized by 
physicochemical techniques (section 5.3).  However, valuable 
preliminary information can be obtained using this experimental 
approach in the absence of such sophisticated analyses. 

    Methods developed to study mutagenicity can also be used for 
the detection of chemically reactive metabolites when radiolabelled 
substrate is not available.  The xenobiotic can be incubated with 
the appropriate subcellular fraction of a tissue, the necessary 
cofactor(s), and one of several detector systems, such as the 
 Salmonella typhimurium tester strains, which have been specifically 
developed for the detection of mutagens (Ames et al., 1975).  This 
approach can also be used with intact cells by co-incubation or co-
culture with appropriate bacterial or human indicator cells (e.g., 
Aune et al., 1985).  One advantage of these procedures is that, 
once established, they are relatively inexpensive.  Thus, it 
becomes feasible to compare various subcellular fractions from 
several tissues and species for the metabolic activation of a 
xenobiotic (and its metabolites) into a mutagenic product(s). 

    Another inexpensive procedure is to compare (both qualitatively 
and quantitatively) the organic extractable metabolites formed in 
a complete incubation mixture (e.g., substrate, NADPH, buffer, 
liver microsomes, incubated at 37 °C for 20 - 60 min) in the 
absence of GSH with those formed in an identical reaction mixture 
containing GSH.  A marked increase in the amount of polar 
metabolites in the incubation mixture containing GSH indicates the 
formation of an electrophile.  Should this be the case, the 
experiment can be repeated with a simpler nucleophile (e.g., 
 N-acetylcysteine), the metabolite-thioether adduct isolated, 

purified, and chemically identified.  On the basis of these data, 
it is possible to predict the structure, or one of several possible 
structures, of the electrophile(s) formed during metabolic 


7.1.  General Considerations

    Chemicals are excreted from the body either unchanged or, more 
frequently, as water-soluble metabolites.  The kidney is an 
extremely efficient organ for the excretion of water-soluble 
compounds.  Other routes are also important for the excretion of 
specific compounds, for instance, the liver and its biliary system 
are important for the excretion of metals, high relative molecular 
mass anions and cations, and most lipophilic substances.  The 
amount of a metabolite excreted in the bile is, to some extent, 
directly proportional to the molecular size.  Enterohepatic 
circulation is responsible for retaining chemicals in the body; 
conjugates may be hydrolysed in the intestine and reasorbed into 
the circulation.  The lungs excrete gases such as carbon oxides, 
and volatile substances such as hydrocarbons, halogenated 
hydrocarbons, alcohols, aldehydes, ketones, and ethers of low 
relative molecular mass.  The stomach and intestine can also act as 
excretory organs for weak organic acids and bases, such as 
4-acetaminobenzoic acid, aniline, nicotine, and acetanilide (Parke, 
1968; Yasuhara et al., 1984).  In addition to the above routes of 
excretion, some chemicals and their metabolites are found, to a 
lesser extent, in saliva, skin glands (sweat), milk, genital 
secretions, hair, pancreatic secretions, and tears (Klaassen, 
1980).  These routes of excretion may indicate localized toxicity. 

7.2.  Important Excretory Mechanisms

    Chemicals are eliminated from the body by various routes.  The 
relative importance of the excretion processes depends on the 
physical and chemical proporties of the compound and its various 
metabolites.  The mechanisms by which a chemical passes through a 
biomembrane can be classified into 2 general types: 

    (a)  diffusion or filtration of the substance, in which
         the cell membrane does not require energy to carry
         out the process; and

    (b)  carrier-mediated transport of the chemical through
         the membrane, in which energy-dependent and
         -independent processes can be involved.

General aspects of diffusion, filtration, and carrier-mediated 
transport have been described earlier (sections 3.1.1, 3.1.2, 
3.1.3).  Aspects that are more closely related to xenobiotic 
excretion are discussed below. 

7.2.1.  Diffusion and filtration

    The kidney and the liver have at least two and three active 
transport systems, respectively.  The systems in the kidney are for 
organic cations and anions, whereas those in the liver are for 
organic cations, anions, and neutral molecules.  It is well known 
that there is a variety of pumps of this type.  Each transports a 
specific type of chemical, e.g., sodium, potassium, magnesium, 

organic acids, and organic bases, and related compounds compete for 
the same transport mechanism.  The system is noncompetitively 
inhibited by interference in energy supply (Meyers et al., 1978). 

    Additional transport systems, phagocytosis and pinocytosis, 
may also be of importance in, for example, the removal of 
particulate matter from the alveoli by alveolar phagocytes, and the 
removal of some large molecules such as the Cd-metallothionein 
complex (Pritchard, 1981) from the body by the reticuloendothelial 
system in the liver and spleen (Klaassen, 1980). 

7.3.  Sites of Excretion

7.3.1.  Kidney

    The kidney is the most efficient organ for the elimination of 
most chemicals from the body.  It receives about 25% of the cardiac 
output, 20% of which is filtered at the glomeruli.  The excretion 
processes involved are passive glomerular filtration, tubular 
reabsorption, and active tubular secretion.  Glomerular filtration

    Small relative molecular mass compounds are readily filtered.  
Some of the chemicals carried by the blood bind tightly to plasma 
proteins, and these binding products and unbound compounds of high 
relative molecular mass with an effective radius greater than 44 A 
are frequently too large to pass through the pores (40 A) of the 
glomerular capillaries (Cafruny, 1971).  Molecules of 20 - 42 A are 
also restricted, the extent of restriction being dependent on 
molecular charge (Brenner et al., 1978; Pritchard & James, 1979).  
Thus, the glomerular wall acts as a size- and charge-selective 
filter.  This can be demonstrated in rats using 3 kinds of dextran 
(neutral, anionic, and cationic) in comparison with albumin 
(Brenner et al., 1978).  The Bowman's capsule filters out 
substances with a relative molecular mass of 66 000 or more, 
including plasma proteins and chemical-protein complexes.  Tubular secretion

    Xenobiotics may be actively taken up by the renal tubular cells 
against high concentration gradients by anion and cation carrier-
mediated processes.  Secretion from these cells into the urine is 
by passive diffusion.  These processes are of relatively low 
specificity, are saturable, and may be inhibited.  Many acidic or 
basic chemicals and their conjugates are removed from the plasma 
by these processes.  It is possible for highly protein-bound 
chemicals to be almost completely cleared in a single passage 
through the kidney, since the dissociaton rate for chemical-albumin 
complex is very high (Renwick, 1982).  Tubular reabsorption

    After the chemical has been filtered at the glomeruli or 
secreted by the tubular cells, it may be excreted or passively 
reabsorbed across the tubular cells of the nephrons into the blood 
stream.  The principles governing the back diffusion of the 
chemical across the tubular cells are the same as those relating to 
any passive membrane transfer.  Therefore, if the material is more 
lipid-soluble, the degree of reabsorption becomes greater.  
Similary, polar compounds and ions will diffuse more slowly and 
therefore will be excreted in the urine.  Since numerous chemicals 
are either weak acids or bases, they exist in a mixture of ionic 
and non-ionic forms, depending on the pKa of the chemicals and the 
pH of the urine.  Generally, the excretion of these chemicals can 
be altered by alkalinization or acidification of the urine. 

    The percentage of the chemical in the ionized form in the 
mammalian urine can be markedly altered by changing the pH.  The 
excretion of acidic compounds is increased if the renal tubular 
fluid is alkaline, because the reabsorption is greatly decreased.  
On the other hand, basic substances are excreted to a greater 
extent if the urine is acidic, because they are then in the ionized 
form (Meyers et al., 1978). 

7.3.2.  Liver-biliary excretion

    In general, lower relative molecular mass anionic and cationic 
compounds are excreted through the kidneys, whereas biliary 
excretion is an important excretion route for many compounds with 
comparatively high relative molecular mass (approximately 300 - 
700).  The metabolites formed in the liver may be excreted directly 
into the bile without entering the blood-stream.  The biliary 
excretion of compounds is influenced not only by hepatic function, 
but also by blood flow.  Current concepts of the formation and flow 
of bile have recently been reviewed (Boyer, 1980; Erlinger, 1981; 
Blitzer & Boyer, 1982; Klaassen & Watkins, 1984). 

    It has been suggested that there are 2 types of bile formation: 
bile salt-dependent and bile salt-independent (Boyer, 1980).  Over 
200 chemicals and/or their metabolites have been detected in the 
bile.  The biliary excretion of chemicals varies considerably among 
species, including human beings (section, and is generally 
high in the dog and the rat (Abou-El-Makarem et al., 1967; Renwick, 
1982; Levine, 1983).  The bile-to-plasma concentration ratios also 
vary markedly from compound to compound.  Enterohepatic circulation

    Some of the compounds excreted as conjugates in the bile are 
hydrolysed in the intestine, reabsorbed in the intestine, and then 
excreted again by the liver into the bile.  This recycling 
phenomenon is referred to as enterohepatic circulation (Plaa, 
1975).  Pancreatic secretions can also contribute to the process, 
which delays the excretion of some chemicals.  Hydrolysis of 
conjugates, particularly beta-glucuronides, by the intestinal flora 

is the most common reaction that contributes to the enterohepatic 
circulation of chemicals.  Administration of a beta-glucuronidase 
inhibitor to rats shortens the duration (and half-time) of the 
pharmacological action of phenobarbital and progesterone, both of 
which are excreted in the bile as glucuronides (Marselos et al., 

    Enterohepatic circulation has been demonstrated for many 
xenobiotics (Levine, 1981); species differences have been seen for 
some compounds (Yesair et al., 1970; Inaba et al., 1974; Sellman et 
al., 1975).  Smith (1973) reported that the main factors that can 
affect the enterohepatic circulation of a chemical are:  the extent 
and rate of excretion of the compound in the bile, the activity of 
the gall bladder, the fate of the substance in the small intestine 
(emptying time, secretion, absorption, potential hydrolysis), and 
the fate of the compound after reabsorption from the gut. 

    The lungs receive the entire cardiac output.  Therefore, 
clearance by the lungs may contribute significantly to the overall 
clearance of some chemicals (Pang, 1983).  The contribution of the 
lung to the metabolic clearance of chemicals from the blood has 
recently been reviewed (Bend et al., 1985).  Chemicals that exist 
predominantly in a gaseous state at body temperature, and volatile 
liquids, are excreted across the pulmonary alveolar membrane by the 
lungs.  The amount of a liquid excreted by the lungs is related to 
its vapour pressure.  Highly volatile liquids, such as ether and 
ethyl chloride, are excreted almost exclusively by the lungs.  They 
are eliminated by simple diffusion.  Elimination of foreign gases 
takes place in almost inverse proportion to the rate of gas uptake. 

    The solubility of the gas in blood is a very important factor 
for the excretion of gases.  Gases with high blood/gas partition 
ratios, such as chloroform, are excreted slowly by the lungs, 
whereas gases with low blood/gas partition ratios, such as 
ethylene, are excreted rapidly.  Trace concentrations of highly 
soluble anaesthetic gases such as halothane and methoxyflurane may 
be present in expired air for a long time after anaesthesia.  This 
prolonged retention results from the accumulation of highly lipid-
soluble chemicals in adipose tissue.  In the case of gases with a 
very low solubility, the rate of transfer is perfusion limited 
(Meyers et al., 1978; Klaassen, 1980). 

7.3.3.  Other excretory sites

    The excretion of chemicals in biological fluids such as saliva, 
milk, tears, and sweat is minor compared with renal excretion.  
However, these fluids are quite important in studies of 
toxicokinetics, because they can be monitored for xenobiotics and 
their metabolites.  Concentrations of chemicals in saliva generally 
reflect the free fractions of the chemical in plasma and can be 
determined by non-invasive techniques.  Because of pH differences 
between saliva (pH 6.7 - 6.9) and plasma, organic bases such as 
nicotine, theobromine, and caffeine tend to be concentrated in the 
saliva, whereas organic acids such as salicylic acid diffuse into 
the saliva less readily (Levine, 1983).  The secretion of chemicals 

into the milk is discussed in another WHO document (WHO, 1985).  
Chemicals e.g., metals (Klaassen, 1980; Scheler, 1980) may also be 
excreted by the mucosal cells of the intestine. 

7.4.  Modulation by Physiological, Environmental, and Pathological 

7.4.1.  Urinary excretion of xenobiotics  pH and urine volume

    As explained previously, the urinary excretion rate can be 
altered by changes in pH (section or urine volume 
(Pritchard & James, 1979; Renwick, 1982) (sections,  Inhibition and stimulation by xenobiotics

    Some inhibitors, for example, 2,4-dinitrophenol, 2,4,5-
trichlorophenoxyacetic acid (2,4,5-T), and 2,2-bis(4-chlorophenyl)
acetic acid (DDA), block the xenobiotic transport and alter renal 
excretion (Anderson & Schrier, 1981; Irish & Grantham, 1981; 
Pritchard, 1981; Hekman & van Ginneken, 1983; Levine, 1983) 

    Renal excretion in the neonate is stimulated by repeated 
administration of the same or other selected xenobiotics. 
Pretreatment of neonatal animals with penicillin or  p-amino-
hippuric acid (PAH) can stimulate the tubular transport of PAH.  
The uptake of PAH in young rabbits is also increased by 
administration of phenobarbital and 3-methylcholanthrene, but this 
is not so in adult rabbits (Kluwe et al., 1978).  This stimulation 
of tubular transport is probaly due to a more intensive synthesis 
of carrier protein (Rennick, 1972).  Age differences

    The renal excretion rate is lower in the neonate and begins to 
increase shortly after birth (Hilligoss, 1980; Braunlich, 1981a,b; 
Irish & Grantham, 1981; Boreus, 1982; Levine, 1983).  Renal 
excretion by glomerular filtration and tubular reabsorption occurs 
to a lesser extent in the neonate.  However, because of the acidic 
urine of neonates, a high reabsorption rate is to be expected for 
organic acids.  Glomerular filtration and tubular secretion may 
diminish in the aged population (Andreasen et al., 1983; Marks, 
1983).  Age differences in plasma-protein binding, blood flow, and 
carrier capacity in the tubular cell may be other variables.  Species differences

    Differences between mammals in the renal excretion of 
xenobiotics tend to be quantitative in nature (Irish & Grantham, 
1981; Renwick, 1982).  Renal dysfunction

    Renal excretion of chemicals is generally decreased in severe 
kidney disease (section 

7.4.2.  Biliary excretion  Species and age differences

    The relative molecular mass and polarity of xenobiotics are 
important in determining biliary transport and excretion (Rollins & 
Klaassen, 1979; Anderson & Schrier, 1981; Levine, 1981, 1983; 
Renwick, 1982).  Species differences exist for the biliary 
excretion of organic anions (Renwick, 1982; Levine, 1983); optimal 
relative molecular masses are 325 ± 50 in the rat, 440 ± 50 in the 
guinea-pig, 475 ± 50 in the rabbit, and 500 - 700 for man.  The 
enterohepatic circulation may also show ontogenetic variations 
(Kitani et al., 1981; Klinger, 1982).  Effects of physiological compounds

    Physiological compounds may influence biliary function; 
aldosterone (decreased bile flow and bile acid excretion), 
hydrocortisone (increased bile formation, decreased hepatic uptake 
of ampicillin), and estrone (decreased bile flow of 
bromosulfophthalein (BSP) clearance) are examples (Levine, 1981, 
1983).  Pregnancy and long-term administration of estrogens are 
accompanied by decreased bile flow and also depress the biliary 
excretion of a number of xenobiotics (Mueller & Kappas, 1964; Reyer 
& Kern, 1979).  Taurocholate facilitates the hepatic uptake of BSP, 
and increases its excretion.  Low doses of bile salts stimulate 
hepatic uptake of BSP, whereas high doses inhibit it (Levine, 
1983).  Effects of xenobiotics

    Phenobarbital, spironolactone, and pregnenolone 16-alpha-
carbonitrile increase bile salt-independent bile flow, and factors 
such as these should be taken into consideration in experimental 
design.  Depletion of hepatic glutathione may indirectly depress 
xenobiotic excretion.  Many other xenobiotics including carbon 
tetrachloride, kepone, TCDD, PCBs, and certain organic metals 
affect biliary function (Levine, 1981).  Chelating agents often 
increase the excretion of metals that are excreted in the bile 
(Levine, 1983; Cherian, 1984).  Hepatic disease and regeneration

    Hepatic disease may increase the retention of xenobiotics that 
are normally excreted into the bile or metabolized by the liver 
(Wills et al., 1983).  Biliary excretion of xenobiotics is often 
diminished during hepatic regeneration.  However, biliary excretion 
of some chemicals per unit weight of residual liver may actually be 
increased (Levine, 1983). 

7.5.  Methods for Assessing Excretion

7.5.1.  Whole animals

    Chemical excretion studies are carried out by putting the 
animal in a standard metabolism cage in which the urine and faeces 
are collected separately.  Most metabolism cages are merely some 
type of urino-faecal separator.  Caution must be exercised, 
however, because cross contamination of urine and faeces can occur.  
Some metabolic cages are also designed to regulate and collect both 
inspired and expired gases.  One type of metabolism cage is 
depicted in Fig. 4. 


    Techniques for the collection of urine from rats are described 
by Kraus (1980) and include: 

    (a)  reflux emptying under periodic stimulation or massage;

    (b)  bladder centesis;

    (c)  cystostomy or urinary bladder fistula;

    (d)  free catch;

    (e)  uretheral catheterization; and

    (f)  external drainage catheterization (White, 1971).

Experimental conditions must be standardized (Rogers, 1983).  Some 
experimental techniques have been used to study the transport of 
organic molecules (cation and anion) in the kidney.  Clearance 
methods (Stitzer & Martinez-Maldonado, 1978), stop-flow (Blantz & 
Tucker 1978), micropuncture (Lang et al., 1978), and Sperber 
techniques are mainly used in  in vivo tests (Irish & Grantham, 
1981; Berndt, 1982).  Faeces may be simply obtained by retrieving 
voided pellets from beneath a suspended cage or from within bedding 
material.  However, workers have begun experimenting with anal cups 
made from a variety of small plastic laboratory bottles, because of 
problems encountered in all caging devices aimed at preventing 

    The Bollman-type cage is useful for relatively short-term 
studies involving collection of specimens from animals by 
cystostomy, thoracic duct cannulae, collection of pancreatic juice 
or bile, and other procedures.  Bile can be collected readily in 
the anaesthetized animal in short-term acute studies.  Although 
more difficult, successful techniques for long-term bile duct 
cannulation and creation of a biliary fistula have been described.  
One anatomical feature must be considered in this procedure:  the 
bile duct carries both biliary and pancreatic fluids.  
Consequently, if bile must be collected uncontaminated by 
pancreatic secretions, the bile duct must be cannulated near the 
hilum of the liver and before the several pancreatic ducts enter 
the common duct.  For a review of surgery of the bile duct, the 
reader is referred to Lambert (1965).  In addition, reviews by 
Mathews (1981) and Mulder et al. (1981) include descriptions of the 
collection of faeces and bile samples. 

    Direct analysis of volatile metabolites in the air is carried 
out by the trapping and desorption of the compound, with silica gel 
and/or Tenax GC as trapping agents (Tanaka & Watanabe, 1982).  An 
aqueous solution of mercuric acetate or mercuric perchlorate 
(Young, et al., 1952) is often useful as a trap for olefinic 
compounds such as ethylene.  To ascertain that these metabolites 
are being expired requires special techniques such as head- or 
mouth-only collectors.  Glass tubes filled with Drierite dessicant 
or activated charcoal are also used to trap breath volatiles 
(Gudzinowicz & Gudzinowicz, 1977).  A multistage cryogenic trapping 
system for trace organic constituents in human respiratory gas is 
also useful for animal studies (Conkle et al., 1975).  For the 
sampling of saliva, milk, and tears, see Kraus (1980).  Gnotobiotic 
or germ-free animals, as well as suppression of the intestinal 
flora by antibiotics (section 6.5.3), can be used to assess the 
enterohepatic circulation of a chemical.  A second procedure is to 
administer bile excreted from one rat directly into the intestine 
of a second animal and monitor biliary excretion. 

7.5.2.   In vitro preparations

     In vitro methods are also useful for studying the mechanisms of 
excretion.  Isolated organs

    Isolated organs are suitable for the study of many 
physiological and biochemical aspects of excretion. 

    (a)   Isolated perfused kidney (IPK)

    Isolated perfused kidney has been used to study the transport 
and/or metabolism of a number of compounds.  Physiological aspects 
of the technique and its application to drug metabolism have been 
reviewed by Maack (1980) and by Newton & Hook (1981), respectively.  
Swanson et al. (1981) indicated that the addition of globulin and 
erythrocytes to the albumin-containing artificial perfusate 
prevented the gradual increase in vascular resistance and reduced 
the decrease in glomerular filtration rate (GFR) seen with albumin 
alone in the perfusate.  Functions of the superficial proximal 
convoluted tubules are well preserved in IPK and well suited to 
micro-puncture study (Bahlman et al., 1967).  A non-filtering 
kidney preparation, which can be obtained easily by increasing the 
albumin concentration and lowering the perfusion pressure, is 
applicable for the study of the basolateral and peritubular sides 
of the renal cells, separately (Collier et al., 1979). 

    (b)   Isolated perfused liver (IPL)

    The isolated perfused liver has been used  to study the 
excretion of xenobiotic chemicals into the bile (Meijer et al., 
1981).  All conjugated metabolites were rapidly excreted in the 
bile but some were also released into the circulation, when benzo
( a)pyrene 4,5-oxide was added to the perfusate (Smith & Bend, 
1979).  Excretion of the glucuronide conjugate of 2,4-dinitrobenzyl 
alcohol into the bile seemed to be faster in the isolated perfused 
liver of the male rat compared with that of the female (Bond et 
al., 1981).  Intestinal preparations

    When chemicals are recovered in the faeces after iv 
administration, it is often assumed that they have undergone 
excretion in the bile.  However, with some compounds, there is 
evidence that their recovery in the faeces may be due, in part, to 
elimination across the intestinal wall (Selden et al., 1974; Ings 
et al., 1975).  Slices of renal cortex

    Using slices of the renal cortex, Berndt & Koschier (1973) and 
Hook et al. (1974) demonstrated extensive, energy-dependent 
accumulation of phenoxyacetic acid derivatives (2,4-D, 2,4,5-T) via 
the organic anion transport systems, similar to PAH.  Accumulation 

of chemicals in slices indicates that the substances are actively 
transported, but, because of the process of reabsorption, the net 
excretion cannot be estimated by this method.  Other kidney preparations

    Useful mechanistic information concerning xenobiotic excretion 
can also be obtained in preparations of isolated tubules, isolated 
cells, and membrane vesicles.  For example, Pritchard et al. (1977) 
and Pritchard & James (1979) described the transport of 2,4-D and 
the polar DDT metabolite, DDA, by the isolated flounder renal 
tubule preparation.  Techniques for the microperfusion of isolated 
tubules are illustrated in Chonko et al. (1978).  Examples of 
experiments in which isolated kidney cells were used for the study 
of transport mechanisms are described in the report of Uehara et 
al. (1983).  More recently, the luminal and basolateral membranes 
of cortical tubules have been segregated for study  in vitro.  With 
this technique, the nature of the transport mechanisms can be 
studied more specifically.  Carrier-mediated transport systems of 
organic anions (Kinsella et al., 1979; Hori et al. 1982) and of 
organic cations (Takano et al., 1984) have been studied by this 
technique.  Purified membrane preparations

    A particular model system for monitoring the interaction(s) of 
biologically active chlorophenols with membranes has been reported, 
using a fluorescent probe and liposomes prepared from dimyristoyl 
lecithin (Danner & Resnik, 1980). 


8.1.  General Considerations

    Kinetic models describe the fate of a xenobiotic in the 
organism in mathematical terms.  The different models are based on 
the transformation of experimental xenobiotic/metabolite 
concentration data from blood, tissue, or urine to mathematical 
functions describing the processes after absorption, distribution, 
and elimination of the xenobiotic in the body.  Usually, the data 
are fitted to a one- or a two-compartment model.  The different 
compartments used in toxicokinetic models do not represent 
anatomical or physiological units, but, on most occasions, it is 
possible to interpret the derived compartments and model parameters 
as being representative of functionally homogenous tissue groups 
possessing common characteristics with respect to xenobiotic 
disposition.  Even though a multiple compartment analysis will 
always give the best fit of experimental data, the information 
obtained from a one- or two-compartment model is, in most cases, 
sufficient for practical use. 

    Most kinetic processes for the disposition of a xenobiotic can 
be described by first order kinetics (dose-independent), meaning 
that the rates of all processes are proportional to the 
concentration of the substance, at the site in question. As the 
concentration increases, either Michaelis-Menten or zero-order 
kinetics may occur, showing process rates less than expected from 
linear first-order kinetics. 

8.2.  Dose-Independent Kinetics

8.2.1.  One-compartment model

    This is the simplest kinetic model, where the whole body is 
thought of as a single compartment in which the xenobiotic 
distributes rapidly, achieving an equilibrium between blood and 
tissues immediately.  Single dose

    (a)   Distribution and elimination

    The change in amount of a xenobiotic with time in the organism 
after a single iv dose can be described by first-order elimination 
kinetics (equation 15). 

    -- = -ke x X or X = Xo x e-ke x t                          (15)

where X is the amount of xenobiotic in the body at any time (t) 
after the administration, Xo the amount of chemical in the body at 
time zero, and ke the elimination rate constant.  On the basis of 
the assumption of an equilibrium of the xenobiotic with all body 
tissues, the concentration in plasma, C, may be related to the 

total amount of compound in the body by the apparent volume of 
distribution (VD), which has a dimension of litre or ml (equation 

        X            X   Xo
    C = --  or  VD = - = --                                    (16)
        VD           C   Co 

    It should be noted that VD is not a real volume, but a 
parameter expressing the ratio between the total amount of 
xenobiotic in the body and its concentration in plasma at any time, 
t, after administration.  For example, chemicals that are 
extensively bound to serum proteins such as the drugs warfarin and 
furosemide, will have small volumes of distribution, of less than 
12 litres in human beings.  Chemicals, such as the drugs digoxin 
and chlorpromazine, which show a low serum-protein binding compared 
to binding in other tissues, have much larger volumes of 
distribution, exceeding 200 litres in human beings (Grahame-Smith & 
Aronson, 1984).  Combining equations (15) and (16) gives the 
variation in plasma concentration of the xenobiotic with time 
(equation 17). 

    C = Co x e-ke x t                                          (17)

where Co is the initial concentration of the xenobiotic in plasma 
at time zero, to. 

    The elimination-rate constant can easily be calculated from a 
plot after changing the exponential function to a linear function 
by taking the natural logarithm on both sides, giving equation 

    InC - InCo = -ke x t                                       (18)

    In the Briggs logarithmic system, equation (18) transforms to 
equation (19): 

                       ke x t
    log C - log Co = - ------                                  (19)

    Most parameters can be calculated for a xenobiotic after a 
bolus iv injection, followed by subsequent blood sampling and 
analysis of plasma or serum concentrations.  An example which 
illustrates the experimental method for the determination of 
kinetic parameters for a chemical eliminated according to a one 
compartment model is shown in Fig. 5.  The plasma concentration 
time-curve of the xenobiotic presented in Fig. 5 is based on values 
given in Table 2. 

     The slope of the linear phase (log C2 - log C1)/t2-t1, is 
equal to -ke/2.303, and the elimination half-time, t, of the 
xenobiotic is given by equation (20). 

         In2   0.693
    t“ = --- = -----                                           (20)
         ke    ke

    Values, for ke and tcan be calculated; they are 0.292/h and 
2.37 h, respectively.  Otherwise, the elimination half-time of the 
xenobiotic, which is the time needed by the organism to decrease 
the plasma concentration by one-half, can be determined directly 
from Fig. 5, as illustrated. 

Table 2.  Plasma and urinea concentrations 
of a xenobiotic after an iv bolus 
injection of 20 mg (one-compartment model)
Time  Plasma         Urine          Urine       
(h)   concentration  concentration  volume
      (µg/ml)        (µg/ml)        (ml)
0     0.0            0.0
1     19.0
2     13.3           31.6           217
3     11.8
4     8.3            18.6           240
5     6.6
6     4.6            14.9           160
7     3.4
8     2.5            14.6           85
a Collected every 2 h starting at zero 


    An extrapolation of the line in Fig. 5 to the intercept with 
the ordinate gives the plasma concentration of the xenobiotic at 
time zero (Co = 26 µg/ml).  As the administered dose (Xo) is known, 
the apparent volume of distribution of the xenobiotic can be 
calculated from equation (16); this value is 769 ml. 

    The time average plasma clearance (Clt) of the xenobiotic, 
which is the volume of plasma that is completely cleared of the 
xenobiotic in unit time by all elimination processes (usually 
dominated by renal excretion and metabolism), is given in equation 
(21) and is expressed as ml/min. 

    Clt = ke x VD                                              (21)

    The calculated plasma clearance of a xenobiotic for the example 
in Fig. 5 is 3.75 ml/min. 

    The renal excretion rate constant (kr) of a xenobiotic is 
determined by the following equation (22): 

    Clr = kr x VD or kr = ---                                  (22)

where renal clearance (Clr) of the xenobiotic is obtained from the 
slope after plotting the amount of xenobiotic excreted in urine per 
unit time (DELTAX/DELTAt) versus the plasma concentration of the 
xenobiotic measured in the mid-point of the urine collection 
interval.  The method is illustrated in Fig. 6, with data from 
Table 2. 


    Knowing the values of the parameters Clt (3.75 ml/min), Clr 
(3.07 ml/min), ke (0.292/h), and kr (0.239/h), two other important 
parameters can be determined, namely the non-renal clearance (Clnr) 
and the non-renal excretion rate constant (knr) according to 
equations (23) and (24). 

    Clt = Clr + Clnr                                           (23)

    ke = kr + knr                                              (24)

    The calculated values for Clnr and knr are 0.68 ml/min and 
0.054/h, respectively, showing that, for this example, renal 
excretion is the major route of elimination for the xenobiotic.  It 
should be pointed out that the non-renal elimination parameters 
include several elimination processes of which metabolism is often 
the major one. 

    Specific knowledge of the major elimination routes of a 
xenobiotic will provide valuable information for the hazard 
assessment of a xenobiotic, with respect to increased accumulation 
in diseased people who have impairment of one or more important 
elimination pathways. 

    The total or overall elimination rate constant can also be 
determined from urinary excretion data either by a plot similar to 
that shown in Fig. 6, in which the rate of excretion is plotted 
against time, or by the use of the Sigmaminus method (Renwick, 
1982).  The first method has the disadvantage of fluctuation due to 
various degrees of bladder emptying; the latter is limited by the 
requirement for total urine collection during all sampling 

    (b)  Absorption

    Including a first order absorption process in the model,
equation (15) becomes equation (25).

                -- = ka x Xa - ke x X                          (25)

where ka and Xa are the absorption rate constant and the amount of 
xenobiotic at the site of absorption, respectively. 

    The absorption rate constant may be determined by the method of 
residuals.  Using the plasma concentration-time data given in Table 
3, the performance of the method of residuals is illustrated in 
Fig. 7 (From:  Gibaldi & Perrier, 1975). 

    From Fig. 7, an elimination rate constant (ke) of 0.069/h and 
an absorption rate constant (ka) of 0.230/h can be calculated.  The 
latter (ka) is determined from the slope of the residuals obtained 
by plotting the differences between the extrapolated line and the 
corresponding plasma concentrations in the absorption phase 
against time.  Both constants are determined from the slopes of the 

linear phases as shown (Fig. 7).  The slower rate constant denotes 
the slower process and, in this case, the elimination process. 


Table 3. Plasma concentration-time data and 
calculated residual concentrations following a 
single oral administration of a xenobiotic 
(one-compartment model)
Time  Plasma         Extrapolated    Residual
(h)   concentration  concentrationa  concentrationb
      (µg/ml)        (µg/ml)         (µg/ml)
0.5   5.36           69.0            63.64
1     9.95           66.5            56.55
2     17.18          62.5            45.32
4     25.78          54.0            28.22
8     29.78          41.2            11.42
12    26.63          31.2            4.57
18    19.40          20.7            1.30
24    13.26
36    5.88
48    2.56
72    0.49
a Obtained from the extrapolated straight line 
  (----) in Fig. 7.
b Calculated as the difference beteween the 
  extrapolated line and the corresponding plasma 

    Ordinarily, the absorption rate is more rapid than the 
elimination rate for most xenobiotics.  However, for some polar 
compounds, elimination may be more rapid than absorption.  The 
slower rate constant now denotes absorption (ka), and the faster 
rate constant the so-called flip-flop state.  The true elimination 
characteristics of the compound can be determined after an iv 
injection as earlier shown in Fig. 5. 

    The peak plasma concentration (Cmax) and the time to reach it 
(tmax) can be calculated as follows (equations 26 and 27): 

    tmax = In (ka/ke) x (ka-ke)-1                              (26)

                     ke x (ka-ke)-1
           F x D  ke
    Cmax = ----- (--)                
            VD    ka

          ka x F x D
        = ---------- (e-ke x tmax-e-ka x tmax                  (27)

where D is the dose and F is the fraction of the dose that enters 
the blood circulation in an unchanged form and is synonymous with 
the term systemic bioavailability (equation 29). 

    When D = 500 mg, F = 1, and VD = 10 litres in Fig. 7, the tmax 
and Cmax can be calculated to be 7.5 h and 29.9 µg/ml, 
respectively.  It should be noted that tmax is independent of the 
dose D, but only depends on ka and ke, and that plasma 
concentration-time curves have an inflection point at 2 times tmax, 
whereafter ke can be determined if ka > 10 ke (Torell, 1937). 

    The variation in xenobiotic plasma concentration with time 
after an oral intake is given by the equation (28): 

        F x D x ka
    C = ------------  (e-ke x t-e-ka x t)                      (28)
        VD x (ka-ke)

    The fraction of a dose that is absorbed as the parent compound 
can be calculated from the ratio between the area under the curve, 
from zero to infinite time (AUCo-infinite), after oral and iv 
administration of the same dose of the xenobiotic (equation 29): 

    F = -------------------                                    (29)

    It should be noted that F includes first-pass effects and is 
synonymous with bioavailability.  If a xenobiotic shows a value of 
F of less than unity, this means that the parent compound is either 
not entirely taken up in the gut or is subject to first pass 
effects or both.  The area under the curves can be approximated by 
the use of the trapezoidal rule (for illustration, see Gibaldi & 
Perrier (1975)) (equation 30). 

                     n-1             Ci+1 + Ci   Cn
    AUCo-infinite = SIGMA (ti+l - ti)--------- + --            (30)
                     i=0                 2       ke

where Ci represents the plasma concentration at time ti, and Cn 
denotes the last measured plasma concentration on the plasma 
concentration-time curve at time tn.  Relative AUCs may also be 
estimated by accurately cutting out the curves and weighing them on 
an analytical balance.  In these procedures, it must be certain 
that Cn represents a true point on the elimination curve that is 
not influenced by absorption (Torell, 1937). 

FIGURE 8  Repeated dosing

    Unlike single dose kinetics, repeated dosing or an intermittent 
regular exposure may cause an accumulation of the xenobiotic in the 
organism.  After a defined period of regular, repetitive exposure, 
the plasma concentration of the xenobiotic will fluctuate between a 
minimum (Cssmin) and a maximum (Cssmax) level.  The time needed to 
reach this "steady-state" level (defined as the state where the 
body eliminates an amount of a xenobiotic that is the same as that 
absorbed during an exposure interval), is 5 times the elimination 
half-time of the xenobiotic (97% of the Css level is reached).  The 
average plateau level, Css at steady state, may be estimated from a 
single dose and is given by equation (31). 

          AUCo-infinite   F x D           F x D
    Css = ------------- = ------------- = ---------            (31)
          tau             VD x ke x tau   Clt x tau

where tau represents the time between two dosage or exposure 
intervals.  In equation 31, the AUCo-infinite calculated after a 

single oral administration could be substituted by the AUC 
calculated during a dosage or exposure interval at steady state, as 
they represent the same areas as shown in Fig. 8. 

    The ratio between the maximum and minimum plasma concentrations 
at steady state can be determined from single-dose data as shown 
below (equation 32). 

    Cssmax   C1max
    ------ = -----                                             (32)
    Cssmin   C1min

where C1max and C1min are the maximum and minimum plasma 
concentrations achieved after the first dose during a dosage 
interval (Fig. 8).  The difference between the maximum and minimum 
concentrations at steady state is determined by the elimination 
rate constant of the xenobiotic, ke, and the time available for 
elimination before the next exposure, tau.  The extent of 
accumulation of a xenobiotic on repeated intake, as measured by the 
average concentration in plasma at steady state, Css, depends on 
the dose of the xenobiotic, its elimination rate constant, exposure 
frequency, extent of absorption as well as apparent volume of 
distribution, but not on the absorption rate of the xenobiotic.  
Accumulation of a xenobiotic in the organism can be expressed by 
dividing the average amount of the chemical in the body at steady 
state with the amount in the body after a single dose.  This 
accumulation ratio (R) depends only on elimination rate constant 
and exposure frequency, and may be expressed as equation (33): 

        1.44 x t
    R = ---------                                              (33)

    From the equation, it can be seen that accumulation of a 
xenobiotic will become significant when tau < 1.44 x t.  If, for 
instance, the administration or exposure frequency is increased 
from once every 12 h to once every 3 h and the elimination half-
time of the xenobiotic is 12 h, then the total body burden at 
steady state will be about six times larger than the total body 
burden after a single administration (unity compared with 5.76). 

    If, however, a person is exposed to a constant concentration of 
a xenobiotic by inhalation for infinite time without any periods 
off exposure, then the total body burden will increase to a level 
given by a plasma concentration equal to ko/(VD x ke), where ko 
represents the amount of xenobiotic absorbed per unit time.  This 
limiting case is illustrated by the dotted line in Fig. 8. 

8.2.2.  Two-compartment model

    The two-compartment model is more complex than the one-
compartment model, introducing an additional compartment for 
distribution and redistribution of the xenobiotic between a central 
(rapidly perfused organs) and a peripheral (not so well perfused 
organs) compartment.  In contrast to the one-compartment model, a 

longer time is usually required in order to achieve an equilibrium 
between blood, tissues, and organs, i.e., to reach the true phase 
of elimination, which is also called the beta-phase or "slow phase" 
in this model.  The alpha-phase is often equated with distribution, 
however, it should be noted that this is a hybrid of absorption, 
distribution, and elimination.  This model is considered to be 
more appropriate than the one-compartment model, though the 
kinetics of several xenobiotics are adequately described by the 
one-compartment model.  Single dose

    (a)  Distribution and elimination

    The rate of change in the amount of a xenobiotic in the central 
(c) and peripheral (p) compartments after a bolus iv injection is 
described by the following equations (34 and 35). 

    --- = k21 x Xp - k10 x Xc - k12 x Xc                       (34)

    --- = k12 x Xc - k21 x Xp                                  (35)

where k12 and k21 are the intercompartment rate constants for 
transfer from the central to the peripheral compartment and vice 
versa, respectively, and k10 is the first-order elimination rate 
constant for elimination from the central compartment.  Xc and Xp 
are the amounts of xenobiotic in the central and peripheral 
compartments, respectively. 

    In analogy to the one-compartment model, the variation in 
plasma concentration (C) of a xenobiotic with time is given by 
equation (36). 
    C = A x e -alpha x t + B x e -beta x t                     (36)

where A is equal to Co (alpha - k21)/(alpha - beta) and B is equal 
to Co (k21 - beta)/(alpha - beta).  Co, the initial plasma 
concentration at time t = 0, is equal to (A + B).  Alpha and beta 
express hybrid rate constants, each influenced by several other 
rate constants according to equations 37, 38, and 39. 

    alpha + beta = k12 + k21 + k10                             (37)

    alpha x beta = k10 x k21                                   (38)

                A beta x B alpha
    k21       = ----------------                               (39)
                     A + B

    From equations (37) and (38), it can be seen that beta is 
different from the elimination rate constant k10, and is referred 
to as the disposition or terminal rate constant.  Analogous to the 

one-compartment model, the elimination half-time of a xenobiotic is 
given by the relationship shown in equation 40. 

    tbeta = ln 2/beta = 0.693/beta                            (40)

    The parameter a is also a combined result of several processes 
and is often referred to as the distribution rate constant; alpha 
is the faster and beta the slower rate constant. 

    A linear relationship is anticipated  between the concentration 
C of a xenobiotic in plasma and the amount in the central 
compartment Xc as follows (equation 41). 

        Xc         Xo   dose(i.v.)
    C = -- or Vc = -- = ----------                             (41)
        Vc         Co     A + B

where Vc is the apparent volume of the central compartment.

    An estimation of the kinetic parameters of a xenobiotic, using 
the two-compartment model after an iv injection, is illustrated in 
Fig. 9.  The figure is based on data from Table 4 (Renwick, 1982).  
A and beta are equal to the negative slopes of the two indicated 
straight lines, when based on natural logarithms (ln).  If the 
Briggs logarithmic system (log) is used, as illustrated earlier in 
Fig. 5 and 7, the slopes will represent - alpha/2.303 and - 
beta/2.303.  Both slopes can be easily calculated by least square 
linear regression analysis, beta from the terminal phase at the 
point where log concentration-time linearity commences (A x e -
alpha x t~0 in equation 36), and alpha from the line of residuals 
as shown in Fig. 9. 

    If the sample data are substituted into equations (37) and 
(38), the rate constants k12, k10, and k21 are calculated to be 
0.539/h, 0.505/h, and 0.291/h, respectively.  As with beta, Vbeta 
is also a hybrid term; it is defined as the apparent volume of 
distribution of the xenobiotic in the body.  The total body 
clearance (Clt) of a xenobiotic from the body and Vbeta are 
independent variables (Equations 42 and 43): 

    beta = -----                                               (42)

    Clt = Vbeta x beta = Vc x k10 = ------------               (43)
    The renal elimination rate constant kr can easily be derived 
from urinary excretion data (equation 44). 

    kr = ------------- x k10                                   (44)

where Xexo-infinite is the cumulative total amount of xenobiotic 
excreted in urine from zero to infinite time. 

Table 4. Plasma concentration-time data and residual 
concentrations obtained following an iv administration 
of a xenobiotic (two-compartment model)
Time  Plasma          Extrapolated     Residual
(h)   concentrations  concentrationsa  concentrationsb
      (µg/ml)         (µg/ml)          (µg/ml)
0.5   1345            326              1019
1     864             307              557
1.5   593             289              304
2     438             272              166
2.5   346             256              90
3     290             241              49
4     228
5     193
6     168
8     131
12    81
16    50
a Obtained from the extrapolated straight line (------) 
  in Fig. 9.
b Calculated as the difference between the extrapolated 
  line and the corresponding plasma concentrations.

    The renal clearance (Clr) of the xenobiotic can be calculated 
according to the relationship (equation 45). 

    Clr = kr x Vc                                              (45)

    As with the one-compartment model, the non-renal elimination 
rate constant (knr) and non-renal clearance (Clnr) can be 
calculated according to equations (24) and (23), respectively. 

    (b)  Absorption

    After oral administration, three separate phases are observed 
for a xenobiotic that distributes in the body according to a two-
compartment model.  The additional phase to the distribution and 
elimination phases is the absorption phase, which introduces the 
concept of an absorption rate constant ka.  The variation in plasma 
concentration with time is complex, but can be expressed 
mathematically as equation 46. 

   C = A1 x e-alpha x t + A2 x e-beta x t + A3 x e-ka x t      (46)

as ke >> alpha and alpha >> beta for most xenobiotics, the 
equation can be reduced to (equation 47). 

   C = A2 x e-beta x t                                         (47)

              ka x F x dose (k21 - beta)
   where A2 = --------------------------
              Vc(ka-beta)(alpha - beta)

    In principle, the calculation of ka in this model is performed 
as in the one-compartment model (Fig. 7); however, in the two-
compartment model, one must also include the contribution of the 
alpha-phase.  Repeated dosing

    Some similarities exist between the one- and two-compartment 
models; a steady state is reached also for a xenobiotic that 
distributes according to a two-compartment model.  The time to 
reach this plateau level depends on the elimination rate, while the 
actual level depends on the dose, the exposure frequency, the 
elimination rate, the extent of absorption, and the volume of 
distribution of the xenobiotic.  The mean steady-state 
concentration after repeated dosing or exposures can be expressed 
as equation (48), which is analogous with equation (31): 
      AUCo-infinite      F x Dose        F x Dose           F x Dose
Css = ------------- = -------------- = ------------------ = ---------           (48)
           tau        Vc x k10 x tau   Vbeta x beta x tau   Clt x tau

8.3.  Kinetics of Metabolites in the Presence of Parent Compound

    After absorption of a xenobiotic (X), metabolites can be formed 
that possess equal (metabolite A) or longer (metabolite B) apparent 
elimination half-times than the parent compound itself, as 

illustrated in Fig. 10.  In the first case, where t“app for the 
metabolite A equals tfor the parent compound, the elimination of 
the parent compound is usually the rate-limiting step in 
elimination of the xenobiotic and its metabolites.  The true 
elimination half-time of the metabolite will be shorter than the 
apparent one.  Generally, the concentration of the metabolite will 
be lower than that of the parent compound.  However, if the volume 
of distribution of the metabolite is smaller than that of the 
parent compound, the metabolite concentration may exceed that of 
the xenobiotic.  In the second case, where t“app for metabolite B 
is greater than tfor the parent compound, the elimination of 
metabolite is indicated to be the rate-limiting step, and the true 
elimination half-time of the metabolite may be equal to the 
apparent half-time.  In the second case, the concentration of the 
metabolite is often higher than that of the parent compound. 


    It is important to know where the rate-limiting step lies in a 
sequence, as clearly all metabolites beyond the rate-limiting step 
decline with apparent elimination half-times equal to that of the 
slowest step. 

    The processes of absorption and elimination (metabolism) of 
xenobiotic (X) and elimination of its metabolite (Xm) are 
illustrated below.  For simplicity, it is assumed that all of the 
xenobiotic is metabolized to Xm.  This means that fm=1.  All 
processes are determined by first-order rate constants (Scheme 1). 

       ka         fm x kx         km
    Xa -----> X  ---------> Xm  ------>               (Scheme 1)


    ka = absorption rate constant of the xenobiotic;
    kX = overall elimination rate constant of the xenobiotic;
    kM = overall elimination rate constant of the metabolite;
    fm = the ratio of formation of the metabolite to the total
         rate of elimination of the parent compound;
    fm x kX = rate constant of metabolite formation;
    Xa = amount of xenobiotic at the site of absorption;
    X = amount of xenobiotic in the body; and
    Xm = amount of metabolite in the body.

    If ka in the above sequence is the rate-limiting step, the 
apparent elimination half-times of both X and Xm will be equal to 
the absorption half-time.  If fm x kx is the rate-limiting step in 
the sequence, the apparent half-time of Xm will be equal to the 
half-time of X. 

    Graphic methods have been presented for the estimation of 
elimination rate constants of metabolites in the presence of the 
parent compound (Pang & Gillette, 1980).  All calculations are 
based on iv administration of the parent compound, first-order 
kinetic processes, and a one-compartment model with the liver as 
the only organ of elimination.  However, the principles may also be 
useful for calculations during the terminal phases of elimination 
in more complex systems. 

    The ratio between the serum concentration of metabolite
(CM) and parent compound or precursor (CX) in such a model is
given by equation (49):

    CM   fm x kX x F(M,X) x VX[1- e(kX - kM) x t]
    -- = ----------------------------------------              (49)
    CX                 VM(kM-kX)

where VM and VX are the apparent volumes of distribution for 
metabolite and parent compound, respectively.  F(M,X) denotes the 
availability of the metabolite from the parent compound, i.e., the 
fraction of the metabolite reaching the hepatic venous blood. 

    The true elimination rate constant of a metabolite, kM, can be 
estimated directly from the terminal phase in the serum metabolite 
concentration-time plot after about four elimination half-times of 
the parent compound if kX >> kM.  If kX and kM are similar, or if 
kX << kM, then a continous and significant formation of 
metabolite will occur throughout the whole period, and calculation 
of kM directly from the serum metabolite concentration-time plot is 
not possible. 

    However, equation (49) can be transformed into equation (50). 

    ---      ---      ---                   ---  
    |     (CM) |      |                       |
    |DELTA(--) |      |                       |
    |     (CX) |      |fM x kX x F(M,X,) x VX |   (kX - kM) x tmid
log | -------- |= log |---------------------- | + ----------------   (50)
    | DELTA t  |      |        VM             |       2.303
    ---      ---      ---                   ---

where tmid is the midpoint of the time interval DELTAt and 
DELTA(CM/CX) is the difference in the ratio of (CM/CX) for the 
interval.  The "delta ratio plot" is obtained by plotting the ratio 
DELTA(CM/CX)/DELTAt versus tmid on semilogarithmic paper.  A 
straight line will emerge with a slope (kX - kM)/2.303 as 
illustrated in Fig. 11. 


    The elimination rate constant of the parent compound, kX, can 
be estimated directly from the serum concentration-time plot of the 
parent compound, thus making a calculation of kM possible from the
"delta ratio plot".  The same procedure can be used when kX < kM, 
but the slope of the plot will be negative.

    The "delta ratio plot" lacks precision, when kM nearly equals 
or greatly exceeds kX, even with rather good analytical data.  For 
this reason, it is recommended that the validity of the kM value be 
confirmed by other methods, such as the "feathered ratio plot" or 
the "log ratio plot" as described by Pang & Gillette (1980). 

8.4.  Non-Linear Kinetics

    At high doses or concentrations, metabolism or renal tubular 
secretion approach the capacity of the elimination system, and the 
parameters discussed earlier are no longer based strictly on first 
order kinetics.  Saturation of a process will proceed with zero 
order kinetics according to the Michaelis-Menten equation (51). 

      dC   V'max x C
    - -- = ---------                                           (51)
      dt    Km + C

where the change in plasma concentration C over time is determined 
by the maximum rate of the process, V'max and the Km, the Michaelis-
Menten constant, the plasma concentration at which the rate of the 
process is 50% of the maximum rate.  V'max in equation (51) is 
corrected for volume, i.e., Vmax/V. 

    The time course in plasma of a xenobiotic described by 
Michaelis-Menten kinetics is shown in Fig. 12 (Renwick, 1982).  The 
xenobiotic with a Km equal to 20 µg/ml is given iv in a high dose 
(200 mg) and in a low dose (5 mg). 


    There are two limiting cases of Michaelis-Menten kinetics.  The 
first is if Km >> C, then equation (51) can be simplified to 
equation (52). 

      dC   V'max x C
    - -- ~ ---------                                           (52)
      dt      Km

which is equal to Equation (15), and describes a first-order 
elimination process.  As shown in Fig. 12, first-order elimination 
kinetics are achieved for both doses when the plasma concentration 
is below 2 µg/ml, when C is only 10% of Km (20 µg/ml), a 
concentration below which the elimination of the xenobiotic is much 
below the point of saturation of the elimination process. 

    The second limiting case is when Km << C; in this case, 
equation (53) is valid. 

    - -- ~ V'max                                                (53)

    Under these conditions, the elimination rate of the xenobiotic 
is independent of the plasma concentration and will proceed 
according to zero order kinetics with a constant elimination rate 
equal to V'max/VD.  In Fig. 12, the high dose initially produces a 
concentration sufficiently high (> 100 µg/ml) and results in 
saturation.  Between these two limiting cases, the xenobiotic 
follows simple Michaelis-Menten kinetics. 

    As seen from Fig. 12, the time required for the concentration 
to decrease by 50% is dependent on dose and increases with 
increasing dose of the xenobiotic.  This particular kinetic 
property is important, because a steady state is theoretically 
never achieved if the exposure frequency is high enough.  A 
continuous accumulation would occur if no alternative pathways were 

    The values of Km and V'max can be obtained by transformation of 
the Michaelis-Menten equation (51) to a linearized form 
(Lineweaver-Burk, equation 54). 

          1             Km         1
    -------------- = --------- + -----                         (54)
    DELTAc/DELTAt    V'max x C   V'max

Plotting the reciprocal of DELTAc/DELTAt versus the reciprocal of C 
at the midpoint of the sampling interval will give a straight line 
relationship with intercepts on the ordinate and abscissa at 1/V'max 
and Km/V'max, respectively. 

    Saturation kinetics occur after administration of certain 
compounds at high doses to experimental animals; 2,4,5-
trichlorophenoxyacetic acid (Sauerhoff et al., 1975) and 1,4-
dioxane (Gehring & Young, 1978) are examples. 

8.5.  Physiological Kinetic Models

    After absorption, a xenobiotic is transported throughout the 
body via the circulation.  The accumulation of a xenobiotic in each 
organ/tissue depends on several factors, including blood flow to 
the organ, the degree of tissue binding, and the elimination by the 

    The kinetics previously described are mainly correlated with 
the variation over time of the xenobiotic or metabolite in blood or 
in the body as a whole.  For a better understanding of toxicity, 
the concentrations of the xenobiotic and/or metabolite(s) in a 
target organ should be known.  Physiological models have certain 
advantages for they make use of information on the anatomy and 
physiology of the affected organ. 

    A physiological compartment, which may represent one organ or 
several organs or tissues with uniform properties, is illustrated 
in Fig. 13.  CA, CV, and CT are the concentrations of xenobiotic in 
the arterial blood, venous blood, and tissue, respectively; VT is 
the tissue volume and Q the blood flow rate. 


    The uptake of the xenobiotic into the tissue/organ is, in most 
cases, assumed to obey first order kinetics.  At equilibrium, a 
partition ratio, R, may be expressed as (equation 55): 

    R = -- equilibrium                                         (55)

    The instantaneous extraction ratio, E, is the loss in blood 
concentration of the xenobiotic during its passage through the 
organ divided by the arterial concentration (equation 56). 

        CA - CV
    E = -------                                                (56)

    The rate changes in concentration of xenobiotic in tissues over 
time is described by equation (57). 

    dCT   Q x CA - Q x CV
    --- = ---------------                                      (57)
    dt         VT

when no elimination occurs.  If the xenobiotic equilibrates rapidly 
between blood and tissue, equilibrium is achieved during transport 
through the tissue, and equations (56) and (57) can be combined, 
resulting in the relationship expressed by equation (58). 

    dCT   Q         CT
    --- = --  (CA - --)                                        (58)
    dt    VT        R

    In this case, the blood flow-rate is the limiting factor for 
tissue uptake, and an increased blood concentration will increase 
tissue uptake.  The equilibrium constant is determined by the 
relative binding affinities of the xenobiotic between blood and the 
tissue in question. 

    In general, the rate of uptake is given by Q(CA - CV), which, 
by definition, gives an organ clearance (ClO) as shown in equation 

          Q(CA - CV)
    ClO = ---------- = Q x E                                   (59)

    Clearance of a xenobiotic by an organ may vary depending on 
plasma protein binding.  This is a developing area of kinetics 
(Gibaldi & Perrier, 1975; Wilkinson & Shand, 1975; Lutz et al., 
1977; Matthews & Dedrick, 1984). 

    The rate of change in the concentration of a xenobiotic in an 
organ usually involves specific organ processes.  In the liver, 
with the volume VH, the rate change of a xenobiotic concentration 
(CH) within the organ is expressed by (equation 60). 

    dCH             QA x CH   Vmax x CH
    --- = QA x CA - ------- - ---------                        (60)
    dt                RH       Km + CH

where Vmax and Km are Michaelis-Menten constants for xenobiotic 
metabolism and RH denotes the liver/blood equilibrium distribution 

    The use of physiological kinetic models has proved to be 
informative with respect to the toxicokinetic evaluation of 
polychlorinated biphenyls and hexabromobiphenyl (Lutz et al., 1977; 
Matthews & Dedrick, 1984), in which several tissue compartments 
were studied simultaneously.  It was clearly demonstrated that, 
after iv administration, these compounds were quickly taken up by 
highly perfused organs such as liver concomitant with a long 
lasting uptake in fat, where the concentration increased for days 
after administration. 

8.6.  Modulation of Kinetics

    As earlier described, the overall kinetics of a xenobiotic are 
the combined result of several processes including absorption, 
distribution, binding, biotransformation, and excretion, each of 
these being a combination of several processes.  A change in a 
single process may alter the overall kinetics of a xenobiotic; 
physiological, environmental, or pathological factors may exert 
such an effect.  For a more detailed overview of the different 
processes mentioned, see sections 6.3 and 7.4 of this document. 

    It should be noted that the apparent volume of distribution 
(VD) and the total clearance (Clt) of a xenobiotic are both 
frequently changed during any modulation by these factors, making 
the elimination half-time (t) of the xenobiotic a poor indicator 
of the elimination capability because of the relationship shown by 
equation (61). 

         0.693 x VD
    t= ----------                                            (61)


9.1.  General Considerations

    A goal of human health care is to reduce exposure to chemicals 
to a level where no untoward effects are expected.  Because chemical 
concentrations are usually higher in occupational settings, and the 
chemical species better defined, much of the information on the 
assessment of human exposure to chemicals stems from studies in the 
workplace.  An integral part in such investigations is an accurate 
estimation of the exposure.  This is traditionally achieved by 
analysis of concentrations of chemicals in workroom air.  Another 
approach is the use of biological monitoring, which aims at an 
evaluation of the health risk, or uptake of the chemical.  This is 
accomplished by systematic, repetitive measurement of the 
concentrations of chemicals and/or their metabolites in biological 
specimens from exposed workers and the assessment of the 
significance of these concentrations (Berlin et al., 1984b).  Thus, 
measurement of an early, reversible effect of exposure is regarded 
as biological monitoring (Zielhuis, 1984).  Since biological 
monitoring measures concentrations in specimens obtained from an 
exposed individual, it is likely to be more closely related to the 
effect of the chemical than the measurement of concentrations of 
the chemical in the ambient air (Fig. 14).  This is because several 
factors, which are not accounted for in these measurements, affect 
the uptake of chemicals (Table 5). 


Table 5. Some factors that affect uptake of chemicals in the body 
not normally considered in industrial hygiene measurements
1.  Variation in the concentration of the chemical at different
    locations and at different times;

2.  Variation in the concentration of the chemical at different 
    points in time;

3.  Particle size and aerodynamic properties of particles;

4.  Solubility characteristics of the chemical;

5.  Alternative absorption routes (skin, gastrointestinal tract);

6.  Protective devices, and their efficiency;

7.  Respiratory volumes - workload;

8.  Personal habits;

9.  Exposure outside the workplace.

    The latest development in biological monitoring is the 
estimation of exposure to mutagenic and clastogenic carcinogens by 
methods that are nonspecific for the chemical.  This has been 
achieved by measuring mutagenicity in the blood or urine, or by 
assessing adducts on normal body constituent molecules of 
electrophilic reactants derived from carcinogenic chemicals, or by 
analyses of clastogenic effects of the chemicals (Sorsa, 1983, 
1985a,b; Vainio et al., 1985). 

9.2.  Analysis of Parent Compounds or Metabolites

9.2.1.  Toxicokinetics and sampling strategy

    The toxicokinetic characteristics of a chemical determine the 
kind of information that can be obtained through biological 
monitoring.  Because most chemicals are distributed in several 
compartments in the body (section 8) and show widely different 
half-times in these compartments, concentrations may be rather 
difficult to interpret in terms of actual amounts absorbed.  
However, a rough estimation can be made of the total amount of 
chemical in the body if the apparent volume of distribution (VD) of 
the chemical and its concentration (C) in plasma are known.  This 
may be expressed as C x VD (section  There are two major 
problems in making such estimations.  Neither the time-point of the 
specimen collection in relation to exposure nor the VD are known 
for most environmental chemicals.  The toxicokinetics are also the 
major determinant of the sampling strategy, i.e., how often the 
analyses have to be repeated in order to obtain meaningful data, 
and when, in relation to exposure, the specimens should be 
collected.  Toxicokinetics should be taken into account, even when 
considering which biological specimens should be used (Aitio et 
al., in press). 

    For chemicals with a long half-time, such as lead, mercury, and 
cadmium, concentrations in the blood reach a plateau that reflects 
the amount being absorbed.  Under stable exposure conditions, the 
variation in the concentration is minor.  Thus, the time of 
specimen collection in relation to the exposure is not important, 
and measurements performed with rather long intervals, up to “ - 1 
year, give a reliable picture of the continuous exposure.  The 
total amount of, e.g., lead accumulated (mainly in bones) has only 
a minor effect on the blood-lead concentration.  The concentration 
of lead in the nervous system, the main target organ for lead 
toxicity, is, however, more closely related to blood-lead than to 
the body burden (WHO, 1980). 

    The situation is rather different for the chemicals with short 
half-times such as organic solvents.  Most of them have several 
consecutive half-times, corresponding to distribution in blood and 
richly vascularized parenchymal organs, muscles, and adipose 
tissue.  The tissue time-constants for these three are, e.g., for 
 m-xylene, a typical aromatic solvent, approximately 3 min, 50 
min, and 70 h, respectively (Riihimäki, 1984).  Thus, immediately 
after exposure, for approximately 15 min, the blood-solvent 
concentrations decrease very rapidly.  If the collection of the 
blood specimen is accurately timed, information concerning a 
preceding short-term peak of exposure can be obtained.  If the 
specimen collection cannot be accurately timed (± 30 seconds), the 
results cannot be interpreted. 

    Analysis of a specimen collected 15 min - 3 h after the 
exposure gives an idea of the exposure during the last 1 - 3 h, 
timing need not be equally accurate, ± 5 min variations are 
acceptable.  In contrast to the first case, this may be achieved in 
routine biological monitoring. 

    Specimens collected several hours after exposure mainly 
represent the concentrations of the solvent in the fat, and thus 
give an idea of the exposure over a whole working day, even several 
days.  The fat thus functions as an integrator of the exposure over 
time.  In this case, a variation of several hours in specimen 
collection does not make any marked difference.  It seems that the 
latter approach is best suited for routine biological monitoring.  
The specimens are collected in the morning before the next day's 
exposure commences. 

    Urine samples are not routinely obtained at short intervals.  
Consequently, they reflect exposure over at least a few hours and 
are unlikely to indicate short-term peaks of exposure. 

    Concentrations of chemicals in the environment are not stable.  
Because of variations in absorption, the amounts taken up may vary 
markedly over time.  Thus, as a general rule, the shorter the half-
time, the more frequent the biological monitoring must be. 

9.2.2.  Dermal absorption

    Several chemicals are effectively absorbed through intact skin 
(section 3).  Dermal absorption cannot be accurately determined 
from a knowledge of the concentration of a chemical in the 
environment, and biological monitoring is very important in the 
evaluation of the exposure to chemicals that effectively penetrate 
the skin. 

    Biological monitoring data may be misinterpreted when dermal 
exposure is not carefully considered.  For example, the error 
generated may be more than 100-fold (Aitio et al., 1984; Hogstedt, 
1984; Riihimäki, 1984).  However, this error is avoided when the 
urinary excretion of the parent compound and metabolites is 
measured, when metabolites in the blood are analysed, or when 
several hours have elapsed since skin contact. 

9.2.3.  Specimens in use

    For practical purposes, blood and urine are the only biological 
media that are used in routine monitoring.  Whether blood, urine, 
or both are chosen, depends on metabolic, kinetic, and analytical 
factors specific for the chemical in question.  Hair cannot be used 
unless external contamination is excluded (Aitio et al., in press).  
Problems in sample collection and storage, as well as in the 
standardization, prohibit the routine use of exhaled air specimens. 

9.3.  Effect Monitoring

    Effect monitoring means that a selective effect is measured 
instead of the substance itself or its metabolite(s).  Monitoring 
of an early effect of the chemical should be ideal for the 
prevention of adverse health effects.  It compensates for 
individual differences in susceptiblity and differences in the 
amounts of the chemical taken up.  It should be realized that a 
single effect is seldom, if ever, specific for a single chemical.  
The best known examples of such effects are the reversible 
depression of 5-delta-amino-levulinate dehydratase (EC 
activity and the elevation of the zinc protoporphyrin concentration 
in the erythrocytes after exposure to lead.  However, not only lead 
exposure is indicated by these effects; the erythrocyte zinc 
protoporphyrin level is also elevated in iron deficiency anaemia.  
Measurement of effects should usually be regarded as part of health 
surveillance, but not as biological monitoring. In practice, 
however, biological monitoring and health surveillance form a 
continuum in human health protection. 

9.4.  Monitoring of Exposure to Carcinogens

    Methods for assessing the exposure of human beings to 
carcinogens was the subject of a recent symposium (Berlin et al., 
1984a).  The genotoxic carcinogens are also mutagens. This property 
has been applied as a nonspecific means of evaluating exposure to 
mutagenic chemicals.  Most mutagens are metabolized in the body to 
reactive, electrophilic species.  The electrophile reacts with 

nucleophilic sites on the genome, which in turn leads to a 
mutation.  The formation of the electrophilic species has also been 
used to indicate exposure to carcinogenic chemicals.  Both of these 
approaches are, in principle, nonspecific with regard to the 
chemical, but are specific for the nature of the effect.  They have 
been most widely applied in the monitoring of complex mixtures, 
where a whole array of chemical analyses would be necessary to 
verify all exposures. 

    Many carcinogenic chemicals are also clastogenic, i.e., they 
cause chromosomal damage that can be detected microscopically.  
Chromosome damage in circulating lymphocytes can indicate human 
exposure to clastogenic compounds, including some carcinogens. 

9.4.1.  Urinary mutagenicity

    Mutagenicity testing of the urine is widely used to assess the 
exposure of human beings to carcinogens by nonspecific means.  
Mutagens have been detected and monitored in the urine of people in 
the rubber, explosives, and plastics industries, and in that of 
persons handling cytostatic drugs, and active and passive smokers 
(Sorsa, 1985).  Urine poses some problems for mutagenicity testing, 
notably, the low concentrations of mutagens in the urine, the 
toxicity of urine constituents for the indicator organisms, and the 
presence of the mutagen in the urine in an inactive form.  The 
problem of sensitivity has been solved by applying concentration 
methods (generally XAD-resins), and by adopting very sensitive 
assay methods, most often the fluctuation modification of the 
classical  Salmonella-reversion assay (Green et al., 1977; Yamasaki 
& Ames, 1977; Falck et al., 1979).  In order to reactivate the 
chemicals in the urine, treatments with enzymes, such as beta-
glucuronidase and sulphatase, have been used.  The toxicity of 
urine usually limits the sensitivity of the assay, and the toxicity 
should always be assayed together with mutagenicity. 

    Smoking is often a problem in mutagenicity assays because 
tobacco smoke contains a variety of mutagenic chemicals.  However, 
most of the mutagens in smoke are frame-shift mutagens, and, in 
situations where the occupational exposures contain mutagens of 
other types, the smoking effect can be removed by using bacterial 
strains not sensitive to frame-shift mutagens (Falck et al., 1980). 

    Only genotoxic carcinogens can be detected by mutagenicity 
assays.  Carcinogenic chemicals may be excreted in the urine in a 
form that is not mutagenic when assayed by standard techniques.  
The concentration methods applied may show very small recovery of 
the mutagens in the urine.  Therefore, negative results in the 
urine mutagenicity testing do not prove the absence of exposure to 
carcinogenic chemicals.  Despite the problems involved, urinary 
mutagenicity monitoring has proved very useful for the detection of 
carcinogen exposure and the subsequent implementation of preventive 
hygienic measures. 

9.4.2.  Alkylation or arylation of proteins, peptides, amino acids, 
and nucleic acids

    The most widely applied technique has been the analysis for 
thioether metabolites in the urine.  Urinary thioethers are mainly 
derived from the reaction of electrophilic chemicals with the 
tripeptide glutathione.  After this initial reaction, glutathione 
thioethers are enzymatically degraded to yield  N-acetyl- S-
substituted cysteines, or mercapturic acids:  these are excreted in 
the urine (section  However, the evaluation of carcinogen 
exposure from urinary mercapturic acid excretion poses several 
serious problems.  Variable amounts of mercapturic acids are 
derived from dietary sources.  Because of variations in the 
chemical structure and the physical and chemical properties of the 
mercapturic acids, their recovery in the purification steps of the 
analysis may be variable and low.  Detection of urinary thioethers 
has to be regarded as a signal of exposure only, and may be used in 
the follow-up of exposure in selected cases.  The development of 
simpler methods for determining specific thioether compounds will 
probably overcome this problem in the future. 

    The alkylation of haemoglobin has also been used as an 
indicator of exposure to alkylating compounds (Ehrenberg et al., 
1974; Calleman et al., 1978; Farmer et al., 1985).  So far, 
experience has been rather limited, and the very high degree of 
sophistication required in the analysis limits wider application of 
this approach.  Estimation of the alkylation of DNA, although 
promising, is not, at present, applicable to routine biological 
monitoring.  The development of immuno-chemical assays for 
metabolite-modified macromolecules, using selected monoclonal 
antibodies, combines high selectivity and sensitivity for the 
analysis of these adducts (Adamkiewicz et al., 1984). 

9.4.3.  Chromosomal damage

    Assays of chromosomal aberrations and sister chromatid exchange 
frequencies have been successfully used to indicate exposure to 
carcinogenic chemicals (Sorsa, 1983, 1984, 1985).  Strictly 
speaking, both represent effects rather than amounts of chemicals.  
However, the relationship between these effects and the final 
carcinogenic outcome is obscure and, at present, both have to be 
regarded as indicators of exposure rather than direct measures of 
risk.  Analysis of structural chromosomal aberrations is a very 
tedious task, requires highly skilled expertise and is also, to 
some extent, subjective.  Analysis of sister chromatid exchanges is 
technically less demanding and less time consuming.  However, the 
circumstances of cell culture have a remarkable effect on the 
frequencies of the sister chromatid exchanges detected.  Therefore, 
at present, both chromosomal aberration and sister chromatid 
exchange examinations should be used for  ad hoc studies only, and 
not in routine monitoring programmes.  When they are used, a 
matched group of unexposed people has to be studied concurrently. 

9.5.  Preanalytical Error

    Analytical methods and errors are dealt with in section 2.  
Because in biological monitoring, preanalytical variation, i.e., 
the variation associated with specimen collection and storage, 
forms a major part of the total variation (Aitio et al., in press), 
these aspects are treated in some detail in the following sections.  
All such errors must be considered by the personnel responsible for 
the sampling.  The analysing laboratory can do little to avoid 

9.5.1.  Physiological and environmental sources of variation

    These sources of variation include body posture (Alström et 
al., 1975), diurnal variation (Piotrowsky et al., 1975), urine 
volume (Tietz, 1976), meals, nature of the diet, and tobacco smoke, 
and may lead to preanalytical error if not considered. 

9.5.2.  Variation associated with specimen collection and storage

    The factors most likely to cause error in specimen collection 
and storage include evaporation, chemical deterioration, 
precipitation and adsorption on vessel surfaces, and contamination.  
As the chemical nature of substances monitored differs widely, the 
relative importance of these processes also varies (Aitio & 
Järvisalo, 1984).  Many organic solvents are volatile and care must 
be exercised to prevent loss (Curtis et al., 1973).  Chemical 
deterioration is a problem typical of organic chemicals and may be 
of special significance for samples containing trace amounts of 
xenobiotics.  Urine is often voided as a supersaturated solution 
and the salts may precipitate upon storage.  Trace elements may 
coprecipitate with the salts, or may be adsorbed on the surface of 
crystals.  Contamination is often the most important source of 
error in the analysis of xenobiotics (Aitio & Järvisalo, 1984; 
Aitio et al., 1985, in press).  This specific problem is well 
illustrated by the changes in reported serum chromium values in 
people over the last 20 years (Table 6) (Versieck & Cornelis, 1980; 
Aitio et al., 1985).  A similar table can be constructed for many 
trace elements (Versieck & Cornelis, 1980). 

    Contamination may come from the environment or the laboratory, 
from the skin or clothes of the exposed individual, from plastic or 
glass specimen containers, additives, reagents, or from the 
instruments used for analysis. 

Table 6.  Evolution of the values regarded 
as normal average serum-chromium 
concentrations in unexposed men
Reference                   Serum-chromium
Monacelli et al. (1956)     3600
Glinsman et al. (1966)      540
Behne & Diehl (1972)        198
Davidsohn & Secrest (1972)  97
Pekarek et al. (1974)       31
Grafflage et al. (1974)     14
Versieck et al. (1978)      3.1
Kayne et al. (1978)         2.7
Kumpulainen et al. (1983)   2.3
Veillon et al. (1984)       2.1


10.1.  General Considerations

    Toxicokinetic studies are performed on experimental animals to 
help understand the chemical and the biological basis for the 
toxicological effects observed.  It is the ultimate goal of such 
studies to aid in the assessment of the toxic effects of the test 
compounds in human beings. 

    The use of toxicokinetic methods will improve the evaluation of 
human health risks associated with environmental exposure to 

    Health risks in exposed groups can be estimated on the basis of 
toxicokinetic data derived from animals and man, together with 
epidemiological studies of adverse effects.  Toxicokinetic data 
concerning xenobiotics other than drugs are limited in human 
beings.  Existing information has been obtained from occupational 
exposures, accidental environmental exposures, and poisonings. 

    With the specificity and sensitivity of recently developed 
immunochemical methods, it may be possible, in some instances, to 
quantify ultimate reactive metabolite-macromolecule adducts in the 
blood cells of human beings.  Since the amount of metabolite-
macromolecule adduct is likely to be closely related to the 
fraction of the dose involved in the initiation of the toxic 
effect, this parameter has considerable potential for risk 
estimation.  Toxicokinetic studies on animals can serve as a basis 
for selecting appropriate sampling times, and it may be possible to 
determine exposure levels of human beings to toxic environmental 
chemicals with such techniques. 

    Ideally, to assess data developed from toxicological effect 
studies, toxicokinetic investigations should be performed at dose 
levels that do not cause biological effects as well as at dose 
levels that cause acute or chronic toxicity. 

    The  in vitro preparations that are used in toxicokinetic
studies are much simpler than integrated animal systems.  They are 
often selected for their ability to provide mechanistic information 
concerning individual aspects of complicated processes.  For this 
reason, they often do not accurately reflect what occurs  in vivo. 

10.2.  Analytical Data

    The assay procedures used in toxicokinetic studies must both 
identify and quantify the xenobiotic and its metabolites.  Careful 
attention must be paid to assure both the accuracy and precision of 
the analytical methods.  The assay methods applied must be accurate 
over a sufficiently wide range of concentrations for the xenobiotic 
and its metabolites. 

10.3.  Absorption Data

    Ideally, the test chemical should not be changed enzymatically 
or non-enzymatically during absorption.  However, as metabolism 
generally starts immediately after absorption, it may be necessary 
to study metabolites as well as the parent compound.  In general, 
basic mechanisms can be investigated in more detail using  in vitro 
methods than using  in vivo procedures. 

    The chemical under investigation may have biological effects 
influencing perfusion, in this way changing the concentration 
gradient.  The chemical may also affect secretion, modify the 
contact time, or it may modify the thickness and characteristics of 
the diffusion barrier by direct or indirect action (e.g., 
irritation, inflammation), altering absorption.  Studies of 
absorption should therefore be performed under conditions in which 
such biolgical effects are controlled.  These biological effects 
are less pronounced in  in vitro systems.  Therefore, a combination 
of  in vivo and  in vitro methods is advisable and final 
assessments should be based on results from both  in vivo and  in 
 vitro studies. 

    It should also be realized that dermal absorption studies in 
fur-bearing animals are unlikely to accurately reflect dermal 
absorption in human beings.  If the skin is wounded during shaving, 
erroneous results (more absorption) will be obtained. 

10.4.  Distribution Data

    In general, the methods used for investigating the circulation 
and partition of an absorbed xenobiotic, within the body, reflect 
distribution.  When xenobiotics are extensively metabolized, 
distribution studies should be performed for the metabolites as 
well as for the parent compound.  Distribution studies yield 
evidence of possible distribution spaces (e.g., intravascular, 
extracellular, total body water) and the accumulation of a given 

    Distribution patterns do not necessarily indicate target organs 
for toxicity; the highest concentrations of a chemical and its 
metabolites are often found in the elimination organs.  If reactive 
metabolites are formed, covalent binding to macromolecules may 
occur.  This may, but need not, indicate a tissue or cellular 
target of toxicity. 

    The distribution of a chemical may be different at different 
dose levels. 

10.5.  Reversible Binding Data

    The free, but not the total, concentration of a xenobiotic in 
plasma reflects the free concentration in other tissues.  If 
metabolites are formed in the organism, their binding 
characteristics can be investigated separately. 

10.6.  Metabolism Data

    Whether a parent xenobiotic or one or more of its metabolites 
is the ultimate toxic species, can be determined by  in vivo 
studies on otherwise untreated experimental animals as well as on 
animals treated with enzyme inducers and/or inhibitors.  It is 
assumed that the chemical nature of an unstable, toxic 
metabolite(s) can be predicted with some accuracy from a thorough 
knowledge of the biotransformation pathways of the parent compound. 

    If toxicity is due to a biotransformation product, and  in vivo 
metabolism studies are performed under conditions in which a toxic 
response is observed, the excreted biotransformation products 
provide knowledge about the metabolic activation pathway(s) 
involved, as long as the ultimate toxicant is a quantitatively 
important product.  Such studies may also give an indication of the 
chemical mechanism(s) of toxicity, but this is not often the case.  
However, with these preliminary studies, it is, almost invariably, 
possible to choose an appropriate experimental system for 
subsequent  in vitro studies. 

    If large amounts of a single conjugated (glucuronide and 
sulfate) phenolic metabolite are excreted in the urine, the primary 
metabolite can be tested as a substrate for subsequent metabolic 
activation.  The presence of large amounts of mercapturic acids 
( N-acetyl- S-substituted cysteines) in urine suggests that the  in 
 vivo formation of an electrophilic metabolite(s) is a major 
biotransformation pathway, and that this electrophile(s) may be the 
ultimate toxicant.  Logical follow-up studies in this situation 
include investigation of the  in vivo metabolism and toxicity of 
the parent compound in animals that have been treated with a 
depletor of hepatic and renal GSH (e.g., diethyl maleate),  in vitro  
incubation of mixtures in the presence and absence of exogenous 
GSH, and the chemical identification of the metabolites formed in 
both cases. 

    With the appropriate experimental system(s), a chemically and 
radiochemically pure xenobiotic of high specific radioactive 
content, and state-of-the-art instrumentation, structural 
elucidation of xenobiotic metabolites can be peformed routinely. 

    It is frequently very expensive, and sometimes impossible, to 
obtain the xenobiotic to be investigated in a high state of purity 
and/or in radioactive form.  This jeopardizes subsequent studies, 
because observed toxicity may be due to an impurity, or the toxic 
metabolite(s), if minor, may go undetected and unidentified.  
Certain chemicals can exert their toxicity via radical mechanisms 
under conditions in which the parent compound is excreted 

    Even after the structures of the major and minor metabolites 
of a xenobiotic have been positively identified, and the chemical 
nature of the ultimate toxic species is known with certainty, the 
actual mechanism of toxicity (e.g., why the cells die) normally 
remains unknown.  This means that a scientist must clearly define 
the objectives of a study before its initiation, and be prepared to 
alter these objectives during the course of experimentation, 
because the more detailed the structural information required the 
more expensive and labour intensive the studies become. 

10.7.  Excretion Data

    Comprehensive excretion data can be very useful in the 
assessment of chemical toxicity.  Species differences in toxicity 
can sometimes be explained by differences in the excretion of 
chemicals and their metabolites. 

    When the absorption and/or metabolism of a compound are minor 
or the chemical is extensively accumulated, there is little 
excretion by the renal, biliary, and pulmonary routes.  Rates and 
routes of excretion can vary with dosage and sampling period. 

    Excretion data are of limited use in assigning  in vivo sites of 
metabolism.  The data cannot delineate the importance of the renal 
conversion of primary metabolites to conjugates, and are of little 
value in studies of reactive intermediates that covalently bind to 

    Faecal elimination data reflect the combined effects of biliary 
excretion, enterohepatic circulation, gastrointestinal secretion, 
metabolism by intestinal bacteria and/or lack of intestinal 

    Isolated perfused liver preparations facilitate recovery of 
biliary metabolites; the presence of significant amounts of GSH 
adducts in bile demonstrates the formation of an electrophilic 
metabolite(s) by liver. 

    The biliary excretion of chemicals is quite variable in 
different species including man, therefore, direct extrapolation 
of data obtained from one species to another is often erroneous. 

    With sensitive analytical procedures, the concentration of 
lipophilic substances can be measured accurately in alveolar air, 
and the pulmonary excretion estimated.  In general, non-invasive 
breath tests can be performed with a stable isotope-containing 
xenobiotic under physiological conditions.  Exhalation of a 
volatile metabolite containing this isotope will give an indication 
of the rate of biotransformation. 

10.8.  Kinetic Model Data

    The absorption, distribution, accumulation, metabolism, and 
excretion of xenobiotics in experimental animals and man can be 
expressed by mathematical functions, used for the construction of 
kinetic models.  Using these models, the fate of xenobiotics and 
metabolites within the body can be described in exact terms and 
information can be obtained that can be used in the estimation of 
xenobiotic and metabolite accumulation in the organism as a whole, 
or, in individual organs.  Data generated by kinetic models provide 
a basis for the extrapolation of toxicological information from  in 
 vitro and animal studies to man.  Such data are essential both for 
the planning of biological monitoring and for the interpretation of 
the results obtained. 

    The development of an accurate kinetic model depends on 
adequate numbers of individual experimental measurements. 

10.9.  Human Data

    For an accurate determination of a dose-response relationship 
for the detrimental effect(s) of an environmental chemical, 
reliable information on dose is required.  Such data can be 
provided by toxicokinetic studies in which all exposure routes and 
sources of exposure are considered.  Data of this kind may indicate 
interindividual differences in chemical uptake.  Studies of 
occupational exposures, accidental environmental exposures, or 
poisonings in human beings, though lacking detailed toxicokinetic 
information, often provide crude relationships between dose and 
toxic response.  The times of specimen collection relative to 
exposure, the chemical species to be analysed in the exposed human 
beings, and indicator media (blood or urine) of preference, can be 
derived from detailed toxicokinetic studies on experimental 

    Toxicokinetic studies are of limited value for effects that 
occur locally at the port of entry. 


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    See Also:
       Toxicological Abbreviations