INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 57
PRINCIPLES OF TOXICOKINETIC STUDIES
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
chemicals.
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CONTENTS
PRINCIPLES OF TOXICOKINETIC STUDIES
1. INTRODUCTION
2. ANALYTICAL METHODS
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. ABSORPTION
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
3.2.2.1 Measurement
3.2.2.2 The site of gastrointestinal absorption
3.2.3. In vitro methods
3.2.3.1 Isolated loops
3.2.3.2 Isolated cells and vesicles
3.3. Pulmonary absorption
3.3.1. General considerations
3.3.2. In vivo methods
3.3.2.1 Methods for the pulmonary exposure
of intact animals
3.3.2.2 Methods for the pulmonary exposure
of anaesthetized animals
3.3.3. In vitro methods
3.3.3.1 Perfused lungs
3.3.3.2 Fluid-filled lung lobes
3.3.3.3 Isolated cells
3.4. Dermal absorption
3.4.1. General considerations
3.4.2. In vivo methods
3.4.2.1 Methods for dermal exposure
3.4.3. In vitro methods
3.4.3.1 Isolated skin preparations
3.4.3.2 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. DISTRIBUTION
4.1. General considerations
4.2. Invasive methods
4.2.1. Qualitative methods
4.2.1.1 Autoradiographic methods
4.2.2. Quantitative methods
4.2.2.1 Radiometric methods
4.2.2.2 Chemical methods
4.3. Non-invasive methods
5. BINDING
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. METABOLISM
6.1. General considerations
6.2. Important enzymatic pathways in xenobiotic
metabolism
6.2.1. Phase I reactions
6.2.1.1 Oxidation reactions
6.2.1.1.1 Cytochrome P-450 monooxy-
genase system (EC 1.14.14.1)
6.2.1.1.2 Microsomal flavin-containing
monooxygenase (EC 1.14.13.8)
6.2.1.1.3 Cooxidation by prosta-
glandin H synthase
(EC 1.14.99.1)
6.2.1.1.4 Miscellaneous peroxidative
pathways
6.2.1.1.5 Alcohol dehydrogenase (EC
1.1.1.1) and aldehyde
dehydrogenase (EC 1.2.1.3)
6.2.1.1.6 Monoamine oxidase (EC 1.4.3.4)
6.2.1.2 Reduction reactions
6.2.1.2.1 Cytochrome P-450-
dependent reactions
6.2.1.2.2 Flavoprotein-dependent reactions
6.2.1.2.3 Carbonyl reductases
6.2.1.3 Hydrolysis reactions
6.2.1.3.1 Epoxide hydrolase (EC 3.3.2.3)
6.2.1.3.2 Carboxylesterases/amidases
6.2.2. Phase II reactions
6.2.2.1 UDP-glucuronosyltransferase (EC 2.4.1.17)
6.2.2.2 Sulfotransferases
6.2.2.3 Mercapturic acid biosynthesis
6.2.2.3.1 Glutathione S-transfer-
ases (EC 2.5.1.18)
6.2.2.3.2 Cysteine conjugate betalyase/
thiomethylation
6.2.2.4 Amino acid N-acyltransferases
6.2.2.5 N-acetyltransferases (EC 2.3.1.5)
6.2.2.6 N- and O-methyltransferases
6.3. Modulation of important metabolic pathways
6.3.1. Physiological factors
6.3.1.1 Age
6.3.1.2 Genetic factors
6.3.1.3 Sex hormones
6.3.1.3.1 Sex-linked differences
6.3.1.3.2 Pregnancy
6.3.1.4 Thyroid hormones
6.3.1.5 Corticoid hormones
6.3.1.6 Pituitary hormones
6.3.1.7 Immune system
6.3.2. Environmental factors
6.3.2.1 Enzyme induction
6.3.2.2 Inhibition
6.3.3. Pathological factors
6.3.3.1 Liver disease
6.3.3.1.1 Acute viral hepatitis
6.3.3.1.2 Chronic hepatitis and cirrhosis
6.3.3.2.3 Obstructive jaundice
and cholestasis
6.3.3.2 Kidney disease
6.3.3.3 Diabetes
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
6.5.1.1 Intact animals
6.5.1.2 Isolated organs
6.5.1.3 Freshly isolated cells
6.5.1.4 Organs and cells in culture
6.5.2. Cell-free systems
6.5.2.1 Subcellular fractions of tissue
homogenate
6.5.2.2 Purified enzymes and/or reconstituted
enzyme systems
6.5.3. Intestinal microflora
6.6. Methods for assessing chemically reactive
metabolites in vitro
7. EXCRETION
7.1. General considerations
7.2. Important excretory mechanisms
7.2.1. Diffusion and filtration
7.3. Sites of excretion
7.3.1. Kidney
7.3.1.1 Glomerular filtration
7.3.1.2 Tubular secretion
7.3.1.3 Tubular reabsorption
7.3.2. Liver-biliary excretion
7.3.2.1 Enterohepatic circulation
7.3.3. Other excretory sites
7.4. Modulation by physiological, environmental, and
pathological factors
7.4.1. Urinary excretion of xenobiotics
7.4.1.1 pH and urine volume
7.4.1.2 Inhibition and stimulation by
xenobiotics
7.4.1.3 Age differences
7.4.1.4 Species differences
7.4.1.5 Renal dysfunction
7.4.2. Biliary excretion
7.4.2.1 Species and age differences
7.4.2.2 Effects of physiological compounds
7.4.2.3 Effects of xenobiotics
7.4.2.4 Hepatic disease and regeneration
7.5. Methods for assessing excretion
7.5.1. Whole animals
7.5.2. In vitro preparations
7.5.2.1 Isolated organs
7.5.2.2 Intestinal preparations
7.5.2.3 Slices of renal cortex
7.5.2.4 Other kidney preparations
7.5.2.5 Purified membrane preparations
8. KINETIC MODELS
8.1. General considerations
8.2. Dose-independent kinetics
8.2.1. One-compartment model
8.2.1.1 Single dose
8.2.1.2 Repeated dosing
8.2.2. Two-compartment model
8.2.2.1 Single dose
8.2.2.2 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. TOXICOKINETIC METHODOLOGY IN THE ASSESSMENT OF
HUMAN EXPOSURE
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. ASSESSMENT OF TOXICOKINETIC STUDIES
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
REFERENCES
NOTE TO READERS OF THE CRITERIA DOCUMENTS
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.
IPCS TASK GROUP ON TOXICOKINETICS
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,
Indonesia
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,
Norway
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
Secretariat
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.
1. INTRODUCTION
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. ANALYTICAL METHODS
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
obtained.
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
tried.
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
safety.
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
compounds.
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
investigated.
(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
experimentation.
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
procedures.
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
detection.
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. ABSORPTION
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 8.2.1.1,
8.2.1.2).
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
kidneys.
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
Charge
- acid or base characteristics
Stability
Reactivity
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,
radiation)
-------------------------------------------------------------------
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
membrane.
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:
non-ionized
for acids: pKa - pH = log -----------
ionized
ionized
for bases: pKa - pH = log -----------
non-ionized
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,
1984).
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
tract.
3.2.2. In vivo methods
3.2.2.1. 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 8.2.1.1).
3.2.2.2. 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
3.2.3.1. 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.
3.2.3.2. 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
3.3.2.1. 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).
3.3.2.2. Methods for the pulmonary exposure of anaesthetized
animals
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
3.3.3.1. 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).
3.3.3.2. 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).
3.3.3.3. 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
3.4.2.1. 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
3.4.3.1. 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.
3.4.3.2. 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
absorption.
4. DISTRIBUTION
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
patterns:
(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 8.2.1.1). 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
4.2.1.1. 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
4.2.2.1. 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.
4.2.2.2. 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. BINDING
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
responses.
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. METABOLISM
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
4-ipomeanol.
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 1.14.14.1) 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
6.2.1.1. 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.
6.2.1.1.1. Cytochrome P-450 monooxygenase system (EC 1.14.14.1)
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
reactions.
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
cofactor:
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 1.6.2.2) 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.,
1980).
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,
1980).
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).
6.2.1.1.2. Microsomal flavin-containing monooxygenase (EC 1.14.13.8)
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).
FMO
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,
1973).
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).
6.2.1.1.3. Cooxidation by prostaglandin H synthase (EC 1.14.99.1)
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).
6.2.1.1.4. 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 1.11.1.7) 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.
6.2.1.1.5. Alcohol dehydrogenase (EC 1.1.1.1) and aldehyde
dehydrogenase (EC 1.2.1.3)
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
6.2.2.1) 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.
6.2.1.1.6. Monoamine oxidase (EC 1.4.3.4)
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
substituents.
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.
6.2.1.2. 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).
6.2.1.2.1. 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
pathway.
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).
6.2.1.2.2. 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 1.6.2.4)
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
mitochondria.
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 1.6.99.2),
NADH or NAD(P)H dehydrogenase, quinone). Several exogenous nitro
compounds are known to serve as electron acceptors for xanthine
oxidase (EC 1.1.3.22) as do certain N-oxides. Aldehyde oxidase
(EC 1.2.3.1) 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.
6.2.1.2.3. Carbonyl reductases
As mentioned earlier (section 6.2.1.1), 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 1.1.1.2) and
carbonyl reductases (EC 1.1.1.184). 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
ketones.
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.
6.2.1.3. 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.
6.2.1.3.1. Epoxide hydrolase (EC 3.3.2.3)
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).
6.2.1.3.2. 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 3.1.1.2; arylesterases) preferentially hydrolyse
aromatic esters, hydrolyse the organophosphate paraoxon, and are
inhibited by Hg2+; B-esterases (EC 3.1.1.1; carboxylesterases)
preferentially hydrolyse aliphatic esters, are inhibited by
paraoxon, and are not influenced by Hg2+; and C-esterases (EC
3.1.1.6; 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
activity.
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.
6.2.2.1. UDP-glucuronosyltransferase (EC 2.4.1.17)
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 7.3.2.1).
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.
6.2.2.2. 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
2.8.2.1) are active with phenols, hydroxylamines (e.g., N-hydroxy-
2-acetylaminofluorene), tyrosine esters, and catecholamines; the
alcohol sulfotransferases (EC 2.8.2.2) are active with primary
and secondary steroid alcohols; and amine sulfotransferases
(EC 2.8.2.3) are active with arylamines (to form sulfamates), an
activity not catalysed by either purified aryl or alcohol
sulfotransferases.
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).
6.2.2.3. 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
N-acetyltransferase.
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.
6.2.2.3.1. Glutathione S-transferases (EC 2.5.1.18)
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
attacks.
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).
6.2.2.3.2. 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 4.4.1.13) (Tateishi & Shimizu, 1980; Jakoby et al., 1984b),
which cleaves the cysteine adduct to a free thiol, ammonia, and
pyruvate (equation 9).
beta-lyase
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
cycles.
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).
6.2.2.4. 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).
6.2.2.5. N-acetyltransferases (EC 2.3.1.5)
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 6.2.1.3); 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).
6.2.2.6. N- and O-methyltransferases
Since S-adenosyl-L-methionine (Ad-Met)-dependent S-methyl-
ation has already been discussed (section 6.2.2.3), 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 2.1.1.6), histamine
N-methyltransferase (EC 2.1.1.8), 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
6.3.1.1. 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 6.2.1.2). 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).
6.3.1.2. 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
metabolism.
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 6.3.2.1). 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).
6.3.1.3. Sex hormones
6.3.1.3.1. 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.,
1977).
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).
6.3.1.3.2. 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
unchanged.
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).
6.3.1.4. 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.
6.3.1.5. 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).
6.3.1.6. 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).
6.3.1.7. 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
6.3.2.1. 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 6.3.1.2).
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).
6.3.2.2. 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
chemical.
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.
6.3.3.1. 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).
6.3.3.1.1. 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.
6.3.3.1.2. 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).
6.3.3.1.3. 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.
6.3.3.2. 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).
6.3.3.3. 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
methods.
Once collected, the fluids containing the chemicals of interest
should be analysed according to the principles outlined in section
2.
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.
6.5.1.1. 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).
6.5.1.2. 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 7.5.2.1).
6.5.1.3. 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.
6.5.1.4. 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
6.5.2.1. 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.
6.5.2.2. 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,
1981).
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
activation.
7. EXCRETION
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.
7.3.1.1. 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.
7.3.1.2. 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).
7.3.1.3. 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 7.4.2.1), 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.
7.3.2.1. 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.,
1975).
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
Factors
7.4.1. Urinary excretion of xenobiotics
7.4.1.1. pH and urine volume
As explained previously, the urinary excretion rate can be
altered by changes in pH (section 7.3.1.2) or urine volume
(Pritchard & James, 1979; Renwick, 1982) (sections 7.2.1.1,
7.3.1.2).
7.4.1.2. 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)
(section 7.5.2.3).
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).
7.4.1.3. 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.
7.4.1.4. Species differences
Differences between mammals in the renal excretion of
xenobiotics tend to be quantitative in nature (Irish & Grantham,
1981; Renwick, 1982).
7.4.1.5. Renal dysfunction
Renal excretion of chemicals is generally decreased in severe
kidney disease (section 6.3.3.2).
7.4.2. Biliary excretion
7.4.2.1. 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).
7.4.2.2. 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).
7.4.2.3. 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).
7.4.2.4. 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
coprophagy.
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.
7.5.2.1. 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).
7.5.2.2. 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).
7.5.2.3. 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.
7.5.2.4. 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.
7.5.2.5. 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. KINETIC MODELS
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.
8.2.1.1. 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).
dX
-- = -ke x X or X = Xo x e-ke x t (15)
dt
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
16).
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
(18):
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)
2.303
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 t“ can 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
time.
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
Clr = kr x VD or kr = --- (22)
VD
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
intervals.
(b) Absorption
Including a first order absorption process in the model,
equation (15) becomes equation (25).
dX
-- = ka x Xa - ke x X (25)
dt
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
concentrations.
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)
VD(ka-ke)
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):
AUCo-infinite(p.o.)
F = ------------------- (29)
AUCo-infinite(i.v.)
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).
8.2.1.2. 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)
tau
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.
8.2.2.1. 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).
dXc
--- = k21 x Xp - k10 x Xc - k12 x Xc (34)
dt
dXp
--- = k12 x Xc - k21 x Xp (35)
dt
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.
t“beta = 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):
Clt
beta = ----- (42)
Vbeta
dose(i.v.)
Clt = Vbeta x beta = Vc x k10 = ------------ (43)
AUCo-infinite
The renal elimination rate constant kr can easily be derived
from urinary excretion data (equation 44).
Xexo-infinite
kr = ------------- x k10 (44)
dose(i.v.)
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.
8.2.2.2. 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 t“ for 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 t“ for 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)
where:
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.
dC
- -- ~ V'max (53)
dt
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
available.
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
organ.
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):
CT
R = -- equilibrium (55)
CV
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)
CA
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
(59).
Q(CA - CV)
ClO = ---------- = Q x E (59)
CA
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
constant.
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)
Clt
9. TOXICOKINETIC METHODOLOGY IN THE ASSESSMENT OF HUMAN EXPOSURE
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 8.2.1.1). 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 4.2.1.24)
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 6.2.2.3). 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
them.
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
(nmol/litre)
------------------------------------------
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. ASSESSMENT OF TOXICOKINETIC STUDIES
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
xenobiotics.
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
chemical.
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
unchanged.
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
macromolecules.
Faecal elimination data reflect the combined effects of biliary
excretion, enterohepatic circulation, gastrointestinal secretion,
metabolism by intestinal bacteria and/or lack of intestinal
absorption.
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
animals.
Toxicokinetic studies are of limited value for effects that
occur locally at the port of entry.
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