Ochratoxin A was evaluated by the Committee at its thirty-seventh meeting (Annex 1, reference 94), when it established a provisional tolerable weekly intake (PTWI) of 112 ng/kg bw, on the basis of deterioration of renal function in pigs, for which the lowest-observed-effect level (LOEL) was 0.008 mg/kg bw per day, and a safety factor of 500. At that time, the Committee recommended that further studies be conducted to elucidate the role of ochratoxin A (and other mycotoxins) in causing nephropathy in pigs and humans, the mechanisms of induction of tumours, and the role of phenylalanine in antagonizing the adverse effects of ochratoxin A. (The present Committee noted that the adverse effects noted at the thirty-seventh meeting consisted of nephrotoxicity.) Ochratoxin A was re-evaluated by the Committee at its forty-fourth meeting (Annex 1, reference 116), when it considered toxicological data that had become available since the previous evaluation, including studies on the epidemiology of nephropathy, on genotoxicity and on experimental nephrotoxicity. At that meeting, the Committee reconfirmed the PTWI, rounding it to 100 ng/kg bw, and reiterated its request for further studies on ochratoxin A.

The Codex Committee on Food Additives and Contaminants at its Thirty-first Session requested the Expert Committee to perform a risk assessment of the consequences of establishing a maximum level of 5 or 20 µg/kg in cereals and cereal products.

Ochratoxin A is produced by a single Penicillium species, P. verrucosum, by Aspergillus ochraceus and several related Aspergillus species, and by A. carbonarius, with a small percentage of isolates of the closely related A. niger. These three groups of species differ in their ecological niches, in the commodities affected, and in the frequency of their occurrence in different geographical regions. P. verrucosum grows only at temperatures below 30 °C and at water activity as low as 0.8. It is therefore found only in cool temperate regions; it is the source of ochratoxin A in cereals and cereal products in Canada and Europe. As cereals are widely used in animal feeds in Europe, and ochratoxin A is relatively stable in vivo, this mycotoxin is also found in some animal products in that region, especially in pig kidney and liver. As P. verrucosum does not occur in the tropics and subtropics, cereals from these regions are unlikely to contain ochratoxin A from this source. A. ochraceus grows at moderate temperatures and at a water activity above 0.8. It is found sporadically in a wide range of stored food commodities, including cereals, but is seldom the cause of substantial concentrations of ochratoxin A. It may also infect coffee beans during sun-drying and is a source of ochratoxin A in green coffee beans. A. carbonarius grows at high temperatures and is associated with maturing fruits, especially grapes. Because of its black spores, it is highly resistant to sunlight and survives sun-drying. It is the source of ochratoxin A in fresh grapes, dried vine fruits, and wine; it is also one source of ochratoxin A in coffee.

The Committee considered several new studies that had become available since the previous evaluation of ochratoxin A. These included further studies of absorption, distribution (including secretion into the milk of experimental animals), metabolism, and excretion; biochemical studies; toxicological studies on genotoxicity, immunotoxicity, neurotoxicity, embryotoxicity, and hepatotoxicity; and studies on the mechanisms of cytotoxicity and nephrotoxicity. The results of epidemiological studies were also reviewed. New data from surveys of food commodities for ochratoxin A and of food consumption were also considered, and intakes were estimated for various countries and regions of the world.

2.1.1 Absorption, distribution, and excretion

(a) Absorption

It has been suggested that, in most species, ochratoxin A is absorbed from the stomach as a result of its acidic properties (pK_a = 7.1) (Galtier, 1978; Roth et al., 1988). In studies of animals with ligated gastrointestinal loops, however, the small intestine was found to be the major site of absorption, with maximal absorption from the proximal jejunum. Absorption from the jejunum can take place against a concentration gradient and depends on the pH at the mucosal surface of the jejunum. Ochratoxin A that is so transferred is lipid-soluble and non-ionized (Kumagai & Aibara, 1982; Kumagai, 1988).

The results of studies in which a low dose of [³H]ochratoxin A was given by intubation to mice were interpreted by the authors as indicating rapid absorption from the stomach, but they could also be interpreted as showing that intestinal absorption is the major route, with rapid transit from the stomach to the intestine. Secondary peaks of ochratoxin A found in the intestinal contents and serum may have been a consequence of enterohepatic circulation, since the biliary excretion of this toxin is very efficient (Fuchs et al., 1988a; Roth et al., 1988).

The overall percentage of ochratoxin A absorbed was 66% in pigs, 56% in rats, 56% in rabbits, and 40% in chickens (Suzuki et al., 1977; Galtier et al., 1981).

In male Wistar rats that received a single intratracheal dose of crystalline ochratoxin A (purity unknown) at 50 ng/g bw, absorption from the lungs was found to be very efficient, the bioavailability being calculated as 98%. The biological half-life of ochratoxin A was estimated to be 127 h. The toxicokinetics of the toxin when given intratracheally, orally, or intravenously were comparable (Breitholtz-Emanuelsson et al., 1995).

Phenylalanine given to mice by gavage with ochratoxin A in a 10:1 molar ratio appeared to increase the absorption of ochratoxin A from the stomach and intestine and to increase gastrointestinal transit. This resulted in an eightfold higher concentration of ochratoxin A in serum and and a fourfold higher concentration in liver during the next 12 h (Roth et al., 1988).

b) Distribution

The bioavailability of ochratoxin A, estimated from a comparison of the maximal serum concentration after oral and intravenous administration, was very low in fish but 44 and 97% for two mammalian species investigated (Hagelberg et al., 1989). Once it reaches the blood, ochratoxin A bound readily to serum albumin (Galtier et al., 1980) and other macromolecules (Hult & Fuchs, 1986). Erythrocytes contained only traces (Galtier, 1978).

The association constant for the binding of ochratoxin A to serum albumin was 7.1 × 10⁴ per mol for pigs, 5.1 × 10⁴ per mol for chickens, and 4.0 × 10⁴ per mol for rats (Galtier et al., 1981). The fraction of ochratoxin A bound to serum albumin and other macromolecules constitutes a mobile reserve of mycotoxin that can be made available for release to the tissues for a long time (Galtier, 1978; Hult et al., 1982). Studies with albumin-deficient rats showed that the main effect of ochratoxin A binding to serum albumin is to retard its elimination by limiting its transfer from the bloodstream to hepatic and renal cells (Kumagai, 1985).

In studies of the stability of ochratoxin A bound to porcine albumin, it was displaced by the acidic drug phenylbutazone, so that more free toxin was available. In male rats, ochratoxin A was more toxic in the presence of phenylbutazone, with a significant decrease in the LD₅₀ value from 33 to 21 mg/kg bw (Galtier et al., 1980).

Ochratoxin A had strong affinity for an unidentified serum macromolecule (relative molecular mass, 20 000), with association constants of 2.3 × 10¹⁰ per mol in human serum and 0.59 × 10¹⁰ per mol in porcine serum. The specific binding of this macromolecule was saturated at concentrations of ochratoxin A of 10–20 ng/ml serum. Significant amounts of serum albumin were bound at higher concentrations of ochratoxin A, with saturation above several hundred micrograms per millilitre of serum (Stojkovic et al., 1984; Hult & Fuchs, 1986).

The fraction of ochratoxin A that remained unbound to two identified plasma proteins was 0.02% in humans and rats, 0.08% in monkeys, 0.1% in mice and pigs, and 22% in fish (Hagelberg et al., 1989).

Once ochratoxin A has been absorbed, the concentrations of the toxin and its metabolites in tissues and plasma residues depend on the length of feeding, the dose, whether the ochratoxin A is naturally occurring or crystalline, the route, the degree of serum binding, the half-life of ochratoxin A, and the duration on an ochratoxin A-free diet before sacrifice. These factors are important in assessing the natural occurrence of residues in animal tissues (Kuiper-Goodman & Scott, 1989).

After a single oral dose, the maximum serum concentrations of ochratoxin A were found within 10–48 h in pigs and rats (Suzuki et al., 1977; Galtier, 1978; Galtier et al., 1981; Mortensen et al., 1983a), at 2–4 h in ruminant calves (Sreemannarayana et al., 1988), after 1 h in rabbits, and after 0.33 h in chickens (Galtier et al., 1981). Maximum concentrations in tissues were found within 48 h in rats.

Wide species differences have been reported in the serum half-life of ochratoxin A. The half-life after oral administration was found to be 510 h in Macaca mulata monkeys (Hagelberg et al., 1989), 72–120 h in pigs (Galtier et al., 1981; Mortensen et al., 1983a), 77 h in pre-ruminant calves (Sreemannarayana et al., 1988), 55–120 h in rats (Galtier et al., 1979; Ballinger et al., 1986; Hagelberg et al., 1989), 6.7 h in quail (Hagelberg et al., 1989), and 4.1 h in chickens (Galtier et al., 1981). In those species tested, the serum half-time was longer after intravenous administration (Hagelberg et al., 1989), perhaps due in part to differences in absorption (Galtier et al., 1981), differences in peak plasma concentrations (see above), and species differences in the degree of binding to serum macromolecules, including albumin.

The rate of disappearance of ochratoxin A was slower from blood than from kidney, liver, and other tissues in pigs (Hult et al., 1979).

Whole-body autoradiography of mice after a single intravenous dose of [¹⁴C]ochratoxin A at approximately 200 µg/kg bw showed that the toxin persisted for > 4 days in the blood, interpreted as showing that the toxin is present mainly in bound form at this dose (Fuchs et al., 1988a). In a similar experiment in rats, the distribution after 24 h was greatest in lung and, in decreasing order, in adrenal medulla, skin, liver, myocardium, kidney, salivary gland, adrenal cortex, muscle, gastric mucosa, and bone marrow (Breitholtz-Emanuelsson et al., 1992). The tissue distribution in pigs, rats, chickens, and goats generally followed the order kidney > liver > muscle > fat (Harwig et al., 1983) or kidney > muscle > liver > fat (Mortensen et al., 1983a; Madsen et al., 1982).

In hens fed ochratoxin A, none was found in eggs (Krogh et al., 1976). In another study, it was found in eggs when the birds were fed 10 mg/kg bw (Juszkiewicz et al., 1982). A study of the tissue distribution of [¹⁴C]ochratoxin A in laying Japanese quail showed specific retention of unidentified radiolabel as a ring-shaped deposition in eggs, indicating that the toxin could be deposited over a short period (Fuchs et al., 1988b). Egg-laying Japanese quail were given a single oral dose of 0, 1, 5, or 20 mg/kg bw. The concentrations of ochratoxin A in abdominal yolk of birds 6 h later were 13 µg/kg in those given 5 mg/kg bw and and 34 µg/kg in those given 20 mg/kg bw. The toxin was still present in abdominal yolks 4 days after administration, and the mean concentration was 10-fold higher than in whole eggs. No ochratoxin A was found in eggs of birds given 1 mg/kg bw (Piskorska-Pliszczynska & Juszkiewicz, 1990).

Lactating rats, treated orally with a single dose of ochratoxin A up to 250 µg/kg bw excreted the toxin in their milk. The milk:blood concentration ratio was 0.4 at 24 h and 0.7 at 72 h. A linear relationship was found between the concentration of ochratoxin A in the dam’s milk and that in the blood and kidneys of pups at 72 h. The pup milk:blood concentration ratio was approximately 1.7. At 72 h, the suckling pups had higher concentrations of ochratoxin A than their dams in both blood and kidney (Breitholtz-Emanuelsson et al., 1993a).

Whole-body autoradiography after intravenous administration of high doses of [¹⁴C]ochratoxin A showed that it could cross the placenta more readily when given on days 8 and 9 than day 10 of gestation, with radiolabel appearing within 20 min in the uterine wall, placenta, and fetal tissues. Ochratoxin A given to mice on day 17 of gestation resulted in very little radiolabel in fetuses (Appelgren & Arora, 1983a,b).

Differences in fetal uptake of ochratoxin A at different times during gestation were suggested to be due to differences in the placenta, which was considered to be completely developed by day 9 of gestation. After intraperitoneal injection of ochratoxin A on day 11 or 13 of gestation, residues appeared more slowly and reached maximum values 30–48 h after dosing. The concentrations in the placenta were high 2–6 h after injection and then decreased more slowly than from other tissues. The serum half-life of ochratoxin A was 29 h at day 11 and 24 h at day 13 of gestation. The authors considered the embryo to be a ‘deep compartment’ (Fukui et al., 1987).

A group of 39 female Sprague-Dawley rats received ochratoxin A orally at 50 µg/kg bw five times a week for 2 weeks before mating, during gestation, and then 7 days a week during lactation. Pups from ochratoxin A-treated dams were cross-fostered at birth to control dams and vice versa. Treatment did not affect maternal body weight nor alter the birthweight or development of pups. The concentrations of ochratoxin A in the blood and kidney of exposed pups were three to four times higher than those in the dams. No differences in weight gain or in body or kidney weight were seen between pups exposed in utero, via lactation, or both. The transfer of ochratoxin A to milk was very efficient (60% of the blood concentration at 8 weeks). The highest blood and kidney concentrations were found in offspring exposed in utero and via milk, but the most significant exposure was via milk (Hallén et al., 1998).

After subcutaneous administration of [³H]ochratoxin A to rats on day 12 of gestation, fetal uptake was delayed, with maximum concentrations 48–72 h after dosing, representing about 0.1% of the dose administered (Ballinger et al., 1986).

Four lactating Blanc de Termonde rabbits received ochratoxin A from feed naturally contaminated at 190 ng/g, equivalent to 16 µg/kg bw, on days 3–19 of lactation. The toxin was effectively transported from blood to milk and subsequently to the offspring. Higher concentrations were found in maternal plasma than in milk, and a linear relationship was found between the concentrations in milk and plasma of offspring. The plasma:kidney concentrations were much higher in offspring than in adults, perhaps due to slower detoxication in the former (Ferrufino-Guardia et al., 2000).

Ochratoxin A given at 0.38 mg/kg bw to pregnant sows on days 21–28 of pregnancy did not cross the placenta (Patterson et al., 1976). Similarly, no residues were found in piglets of sows fed diets containing ochratoxin A at 7–16 µg/kg bw per day throughout gestation (Mortensen et al., 1983b). In a more recent study, however, ochratoxin A was transmitted to six piglets in utero when the sow was fed naturally contaminated feed; the blood concentrations in the newborn piglets were 0.075–0.12 ng/ml, whereas that in the sow was 0.20 ng/ml (Barnikol & Thalmann, 1988).

(c) Excretion

Both biliary excretion and glomerular filtration play important roles in the plasma clearance of ochratoxin A in rats. This is related to its relative molecular mass of 403.8, since both pathways are used in this species for substances with relative molecular masses between 350 and 450. Thus, in rats, both the urinary and faecal excretory routes are important, the relative contribution of each depending on factors such as route of administration and dose (Kuiper-Goodman & Scott, 1989).

In all species, the relative contribution of each excretory route is also influenced by the degree of serum macromolecular binding and differences in the degree of enterohepatic recirculation of ochratoxin A (Hagelberg et al., 1989).

In rats, the major excretory products were ochratoxin alpha (in urine and faeces), ochratoxin A, and the 4R-OH-ochratoxin A epimer. In urine, these represented 25–27%, 6%, and 1–1.5% of the administered dose, respectively (Storen et al., 1982a).

Up to 33% of the radiolabel on an orally administered dose of ochratoxin A was excreted into the bile of rats up to 6 h after dosing; only trace amounts of ochratoxin alpha were detected in the bile (Suzuki et al., 1977).

Biliary excretion of ochratoxin A was increased and urinary excretion of ochratoxin A and ochratoxin alpha was decreased in mice pretreated with phenobarbital (Moroi et al., 1985).

When ochratoxin A was administered to rats intraperitoneally, only traces of ochratoxin A and ochratoxin alpha were identified in faeces, whereas after oral administration 12% ochratoxin A and 9% ochratoxin alpha were found in faeces (Storen et al., 1982a).

In pre-ruminant and ruminant calves, 85–90% of orally administered ochratoxin A was excreted as ochratoxin alpha, most of it in the urine (Sreemannarayana et al., 1988).

2.1.2 Biotransformation

Ochratoxin A is hydrolysed to the non-toxic ochratoxin alpha at various sites. In rats, detoxication by hydrolysis to ochratoxin alpha is a function of the bacterial microflora of the caecum (Galtier, 1978). The enzymes responsible for hydrolysis to ochratoxin alpha in cows and rodents are carboxypeptidase A and chymotrypsin (Pitout, 1969a,b; Pitout & Nel, 1969). Other mycotoxins such as penicilloic acid inhibit this reaction (Parker et al., 1982). Inhibition of the flora of the lower gastrointestinal tract of rats by neomycin reduced hydrolysis of ochratoxin A to ochratoxin alpha and increased the blood concentration of ochratoxin A (Madhyastha et al., 1992).

Studies with rat tissue homogenate showed that the duodenum, ileum, and pancreas also have a high capacity to carry out this reaction, whereas the activity in the liver and kidney was low (Suzuki et al., 1977). It was non-existent in rat hepatocytes (Hansen et al., 1982) and rabbit and rat liver (Kanisawa et al., 1979; Stormer et al., 1983).

In rats given [¹⁴C]ochratoxin A, most of the radiolabel was attached to ochratoxin A, indicating that efficient metabolism of this toxin is lacking in most tissues other than the intestine (Galtier et al., 1979).

Incubation of the contents of the four stomachs of cows indicated effective hydrolysis of ochratoxin A to ochratoxin alpha by the ruminant protozoa. Assuming a similar reaction velocity in vivo, it was estimated that up to 12 mg/kg of feed could be degraded (Hult et al., 1976; Pettersson et al., 1982), so that this species is assumed to be relatively resistant to the effects of ochratoxin A in feed. Sheep also have a good capacity to detoxify ochratoxin A before it reaches the blood (Kiessling et al., 1984).

Studies in mice suggest that ochratoxin A circulates from the liver into the bile and into the intestine, where it is hydrolysed to ochratoxin alpha (Moroi et al., 1985).

About 25–27% of ochratoxin A, given either intraperitoneally or orally to rats, was present as ochratoxin alpha in the urine. Its presence in the urine can be explained by reabsorption from the intestine (Storen et al., 1982a). A similar mechanism of intestinal reabsorption of ochratoxin alpha has been suggested to occur in ruminant calves (Sreemannarayana et al., 1988).

Other minor urinary metabolites of ochratoxin A are 4-OH (4R-and 4S) epimers produced in rat and rabbit liver (Størmer et al., 1981) and rat kidney (Stein et al., 1985) by the action of cytochromes P450 (CYPs; Størmer et al., 1981, 1983). The 4R-OH epimer, which is considered less toxic than ochratoxin A, is the main one formed in human and rat liver microsomal systems (Størmer et al., 1981), whereas the 4S-OH epimer is more prevalent in pig liver microsomes. No data were available on its toxicity (Moroi et al., 1985).

The biotransformation of ochratoxin A has also been studied in various microsomal preparations and in recombinant human and rat CYP preparations (Gautier et al., 2001; Zepnik et al., 2001). Incubation of ochratoxin A with liver microsomes from rats and mice produced 4R- and 4S-hydroxyochratoxin A, but at very low rates, whereas oxidation of ochratoxin A was not observed in kidney microsomes from these species. 4R-Hydroxyochratoxin A was also formed at low rates by recombinant human CYP 3A4 (both studies), CYP 1A1 and CYP 2C9-1 (both in single studies), while conflicting results were obtained with CYP1A2. Oxidation was not observed with recombinant human CYP 2E1 or rat CYP 1A2 or the male rat-specific CYP 2C11 (all in one study). Prostaglandin H-synthase produced small amounts of a non-polar product.

The 10-OH derivative was formed from ochratoxin A in a rabbit liver microsomal system (Størmer et al., 1983). Ochratoxin C, a metabolite of ochratoxin A produced in rumenal fluid, is as toxic as ochratoxin A (cited by Galtier et al., 1981). Ochratoxin B, a dechloro derivative of ochratoxin A, may occur with ochratoxin A in cereal products. In rats, it is less toxic than ochratoxin A and is metabolized to 4-OH-ochratoxin B and ochratoxin beta (Størmer et al., 1985).

Ochratoxin B was not antagonistic to ochratoxin A with respect to effects on the formation of phenylalanyl-tRNA and protein synthesis (Roth et al., 1989).

Many researchers consider that the toxicity of ochratoxin A is due to one of its metabolites. The studies cited above indicate, however, that, in rats, ochratoxin A itself, rather than one of its metabolites, is the active toxic agent, since the known metabolites are less toxic than or as toxic as ochratoxin A. This conclusion concurs with findings in mice, in which the LD₅₀ of ochratoxin A increased by 1.5- to 2-fold after the animals were fed phenobarbital at 500 mg/kg of diet for 1 week before oral or intraperitoneal administration (Moroi et al., 1985).

Similarly, pretreatment with sodium phenobarbital at 80 mg/kg bw per day by gavage for 5 days or 3-methylcholanthrene at 20 mg/kg bw per day by gavage for 2 days resulted in increased LD₅₀ values for ochratoxin A. With phenobarbital, the difference was smaller 144 h after dosing with ochratoxin A than at 48 h. Administration of piperonyl butoxide, an inhibitor of microsomal monooxygenases, decreased the 144-h LD₅₀ of ochratoxin A from 40 to 19 mg/kg bw (Chakor et al., 1988). In contrast, preliminary studies in mice showed that simultaneous feeding of phenobarbital slightly increased the incidence of liver tumours seen with ochratoxin A alone, and that the mice developed large, multiple hepatomas (Suzuki et al., 1986).

Few data are available on the metabolic disposition of ochratoxin A in humans. It has been suggested that it has a long serum half-life, on the basis of its strong binding to human serum macromolecules (Bauer & Gareis, 1987; Hagelberg et al., 1989).

2.1.3 Effects on enzymes and other biochemical parameters

The activities of glycolytic enzymes were reduced, whereas those of gluconeo-genic enzymes were increased. The diabetogenic effect of ochratoxin A was thought to be due to inhibited synthesis and/or release of insulin from pancreatic cells, thereby suppressing glycolysis and glycogenesis and enhancing gluconeogenesis and glycogenolysis (Subramanian et al., 1989).

Calcium homeostasis was studied in rats treated intraperitoneally with ochratoxin A at a single dose of 10 mg/kg bw or multiple doses of 0.5–2 mg/kg bw per day. An increase in renal endoplasmic reticulum calcium pump activity was observed, suggesting an association with ochratoxin A-induced renal cytotoxicity (Rahimtula & Chong, 1991).

Studies with pig renal cortical explants indicated that inhibition of the biosynthesis of macromolecules (protein, RNA and DNA) by ochratoxin A was not due to impairment of cellular respiration (Braunberg et al., 1992).

The biochemistry and molecular aspects of the action of ochratoxin A in both prokaryotes and eukaryotes have been reviewed (Röschenthaler et al., 1984). The findings are inconsistent, owing to differences and limitations in experimental models and procedures as well as interfering factors, especially in more complex organisms. In prokaryotes (Konrad & Röschenthaler, 1977), eukaryotic microorganisms (Creppy et al., 1979a), mammalian cells (Creppy et al., 1980a, 1983a), and experimental animals in vivo (Creppy et al., 1980b, 1984), the primary effect of ochratoxin A is inhibition of protein synthesis; secondarily, RNA and DNA synthesis may be inhibited.

The inhibition of protein synthesis is specific and occurs at the post-transcriptional level, ochratoxin A having a direct effect on the translation step in protein synthesis. This involves competitive inhibition of phenylalanine-tRNAPhe synthetase, so that amino-acylation and peptide elongation are stopped. This reaction is fundamental for all living organisms. In yeast, the first part of this reaction, phenylalanine-dependent pyrophosphate exchange, was inhibited five times more than transfer to tRNA, the second part. In this reaction ochratoxin A may be regarded as an analogue of phenylalanine, and in cell cultures the competitive inhibition could be reversed by an increase in phenylalanine concentration (Creppy et al., 1979a). Similarly, in mice, the lethality of a single dose of 0.8 mg of ochratoxin A injected intraperitoneally was completely prevented by simultaneous injection of 1 mg of phenylalanine (Creppy et al., 1980b).

In yeast, the effect on protein synthesis of the rR-OH-ochratoxin A epimer was similar to that of ochratoxin A, but ochratoxin alpha, which lacks the phenylalanine moiety, had no effect (Creppy et al., 1983a). Analogues of ochratoxin A in which phenylalanine has been replaced by other amino acids, such as tyrosine, inhibit the respective amino acid-specific tRNA synthetases similarly (Creppy et al., 1983b).

The binding affinity of phenylalanine-tRNAPhe synthetase for ochratoxin A is weaker than for phenylalanine and ranges from 1/300 in yeast (K_M = 1.3 mmol/L for ochratoxin A; 3.3 µmol/L for phenylalanine) (Creppy et al., 1983a) to 1/20 in rat liver (K_m = 0.28 mmol/L for ochratoxin A; 6 µmol/L for phenylalanine) (Röschenthaler et al., 1984). Despite these differences in binding affinity, the inhibition of phenylalanine-tRNAPhe by ochratoxin A is very effective, since the toxin is more readily concentrated by cells than phenylalanine. The concentration of ochratoxin A inside hepatoma cells was 200- to 300-fold that in the medium (Creppy et al., 1983a).

A dose-related inhibition of protein synthesis was found in mice given ochratoxin A intraperitoneally at a dose > 1 mg/kg bw. The degree of inhibition of protein synthesis 5 h after administration of ochratoxin A at 1 mg/kg bw was 26% in liver, 68% in kidney, and 75% in spleen as compared with controls (Creppy et al., 1984).

Ochratoxin A may also act on other enzymes that use phenylalanine as a substrate, although no direct effect on the activity of other isolated enzyme systems has been demonstrated (Röschenthaler et al., 1984). In kidney slices from rats 2 days after they had been fed ochratoxin A at 2 mg/kg bw, the activity of renal phosphoenolpyruvate carboxykinase, a key enzyme in the gluconeogenic pathway, was lowered by 50% (Meisner & Krogh, 1986). The inhibition was due indirectly to specific degradation of the mRNA coding for this enzyme. A similar effect was not seen in rat liver (Meisner et al., 1983).

The effect of ochratoxin A on phenylalanine metabolism was studied in isolated hepatocytes and in liver homogenates from male rats treated in vivo. Both the hydroxylation of phenylalanine to tyrosine and the subsequent metabolism of tyrosine, as measured by homogenate oxidation, were inhibited when ochratoxin A at a concentration of 0.12–1.4 mmol/L was incubated with isolated hepatocytes (Creppy et al., 1990).

Ochratoxin A enhanced NADPH- or ascorbate-dependent lipid peroxidation in rat liver microsomes and NADPH-dependent lipid peroxidation in kidney microsomes in vitro, as measured by malondialdehyde formation or oxygen uptake. It was suggested that ochratoxin A stimulates lipid peroxidation by complexing Fe³⁺ and facilitating its reduction. Subsequent to oxygen binding, an iron–oxygen complex initiates lipid peroxidation. Cytochrome P450, free active oxygen species, and free hydroxy radicals do not appear to be involved in Fe³⁺–ochratoxin A- stimulated lipid peroxidation. Oral administration of ochratoxin A at 6 mg/kg bw to rats appeared to increase lipid peroxidation in vivo, causing a sevenfold increase in ethane exhalation (Rahimtula et al., 1988; Omar et al., 1990).

In pig renal cortical tissue, ochratoxin A and citrinin added singly or in combination at a concentration of 10^–6 or 10^–3 mol/L did not elicit consistent or strong synergistic effects, as measured by transport of tetraethylammonium and paraaminohippurate ions, or protein synthesis measured with [³H]leucine (Braunberg et al., 1994).

The effects of superoxide dismutase and catalase on ochratoxin A-induced nephrotoxicity were studied. Superoxide removes oxygen by converting it to hydrogen peroxide; this enzyme works in conjunction with catalase, which removes hydrogen peroxide within cells. Rats were given 20 mg/kg bw of each enzyme by subcutaneous injection every 48 h, 1 h before gavage with ochratoxin A at 290 µg/kg bw every 48 h, for 3 weeks. Superoxide dismutase and catalase prevented most of the nephrotoxic effects induced by ochratoxin A, observed as enzymuria, proteinuria, and creatinaemia, and increased the urinary excretion of ochratoxin A. The results indicated that superoxide radicals and hydrogen peroxide are likely to be involved in the nephrotoxic effects of ochratoxin A in vivo (Baudrimont et al., 1994).

After short-term administration of ochratoxin A to rats, the renal proximal tubule did not appear to be the main target for nephrotoxicity, although decreased capacity to eliminate the toxin may result in a self-enhancing effect (Gekle & Silbernagl, 1994). The main renal effect of ochratoxin A in rats was found in the ‘postproximal’ nephron, as measured by a reduced glomerular filtration rate, increased fractional water, Na⁺, K⁺, and Cl^– excretion, and increased dependence of osmol clearance on urine flow. In addition, ochratoxin A blocked membrane anion conductance in canine kidney cells in vitro (Gekle et al., 1993).

2.2.1 Acute toxicity

The LD₅₀ values found in various species treated by various routes are shown in Table 1. Dogs and pigs were the most sensitive species and rats and mice the least sensitive. Simultaneous oral administration of phenylalanine at 100 mg/kg bw to mice increased the oral LD₅₀ from 46 mg/kg bw to 71 mg/kg bw (Moroi et al., 1985). As is the case with many xenobiotics, neonatal rats were considerably more susceptible than adults.

Histopathological and electron microscopic studies were conducted with groups of 10 male Long-Evans and Sprague-Dawley rats given benzene-free ochratoxin A at a single dose of 0, 17, or 22 mg/kg bw in 0.1 mol/L sodium bicarbonate by gavage and examined for up to 48 h afterwards. The earliest changes were multifocal haemorrhages in many organs and fibrin thrombi in the spleen, the choroid plexus of the brain, liver, kidney and heart, suggesting disseminated intravascular coagulation. The effect was postulated by the authors to be due to activation of extrinsic and intrinsic systems of coagulation. Other changes were hepatic and lymphoid necrosis, enteritis with villous atrophy, affecting the jejunum most severely, and nephrosis. The myocardial changes were considered to be related to shock and subsequent ischaemic injuries (Albassam et al., 1987).

2.2.2 Short-term studies of toxicity

Ochratoxin A had nephrotoxic effects in all monogastric mammalian species tested so far (Kuiper-Goodman & Scott, 1989). The results of short-term studies with this toxin are shown in Table 2.

Groups of 10 male weanling Wistar rats were fed semi-purified diets containing ochratoxin A at a concentration of 0, 2.4, 4.8 9.6, or 24 mg/kg, equivalent to 0, 0.24, 0.48, 0.96, and 2.4 mg/kg bw per day, for 14 days. At the two higher doses, growth retardation, reduced food consumption, and increased serum urea nitrogen were seen. At the highest dose, the relative kidney weight was increased. Renal lesions, involving degenerative changes in the entire tubular system, and a decrease in urine volume were seen at all doses. Increased eosinophilia and karyomegaly in cells of the proximal convoluted tubules were noted at all doses (Munro et al., 1974).

Semi-purified diets containing ochratoxin A at 0, 0.2, 1, or 5 mg/kg, equivalent to 0, 0.015, 0.075, or 0.37 mg/kg bw per day, were fed to groups of 15 weanling Wistar rats of each sex for 90 days. At that time, eight animals from each group were killed, and the remaining rats were fed control diet for an additional 90 days. No changes in blood urea nitrogen or urinary or haematological parameters were seen at any dose. After 90 days at the two higher dietary concentrations, the relative kidney weights were reduced in animals of each sex; these had returned to control values after the 90-day recovery period, except in males at the highest dose. Dose-related changes in morphological appearance were seen after 90 days of treatment at doses > 0.2 mg/kg of diet and involved karyomegaly and increased eosinophilia in cells of the proximal convoluted tubules. The authors considered the latter change to be a phenomenon of ageing which had been accelerated by administration of ochratoxin A. Desquamation of proximal tubular cells, autolysis, changes in the rough and smooth endoplasmic reticulum, and tubular basement membrane thickening up to 4 µm were noted at the highest dose. In animals at the highest dose that were subsequently given control diet for 90 days, the karyomegaly and tubular basement membrane thickening persisted, but otherwise the kidneys appeared normal (Munro et al., 1974).

Similar effects were seen when ochratoxin A was administered to groups of four to six adult Sprague-Dawley and Wistar rats by intraperitoneal injection for 5–7 days at a dose of 0, 0.75, or 2 mg/kg bw per day. Decreased body weight, increased urine flow, increased urinary protein, increased urinary glucose, and impaired urinary transport of organic substances were seen at all doses. Sprague-Dawley rats were more sensitive than Wistar rats, and males were more sensitive than females. It was suggested that the increased urinary protein indicated interference with protein reabsorption by cells of the convoluted tubules (Berndt & Hayes, 1979).

Ochratoxin A was administered by gavage in maize oil to groups of five weanling male and female Fischer 344/N rats at a dose of 0, 1, 4, or 16 mg/kg bw per day on 5 days per week for a total of 12 doses over 16 days. All rats that received the highest dose had diarrhoea and nasal discharge and died before the end of the study. Increased relative weights of kidneys, heart, and brain, thymus atrophy, forestomach necrosis and/or hyperplasia, and haemorrhage of adrenal glands were seen at the two higher doses. Bone-marrow hypoplasia and nephropathy were seen at all doses, involving renal tubular degenerative and regenerative changes (National Toxicology Program, 1989).

Ochratoxin A was administered by gavage in maize oil to groups of 10 male and female weanling Fischer 344/N rats at a dose of 0, 0.06, 0.12, 0.25, 0.5, or 1 mg/kg bw per day for 5 days/week for 91 days. Growth retardation and a reduced relative kidney weight were seen in males at the two higher doses. The NOEL for renal tubular necrosis was 0.062 mg/kg bw, but karyomegaly of dose-related severity was observed in the proximal tubules at all doses. Milder renal changes consisting of tubular atrophy were seen at lower doses (National Toxicology Program, 1989).

Groups of 15 weanling rats were given ochratoxin A in 0.1 mol/L sodium bicarbonate at a dose of 0 or 100 µg/rat (equivalent to 1.25 mg/kg bw per day) by gavage for 8 weeks. Blood samples from fasted treated rats contained about twice the amount of glucose as those of controls. In a glucose tolerance test, the insulin concentration did not reach that in control rats. Total carbohydrate and glycogen concentrations in liver of treated rats were reduced, as seen earlier (Suzuki et al., 1975; T. Kuiper-Goodman, personal observation).

Groups of three to six young beagle dogs were given ochratoxin A by capsule at a dose of 0, 0.1, or 0.2 mg/kg bw per day for 14 days. No changes were observed in renal function, but tubular necrosis and ultrastructural changes in the proximal tubules were observed at all doses. Necrosis of lymphoid tissues of the thymus and tonsils was also seen at all doses (Kitchen et al., 1977a,b,c).

In a series of experiments, groups of three to six sows were given feed containing ochratoxin A at a concentration of 0, 0.2, 1, or 5 mg/kg, equivalent to 0, 0.008, 0.04, and 0.2 mg/kg bw per day, for periods of 5 days, 8 or 12 weeks, or up to 2 years. Decreased renal function, nephropathy, and reduced renal enzyme activity were reported. Progressive nephropathy but no renal failure was seen in female pigs given feed containing 1 mg/kg for 2 years; no results were reported for male pigs (Krogh & Elling, 1977; Elling, 1979a,b, 1983; Elling et al., 1985; Krogh et al., 1988).

In groups of 10 broiler chicken given ochratoxin A at a dietary concentration of 4 mg/kg for 2 months, the mortality rate was 42%. When the feed was supplemented with 0.8 or 2.4% L-phenylalanine, the mortality rate decreased to 12 and 15%, respectively (Gibson et al., 1990).

2.2.3 Long-term studies of toxicity and carcinogenicity

Diets containing ochratoxin A at 0 or 40 mg/kg, equivalent to 5.6 mg/kg bw per day, were fed to groups of adult male 10 ddY mice for 44 weeks, followed by 5 weeks of basal diet. Of the nine surviving treated mice, five had hepatic-cell tumours, nine had renal cystic adenomas, and two had solid renal-cell tumours (terms used by the authors). No hepatic or renal tumours were observed in control mice, and no data on the incidence of these tumours in other control groups of this strain of mice were presented. It was not clear indicated whether the liver tumours were benign or malignant (Kanisawa & Suzuki, 1978).

In a second study from the same laboratory, diets containing ochratoxin A at 0 or 25 mg/kg, equivalent to 3.5 mg/kg bw per day, were fed to groups of 20 6-week-old male DDD mice for 70 weeks. All 20 surviving treated mice had renal cystic adenomas, six had solid renal tumours, and eight had hepatic-cell tumours. One of the 17 control mice had a hepatic-cell tumour (Kanisawa, 1984).

In a third study from the same laboratory, the mice were not exposed for life but for 70 weeks. Diets containing ochratoxin A at 0 or 50 mg/kg, equivalent to 7 mg/kg bw per day, were fed to groups of 16 adult male ddY mice for 5–30 weeks, followed by control diet for 40–65 weeks. No renal or liver tumours were observed in control mice or in mice fed ochratoxin A for ­ 10 weeks. The incidences of renal-cell tumours were 3/15, 1/14, 2/15, and 4/17 after 15, 20, 25, and 30 weeks on the ochratoxin A-containing diet, respectively. The incidence of renal cystic adenomas was not indicated. A significant increase in the incidence of liver tumours was observed after mice had been fed ochratoxin A for 25 weeks (5/15) or 30 weeks (6/17). These results indicated that the renal and liver tumours persisted through subsequent feeding of control diet (Kanisawa, 1984).

In these studies, two types of renal tumour were distinguished by the authors: papillary cyst adenomas (benign) and solid renal-cell tumours, which contained atypical cells, displayed infiltrative growth, and were interpreted by the Committee as malignant. Preneoplastic renal lesions were frequent and multiple and consisted of distended tubules with atypical epithelial cells. No metastases attributable to the kidney or liver tumours were found.

Diets containing ochratoxin A at a concentration of 0, 1, or 40 mg/kg were fed to groups of 50 weanling B6C3F₁ mice of each sex for 24 months. The test compound contained about 84% ochratoxin A, 7% ochratoxin B, and 9% benzene. Dead and moribund mice were identified daily. The mice were examined and weighed, and their food consumption was recorded weekly for the first 4 weeks, then monthly. Animals at the high dose showed decreased body weights, by 25% for females and 33% for males, indicating that the maximum tolerated dose was exceeded, although no other signs of toxicity were observed. Nephropathy, characterized by cystic dilatation of renal tubules often with hyperplasia of the lining epithelium, was seen only in mice fed diets containing the highest concentration and was more severe in males than in females. No nephropathy was found in males or females given a control diet or the lower concentration of ochratoxin A. Benign and malignant renal tumours were seen only in male mice fed diets containing the high concentration, at incidences of 53% and 29%, respectively (combined incidence, 63%). No metastases from the renal tumours were found.

When the combined incidence of hepatocellular adenomas and carcinomas in treated mice was compared with that in concurrent controls, the increase was statistically significant in both male and female mice given the high dose; however, the 20% incidence in males was within the range of past controls of 0–22% for this strain of mouse, but the 14% incidence in females was greater than the incidence of 0–3.9% in previous controls (Ward et al., 1979). The authors noted that the ochratoxin A used in their study contained 9% benzene, a proven carcinogen, and thus the possibility of synergism must be considered. The presence of renal tumours in males did not decrease their survival rate. In fact, the survival rates of males at 18 months were 75% in the controls and 65% among those at 1 mg/kg of diet, compared with 98% for those at 40 mg/kg of diet, owing to a high incidence of fatal obstructive urinary-tract disease among the controls and low-dose mice, with onset as early as 4 months (Bendele et al., 1985a). It was suggested that the apparent protective effect of ochratoxin A at 40 mg/kg of diet was due to inhibition of the growth of gram-positive bacteria and to the induction of polyuria as a result of renal proximal tubule damage (Bendele & Carlton, 1986). Group caging and fighting-related lesions of the prepuce and penis may have contributed to the chronic uropathy (Rao, 1987).

Groups of 80 male and female Fischer 344/N rats were given ochratoxin A by gavage in maize oil at a concentration of 0, 21, 70, or 210 µg/kg bw per day, 5 days/week for 9 months, 15 months, or 103 weeks. The rats were observed twice daily, and body weights and food consumption were recorded weekly for the first 13 weeks and then monthly. Feed and water were available ad libitum. Groups of 15 rats of each sex were killed after 9 and 15 months. The body weight of rats at the highest dose was decreased by 4–7% between 18 and 77 weeks for male rats and between 6 and 89 weeks for female rats. No compound-related clinical signs were seen, and the results of haematological and serum chemical analyses showed no effects of biological significance. Urinary analysis indicated a mild to moderate change in the ability to concentrate urine, with no other change in renal function.

The incidences of renal adenomas in males were 1/50, 1/51, 6/51, and 10/50 and those of renal carcinomas were 0/50, 0/51, 16/51, and 30/50, in the four groups, respectively. The combined incidences of renal tubule-cell adenomas and carcinomas were 20/51 and 36/50 at the two higher doses. At the highest dose, many of the renal adenomas and carcinomas were multiple or bilateral. There was a dose-related increase in the number of males found dead or moribund before the terminal sacrifice (7, 19, 23, and 26, respectively, at 0, 21, 70, and 210 µg/kg bw per day). The decreased survival rates among rats at the two higher doses were attributed by the authors to the presence of kidney tumours, since 15/23 and 18/26 rats that died at these two doses had kidney tumours. In addition. a larger proportion of animals that died before the terminal sacrifice had carcinomas that had become metastatic (3/8 and 11/15 at the intermediate and high doses, respectively) than of animals killed at terminal sacrifice (0/7 and 3/15 at the intermediate and high doses, respectively). In male rats given the low dose of ochratoxin A, only one kidney tumour was present, although the decrease in survival was similar to that of rats at the two higher doses. The reduced survival of this group must therefore be attributed to a non-neoplastic treatment-related effect. In females, the combined incidences of renal adenomas and carcinomas were 0/50, 0/51, 2/50, and 8/50 at 0, 21, 70, and 210 µg/kg bw per day, respectively. The significance of the ochratoxin A-induced renal carcinomas in rats is increased by the high frequency of metastases, attributed to renal-cell carcinomas, mainly in the lungs and lymph nodes. Females at the high dose also had a greater multiplicity of fibroadenomas in the mammary gland (14/50) than controls and rats at lower doses (4–5/50).

The non-neoplastic lesions involved mainly the kidney. Chronic diffuse nephropathy, common to old rats, was seen at about the same incidence in all groups, but the extent and grade were not reported. Karyomegaly or karyocytomegaly (large kidney epithelial cells with giant polyploid nuclei and prominent nucleoli) was seen in all males and females at the two higher doses, and it was the most consistent finding in these groups at the the 9- and 15-month interim sacrifices as well as in a preliminary 13-week study (National Toxicology Program, 1989).

In reviewing these data at its forty-fourth meeting, the Committee noted that renal carcinomas were found in 16/51 male rats at 70 µg/kg bw per day and in 30/50 at 210 µg/kg bw per day; no carcinomas were found at the lower doses. In female rats, renal carcinomas were less common, with 0/50, 1/50, and 3/50 animals showing carcinomas at the low, intermediate, and high doses, respectively. Renal adenomas were found in all groups of male rats, increasing in frequency with increasing dose. In the female rats, renal adenomas were found only at the two higher doses. Fibroadenomas in the mammary gland were found in 45–56% of treated females, a significantly higher percentage than in the control group (Annex 1, reference 117).

The slides of the kidneys from the National Toxicology Program study were reviewed subsequently (Hard, 2000). The review confirmed that the site of injury was the straight segment of proximal tubule S3 in the outer stripe of the outer medulla. In the 2-year bioassay, the lesion consisted of contraction and disorganization of the normal linear pattern of the S3 tubules due to marked development of karyomegaly and cytomegaly. This change showed a clear dose–response relationship in both males and females. The 16-day and 13-week studies showed that this response was preceded by focal tubule basophilia involving mainly the outer stripe of the outer medulla, associated with single-cell death, increased mitotic activity, and some simple tubule hyperplasia. Other non-neoplastic lesions involving only the outer stripe of the outer medulla in the 2-year bioassay were dilated atypical tubules, chromophobic tubules, and cystic tubules, the latter being more prominent in females than in males. The review also confirmed that low (microgram) concentrations of ochratoxin A induced a high incidence of renal tubule tumours (74% in males at the high dose), with carcinomas predominating over adenomas. The carcinomas had a relatively rapid onset, progressing with malignant and aggressive behaviour, some tumours showing a tendency towards an uncommon anaplastic phenotype. There was a relatively high incidence of metastasis, and some tumours were undoubtedly the cause of death. These various features of the ochratoxin A-induced tumours distinguish them from the kidney tumours induced by model non-genotoxic renal carcinogens such as d-limonene and chloroform. However, the tendency towards anaplasia and their aggressive nature were reminiscent of renal tubule tumours induced by fumonisin B₁. Renal tumour development was clearly related to the site of ochratoxin A-induced tubule damage, in that preneoplastic atypical tubule hyperplasia, adenomas, and very early carcinomas developed within the outer stripe of the outer medulla. However, a mode of action of sustained cytotoxicity and compensatory cell regeneration coupled with simple tubule hyperplasia, although a possibility, could not be established within the limits of conventional histology alone. Nevertheless, the very high incidence of renal neoplasms, their relatively rapid onset and highly malignant behaviour, coupled with a tendency towards an aggressive anaplastic phenotype and their contribution to death all favour a conclusion that ochratoxin A-induced renal tumour development occurs via DNA reactivity.

The Committee noted that the long-term effects were preceded by evidence of renal toxicity in the 16-day and 13-week studies. It is unclear whether the malignancy and aggressive nature of the tumours is a secure indication that the mechanism of induction is via DNA reactivity. The analogy with tumours induced by fumonisin B₁ is not evidence of a genotoxic mechanism, since it has been postulated that the mechanism by which fumonisins induce tumours may be indirect, involving altered sphingolipid metabolism.

2.2.4 Genotoxicity

The results of studies of genotoxicity with ochratoxin A are summarized in Table 3.

Almost all the available studies in which DNA adducts were detected by ³²P-postlabelling after exposure to ochratoxin A were from one laboratory (Pfohl-Leszkowicz et al., 1991, 1993; Grosse et al., 1995, 1997; Castegnaro et al., 1998; Pfohl-Leszkowicz et al., 1998). All showed positive results in rats and mice given 0.4–2.5 mg/kg bw for 1–16 days or even up to 2 years. The number of adducts ranged from 1 to 200/10⁹ nucleotides in kidney DNA. However, the nonspecific postlabelling technique used may have resulted in adducts that did not contain an ochrotoxin A or ochratoxin A metabolite moiety. At least some of the adducts might have been due to ochratoxin A-induced cytotoxic effects that generate reactive oxygen species. Thus, Grosse et al. (1997) found that prior treatment of rats with superoxide dismutase or catalase, ascorbic acid, or alpha-tocopherol significantly decreased the number of adducts.

Indications that oxidative damage to DNA is not the only source of the presumed adducts are provided by the results of experiments in vitro with purified DNA and mononucleotides incubated with kidney or liver microsomes from mouse and rabbit, ochratoxin A, and either NADPH or arachidonic acid as cofactors (Obrecht-Pflumio & Dirheimer, 2000). Presumed adducts were obtained in all cases but particularly with mouse and rabbit kidney microsomes and arachidonic acid as the cofactor. Liver microsomes were much less active. With NADPH as the cofactor with mouse kidney microsomal enzymes, the adduct level was only 44% that obtained with arachidonic acid. When dAMP, dGMP, dTMP, and dCMP were used as substrates, three adducts were formed with dGMP, mouse kidney microsomes, and either cofactor. However, only one of these adducts was common to the two cofactors. Inhibition of lipid peroxidation and the generation of hydroxyl radicals with desferrioxamine B methanesulfonate did not change the adduct profile. The major adduct obtained with dGMP co-chromatographed with the major adduct obtained with purified DNA. No adducts were obtained with the other three mononucleotides.

In contrast to these results with ³²P-postlabelling methods, Schlatter et al. (1996) and Rasonyi (1995) reported that the level of covalent binding of [³H]ochratoxin A to DNA was below the limit of detection (LOD) of scintillation counting in kidney and liver (< 1.3/10¹⁰ and 5.6/10¹¹ DNA bases, respectively). In addition, Gautier et al. (2001), using scintillation counting, did not find covalent binding of [³H]ochratoxin A to the DNA of the kidneys of male Fischer 344 rats dosed by gavage 24 h earlier. The LOD was 2.7 adducts/10⁹ purified DNA bases. The authors also used a ³²P-postlabelling method with these rat kidney DNA samples and found adducts at levels ranging from 31 to 71/10⁹ DNA bases 24 h after dosing, compared with 6–24/10⁹ DNA bases in untreated controls. Since the adducts occurred at a level 3–17 times higher than the detection limit for scintillation counting and there was no evidence of tritium exchange, most, if not all, of the adducts observed by the ³²P-postlabelling method would not have contained an ochratoxin A moiety.

Furthermore, no adducts were found (detection limit, 20 adducts/10⁹ DNA bases) by scintillation counting when DNA and [³H]ochratoxin A were incubated in the presence of male rat kidney microsomes with NADPH, mouse kidney microsomes with NADPH, rat seminal vesicle microsomes with arachidonic acid, or horseradish peroxidase with hydrogen peroxide.

There was no evidence of DNA repair as a result of possible DNA damage in bacteria, whereas DNA single-strand breaks were consistently induced in cultured mammalian cells. DNA single-strand breaks were also observed in vivo in spleen, liver, and kidney cells of mice after intraperitoneal injection of ochratoxin A. DNA repair, manifested as unscheduled DNA synthesis, was observed in most studies with primary cultures of rat and mouse hepatocytes, porcine epithelial cells from bladder, and human urothelial cells.

Most tests for gene mutation induction in bacteria showed no effect of exposure to ochratoxin A. Two studies showed positive results. One was in S. typhimurium strains TA1535 and TA1538 treated in the presence of mouse kidney microsomes (Obrecht-Pflumio et al., 1999), while the other was in S. typhimurium strains TA1535, TA1538, and TA100 treated with the culture medium of rat hepatocytes exposed to ochratoxin A (Hennig et al., 1991). Both papers described preliminary results that required further investigation before they could be readily accepted. It should be noted, however, that Hennig et al. (1991) obtained negative results with the same bacterial strains when rat liver microsomes were used as the exogenous metabolic activation system. This portion of the results has been confirmed in independent studies in other laboratories.

Gene mutations were not induced in the yeast Saccharomyces cerevisiae D3 (Kuczuk et al., 1978). In mammalian cells, gene mutations were not induced in two studies, while positive results were observed in a third. The last study was performed with NIH 3T3 cells transfected with a human CYP gene (De Groene et al., 1996) at a concentration of 25 µg/ml. In the studies with negative results, concentrations of 10 µg/ml (C3H mouse mammary cells) and > 12 µg/ml (mouse lymphoma L5178Y cells) were cytotoxic. The positive result therefore requires confirmation. No studies of mutation in vivo have been reported.

Sister chromatid exchange was induced in two of four studies in vitro but not in a single study in vivo after gavage of a range of doses that included cytotoxic doses.

Chromosomal aberrations were not induced in Chinese hamster ovary cells (National Toxicology Program, 1989) but were induced in cultured human lymphocytes (Manolova et al., 1990), and micronuclei were induced in ovine seminal vesicle cells and Syrian hamster embryo fibroblasts. In vivo, chromosomal aberrations were induced in mouse cells, an effect that was reduced by treatment of the mice with either ascorbic acid (by gavage) or vitamin A (in the diet). These protective effects are consistent with the observation that the formation of ³²P-postlabelling spots was prevented in some studies in which mice were treated with ochratoxin A (Grosse et al., 1997).

2.2.5 Reproductive toxicity

No adequate studies on the reproductive toxicity of ochratoxin A were available for review. Several studies of effects on developmental toxicity are summarized.

Groups of 4–26 pregnant CBA mice were given a single dose of ochratoxin A in maize oil by gavage at 0, 1, 2, or 4 mg/kg bw on day 8 or 9 of gestation (day of vaginal plug considered to be day 1 after conception) or 4 mg/kg bw per day 2 days before mating and on days 2, 4, 6, 7, 10, and 14 of gestation, and observed until day 19. At this time, the numbers of viable and dead fetuses and the number of resorption sites were determined, and fetuses were weighed and examined for morphological changes. No mention was made of maternal toxicity. Prenatal survival was decreased in groups that had received 4 mg/kg bw on days 7 (24% deaths), 8 (17% deaths), and 9 (22% deaths) of gestation. Overt craniofacial anomalies were seen only after treatment on day 8 or 9; their incidence, multiplicity, and severity increased with increasing dose, the peak effect being seen on day 9. The incidences of malformed pups among surviving pups were 0%, 0%, 8.1%, and 16% of mice given ochratoxin A at 0, 1, 2, or 4 mg/kg bw on day 8 of gestation, and 0%, 29%, 42%, and 91% of mice given the same doses on day 9 of gestation. The mean number of malformations per fetus was 0.3 and 2.3 on days 8 and 9 at 4 mg/kg bw, and 1.7 and 3.9 in animals given 8 mg/kg bw in a separate study. The central nervous system, the eye, and the axial skeleton were the main systems affected. The most important malformations were those affecting the craniofacial structures, including aplasia and dysplasia of the upper facial structures, such as exencephaly, microcephaly, blunt jaws, anophthalmia, microphthalmia, and median cleft face. In animals treated on day 9 of gestation at 4 mg/kg bw, the incidences of the various major anomalies were exencephaly, 89%; anophthalmia, 45%; microphthalmia, 27%; open eyelids, 16%; agenesis of external nares, 21%; cleft lip, 7.1%; median cleft face, 8.9%; and malformed jaws or short maxilla with protruding tongue, 41%. The craniofacial anomalies were considered by the authors to have arisen from failure of closure of the neurocranium, resulting in abnormal configuration, position, and size of the bones of the base and lateral walls of the skull (Arora & Frölen, 1981).

The effects of protein deprivation on the teratogenic effects of ochratoxin A were studied in groups of 10–13 CD-1 mice, maintained on diets providing 26% (control), 16%, 8%, and 4% purified protein (casein), after mating and throughout gestation. A single dose of ochratoxin A in 0.1 N sodium bicarbonate was administered by gavage at a dose of 0, 2, or 3 mg/kg bw on day 8 of gestation (vaginal plug considered to be day 1), and the mice were killed on day 18 of gestation for examination. The dams were monitored twice daily, and food consumption was monitored. Diets and water were available ad libitum.

Ochratoxin A treatment did not affect maternal food consumption, but maternal deaths were significantly more frequent in the group receiving ochratoxin A at 3 mg/kg bw and 26% protein (five deaths), in that given the same dose and 4% protein (four deaths), and in that given 2 mg/kg bw and 4% protein (nine deaths), with no deaths in the untreated groups. The percentages of litters with grossly malformed fetuses and the percentages of malformed fetuses (in brackets) for each of the four diets (26, 16, 8, and 4% protein, respectively) were 58 (25), 50 (17), 75 (45), and 100 (81) at 3 mg/kg bw; 25 (5), 50 (21), 30 (13), and 100 (78) at 2 mg/kg bw; and 0 (0), 0 (0), 18 (3), and 31 (9.8) without ochratoxin A. The fetal weights were reduced as a result of treatment and protein deprivation. Cranofacial malformations were the commonest abnormality, but at lower protein concentrations gross malformations affecting the limbs and tail were also seen (Singh & Hood, 1985).

In microcephalic mice derived from females treated intraperitoneally with ochratoxin A at 3 mg/kg bw on day 10 of gestation, a quantitative assessment of neurons and synapses at 6 weeks of age showed that the somatosensory cortices of treated mice had fewer synapses per neuron than those of controls, indicating reduced dendritic growth (Fukui et al., 1992).

Five groups of 12–20 pregnant Wistar rats were given ochratoxin A at a total dose of 5 mg/kg bw in 0.16 mol/L sodium bicarbonate by gavage, as follows: a single dose of 2.5 mg/kg bw on each of days 8 and 9 of gestation (vaginal plug considered to be day 1), a dose of 1.2 mg/kg bw on each of days 8–11 of gestation, a dose of 0.83 mg/kg bw on each of days 8–13 of gestation, or a dose of 0.63 mg/kg bw on each of days 8–15 of gestation. A control group was given the vehicle only. In a similar way, three groups of 20 rats were given ochratoxin A at a single dose of 2.5 mg/kg bw on each of days 8 and 9 of gestation or a dose of 1.7 mg/kg bw on each of days 8–10 of gestation. The rats were killed on day 20 of gestation. No significant difference was seen in the number of implantations per female in the various groups. Females that had received the same total amount of ochratoxin A but divided into fewer single doses and early in gestation were most affected. There was a dose-related increase in the number of resorptions per female and decreases in the mean number of fetuses per female, mean fetal weight, and mean placental weight. A high dose-related incidence of fetal haemorrhages (seen at 2, 2.5, and 4 times the 1.2 mg/kg dose) and coelosome with or without oedema were considered to be teratogenic responses (Moré & Galtier, 1974).

In a study from the same laboratory, a similar protocol for administration of ochratoxin A was used, but the rats were observed until 82 days after birth. Dose-related decreases in the mean number of newborn rats, the mean number of rats alive at 4 days, and the viability index were seen, but not in the lactation index. In the group given 2.5 mg/kg bw twice, the mean body weights of male and female offspring at 82 days were reduced by 12 and 8%, respectively. Hydrocephalus was observed on day 15 after birth in 26% of the male offspring at that dose, and 40% of these animals died by 20 days after birth. A second generation was bred to examine residual maternal or paternal effects, without further administration of ochratoxin A. No differences in reproductive parameters were noted, and few details were given (Moré & Galtier, 1975).

A dose of 0.5 mg/kg bw given by gavage to rats on days 11–14 of gestation caused learning deficits in pups tested for 26 weeks (Kihara et al., 1984).

Oral administration of ochratoxin A to pregnant rats at 1 mg/kg bw per day on days 6–15 of gestation resulted in decreased fetal weight and increased numbers of resorptions but no overt adverse effects on the dams. Skeletal and/or lung malformations were reported in up to 20% of the fetuses; the incidence of renal malformations was 40%. Concurrent administration of methionine at 43 mg/kg bw protected against these adverse effects (Abdel-Wahhab et al., 1999).

Other studies on the teratogenicity of ochratoxin A in mice and rats treated intraperitoneally or subcutaneously were reviewed by Kuiper-Goodman & Scott (1989).

The embryotoxic potential of ochratoxin A was tested in chicks by injecting hens’ eggs on day 3 and incubating them until day 13 or 18, when visible abnormalities, weight, and length of chicks were recorded. A dose-related increase in the mortality rate was seen after injection of 1–2 µg of ochratoxin A. An increased frequency of abnormalities was seen in one of the two reported experiments (Edrington et al., 1995). The Committee noted that this is not a validated method, and the results could not be used in risk assessment.

Prechondrogenic mesenchymal cells from the limb buds of 4-day-old chick embryos were cultured with ochratoxin A for 6 days. Ochratoxin A inhibited the accumulation of cartilage proteoglycans and general protein synthesis in a dose-related manner (Wiger & Stormer, 1990).

Rat embryos explanted on day 10 of gestation were cultured in a medium containing ochratoxin A at concentrations up to 300 µg/ml. Dose-dependent reductions in the protein and DNA content of the embryos were seen. The malformations induced included hypoplasia of the telencephalon, stunted limb bud development, and decreased size of mandibular and maxillary bones. Cellular necrosis of mesodermal and neuroectodermal structures was observed (Mayura et al., 1989).

2.2.6 Special studies

(a) Covalent binding to nucleic acids and/or proteins

Preliminary observations indicated no specific binding of ochratoxin A to macromolecules in porcine kidney cytosol (Stojkovic et al., 1984).

Subcellular fractions of a number of kidney-derived cell lines and rat intestine, liver, spleen, kidney, and plasma were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then incubated with ochratoxin A coupled with horseradish peroxidase in order to locate the ochratoxin A-binding proteins. The toxin was shown to bind to virtually all rat blood serum proteins and to some proteins in rat intestine, liver, spleen, and kidney, particularly at 60, 40, and 27 kDa. Binding of ochratoxin A to the 60- and 27-kDa proteins, but not the 40-kDa protein, was inhibited by phenylalanine and aspartame in liver but not in the other organs. The binding of ochratoxin A to cytosolic or organelle proteins was comparable in all the kidney cell lines, which were derived from various species and from various regions of the kidney. Phenylalanine and aspartame had no effect on the binding. The authors concluded that ochratoxin A can bind to several cellular proteins, and that this accounts for its accumulation in cells, but that the results do not explain the protective effects of phenylalanine and aspartame described previously (Schwerdt et al., 1999a).

(b) Immunotoxicity

The size of the mouse thymus was reduced to 33% that of controls after four intraperitoneal injections of ochratoxin A at 20 mg/kg bw on alternate days, a dose which caused minimal nephrotoxicity. Bone marrow depression was shown as dose-related, significantly (p < 0.01) decreased marrow cellularity, including a reduction in bone marrow macrophage–granulocyte progenitors, a decreased number of haematopoietic stem cells and a significant decrease in erythropoiesis as measured by ⁵⁹Fe uptake; increased phagocytosis by macrophages was also observed (Boorman et al., 1984).

Residual damage was seen 3 weeks after exposure as increased sensitivity to radiation, even though bone-marrow cellularity and the peripheral blood count had returned to normal (Hong et al., 1988; National Toxicology Program, 1989).

Ochratoxin A administered to 8–10-week-old Swiss mice at 5 mg/kg bw per day by intraperitoneal injection for 50 days reduced the antibody response to Brucella abortis, a cell-mediated immune response. This was postulated to be due to suppression of immunoglobulin (Ig)M synthesis. The same treatment also reduced mitogen (concanavalin A)-induced blast formation in lymphocytes derived from mouse spleen (Prior & Sisodia, 1982).

Groups of eight female BALB/c mice were fed diets containing ochratoxin A at a concentration of 6, 250, or 2600 µg/kg for 28 or 90 days, equivalent to 1, 40, and 400 µg/kg bw per day. Treatment did not cause changes in body or lymphoid organ weights. Kidney weights were reduced at the two higher doses at 28 days and at the highest dose at 90 days. The concentrations of ochratoxin A in kidney were clearly dose-related. No differences in leukocyte count were observed, but a significant reduction in the number of spleen cells (by about 20%) was observed at the highest dose after 90 days. No changes were observed in blood or thymic T lymphocytes at 28 days, but a decreased proportion of mature CD4⁺ and CD8⁺ cells was seen with a corresponding increase in the immature double-positive sub-population at the two higher doses after 90 days. After 28 days, the primary (humoral) antibody response to sheep red blood cells was significantly suppressed in a dose-dependent manner at the two higher doses. The antibody response to another T-cell-dependent antigen (viral antigen PR8) was not affected, suggesting that exposure to ochratoxin A alters certain immune functions in mice, and, as previously demonstrated, the spleen may be the most sensitive immune tissue to ochratoxin A. Differences in the proportions of mature and immature CD4⁺ and CD8⁺ populations suggest that ochratoxin A may affect late-stage differentiation of T cells (Thuvander et al., 1995).

Female BALB/c mice were given diets containing ochratoxin A to provide a calculated average dietary intake of 5–30 µg/kg bw per day for 2 weeks before mating. At birth, the pups were cross-fostered to unexposed dams. Exposed and control pups were killed at 14 and 28 days of age. Ochratoxin A did not effect the reproductive outcome or body weight of pups. No differences in spleen or thymus weight or cell numbers were observed on day 14, but significant increases were seen in both thymus weights (by 20%) and cell number (by 67%) in the offspring of dams at the high dose on day 28. Although the percentages of splenic CD4⁺ and CD8⁺ cells were decreased in pups at the high dose, there were no alterations in absolute numbers. No significant differences were observed in the proliferative responses of splenic or thymic lymphocytes to mitogens nor in the production of interleukin-2 in concanavalin A-stimulated cell cultures. No significant differences in the humoral antibody response to sheep red blood cells or viral antigen PR8 were found. Natural killer cell activity on day 28 was not affected by prenatal exposure to ochratoxin A. Thus, the treatment did not suppress immune function but altered the absolute and relative numbers of lymphocyte subpopulations in lymphoid organs (Thuvander et al., 1996a).

Groups of 10 Han-NMRI mice (sex not specified) received commercial (Serva) or ‘raw’ ochratoxin A at a dose of 1, 3, or 6 mg/kg bw per day by intraperitoneal injection for 8–17 days and were then monitored for up to 20 days. Animals receiving ‘raw’ ochratoxin A at 3 mg/kg bw per day had a significantly lower body weight than controls on days 5–17; however, this correlated with a reduction in feed consumption. No significant change in body weight was noted in the groups receiving crystalline ochratoxin A. The total leukocyte count was unchanged in all groups; however, lymphopenia, neutrophilia, and eosinophilia were observed at 3 and 6 mg/kg bw per day. The blood IgM titre was suppressed at these doses in a dose-dependent manner. The authors concluded that ochratoxin A has a nonselective suppressive effect on various immune reactions, but the paper contains inadequate detail to verify their conclusion (Müller et al., 1995).

Bone-marrow hypocellularity and a reduced thymic size were also seen in Fischer rats given ochratoxin A at 1 or 4 mg/kg bw per day by gavage for 16 days (National Toxicology Program, 1989).

Necrosis of germinal centres in the spleen and lymph nodes was seen in Wistar rats given a single dose of ochratoxin A at 5–50 mg/kg bw (Kanisaw et al., 1977) and in dogs given ochratoxin A by capsule at doses of 0.1–0.2 mg/kg bw per day for 14 days (Kitchen et al., 1977c).

The effects of ochratoxin A on the bone marrow and lymphatic cell population may reflect the sensitivity of these cells to the inhibition of protein synthesis induced by ochratoxin A. These effects on the structural components of the immune system indicated that ochratoxin A is likely to have an effect on immune function.

The immunotoxic effects of perinatal exposure to ochratoxin A were investigated in the offspring of Sprague-Dawley rats treated singly or repeatedly. In a short-term study, dams received a single oral dose of 10, 50, or 250 µg/kg bw on day 11 of lactation, and the pups were examined on day 14. Dose-dependent uptake of ochratoxin A was observed in both dams and pups. The toxin did not induce consistent changes in the weights of the lymphoid organs of pups. A small but significant increase in the number of thymocytes was observed in offspring of dams dosed at 50 µg/kg bw, but it was not dose-dependent. A small but significant decrease in the proliferative response of splenocytes to T-cell mitogen lipopolysaccharide was seen in pups of dams given 250 µg/kg bw. In contrast, exposure to 10–50 µg/kg bw per day resulted in significant increases in the proliferative responses of both splenocytes and thymocytes of pups to concanavalin A. This was not seen at the higher dose. The authors proposed that short-term exposure of suckling pups via the milk stimulates the immune response, measured as proliferation of lymphocytes in response to concanavalin A and lipopolysaccharide (Thuvander et al., 1996b).

In a long-term study, dams received repeated oral doses of ochratoxin A at 50 µg/kg bw on 5 days/week for 2 weeks before mating, during gestation, and then 7 days/week until weaning. At parturition, the number of pups was reduced to eight per litter and they were cross-fostered to produce groups of prenatally, postnatally, and pre- and postnatally exposed pups. The highest blood concentrations of ochratoxin A were detected in pups exposed both pre- and postnatally, but exposure via the milk appeared to account for most of the content. Long-term exposure to 50 µg/kg bw per day did not induce any consistent changes in body or lymphoid organ weights of pups, but prenatal exposure suppressed the lymphocyte response to both B- and T-cell mitogens at 14 days of age. The background proliferation of unstimulated cells was significantly suppressed in cultures from prenatally exposed pups. These effects were not observed in pups exposed during lactation, although the blood concentrations were higher in pups exposed postnatally. Prenatally exposed pups showed a significantly lower primary antibody response to PR8 viral antigen (± 0.36). No significant difference in the natural killer cell activity of splenocytes was measured in exposed pups at 13 weeks of age. The authors concluded that long-term prenatal exposure to ochratoxin A, but not postnatal exposure via milk, may cause immunosuppression; however, short-term postnatal exposure may stimulate proliferation of lymphocytes in response to mitogens (Thuvander et al., 1996b).

The Committee noted that no details were given about the ochratoxin A used. These authors previously used commercial ochratoxin A, but in this paper they quoted a 1984 reference for details of how the ochratoxin A was produced. The size of the groups was not given, but they seem to have consisted of four to five dams.

Groups of six weanling hybrid pigs received either pure or crude ochratoxin A at doses of 7–50 µg/kg bw per day by subcutaneous injection for 19–39 days. The animals were immunized 8 days after ochratoxin A challenge with Pasteurella by inhalation. The authors stated that ochratoxin A had no effect on body-weight gain and that the serum concentrations were dose-dependent. The concentrations were reported to be lower after administration of crude ochratoxin A than pure material. A reduction in relative lymphocyte count and increases in total leukocyte, relative neutrophil, and eosinophil counts were seen. Crude toxin had a greater effect than pure toxin. Ochratoxin A decreased the phagocytosis index of individual cells and decreased expression of SWC1 (a lymphocyte cell surface marker) but did not change lymphocyte proliferation (Müller et al., 1999).

The Committee noted that many of the results were conflicting and the study was inadequately reported.

In chickens fed diets containing ochratoxin A at a concentration of 2–4 mg/kg for 20 days, the lymphoid cell population of immune organs was decreased (Dwivedi & Burns, 1984a).

Several studies have shown that ochratoxin A affects both humoral and cell-mediated immunity. In chickens fed a diet containing ochratoxin A at 5 mg/kg for 56 days, the contents of alpha1-, alpha2-, beta-, and gamma-globulins in plasma were reduced (Rupic et al., 1978).

In chickens fed diets containing ochratoxin A at a concentration of 2–4 mg/kg for 20 days, immunoglobulin (Ig)G, IgA, and IgM in lymphoid tissues and serum were depressed (Dwivedi & Burns, 1984b), and complement activity was slightly affected in birds fed at diets containing 2 mg/kg for 5–6 weeks (Campbell et al., 1983).

Ochratoxin A also reduced IgG and increased IgM in the bursa of Fabricius in chick embryos that had been injected with 2.5 µg of the toxin on day 13. This did not affect their immunocompetence, however, as seen after challenge of the hatched chickens with E. coli at 1, 2, and 4 weeks of age, indicating that the effect on immunoglobulins may have been transient (Harvey et al., 1987).

Immunosuppression was observed in chickens fed diets containing ochratoxin A at 0.5 or 2 mg/kg for 21 days. When compared with controls, the treated animals had reduced total serum protein, lymphocyte counts, and weights of the thymus, bursa of Fabricius, and spleen (Singh et al., 1990).

The effects of ochratoxin A on T-cell activation were investigated in purified (> 95%) human lymphocytes cultured in medium containing 1% bovine serum albumin. Intracellular free Ca²⁺ and activation of protein kinase C were measured as indicators of the early stages of activation; the effect on phytohaemagglutinin-induced proliferation was measured as a late event mediated by expression of functional interleukin-2 receptors. The early-stage events were not inhibited by ochratoxin A at a concentration of 12 µmol/L. In contrast, incubation of ochratoxin A with phytohaemagglutinin-stimulated lymphocytes resulted in inhibition of DNA synthesis at concentrations > 6.4 µmol/L. Protein synthesis in resting lymphocytes was markedly inhibited by 12 µmol/L but to a lesser extent in phytohaemagglutinin-stimulated lymphocytes. The authors concluded that ochratoxin A can block DNA synthesis at a late stage in lymphocyte activation and that this effect may be partially mediated by inhibition of protein synthesis (Størmer & Lea, 1995).

Ochratoxin A also inhibited the proliferative response of bovine peripheral blood mononuclear cells cultured in 10% fetal calf serum. The ID₅₀ value varied from 0.1 to 4 µg/ml, depending on the mitogen used to stimulate the cells and the incubation time. The authors considered these results indicative of immunosuppressive potential (Charoenpornsook et al., 1998).

(c) Neurotoxicity

Three male Wistar rats received 1 nmol (about 400 ng) of ochratoxin A by intracerebral administration and four received a diet containing 290 µg/kg by oral gavage for 8 days. The animals were killed 24 h after dosing. Although ochratoxin A was detected in areas of the central nervous system after intracerebral injection, it was not detected in the periphery or blood, kidney, or urine, indicating that it is transferred poorly or not at all from the spinal fluid to blood, kidney, or urine. After administration in the diet, the ventral mesencephalon, hippocampus, striatum, and cerebellum were the main targets of cytotoxicity in rat brain (Belmadani et al., 1998a)

Four male Wistar rats received ochratoxin A at 290 µg/kg bw orally every 48 h for 1–6 weeks. The treated animals had a slight reduction in body weight after 4 weeks, but feed and water consumption were not significantly different from those of controls. Ochratoxin A accumulated in the brain in a linear time-dependent manner, to reach about 100 ng/g of brain after 6 weeks. The toxin was shown to change the concentrations of the amino acids tyrosine and phenanthrene and to damage tissues in the hippocampus (Belmadani et al., 1998b)

Ten adult female Fischer rats received ochratoxin A at 120 µg/kg bw per day by oral gavage for 10, 20, or 35 days. Treatment altered the activity of all enzymes tested. Significant increases in gamma-glutamyl transferase activity were observed in the three brain regions examined. The changes in the other enzyme activities were regionally selective, but most of the activities had returned to control levels by day 35 of dosing (Zanic-Grubisic et al., 1996).

The neurotoxicity of ochratoxin A has been investigated in nerve tissue cell cultures (embryonic chick neural retina and brain) and cultured meningeal fibroblasts. The cells were incubated with ochratoxin A in serum-free medium for 8 days. The median inhibitory concentration (IC₅₀) for a number of parameters of cytotoxicity (cellular protein, 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide [MTT] reduction, neutral red uptake) were found to be about 170 nmol/L in all three culture systems, indicating that ochratoxin A did not have cell-specific effects. Ochratoxin B and the heat-induced 3S-epimer of ochratoxin A induced comparable effects at 19- and 10-fold higher concentrations, respectively (Bruinink et al., 1997).

In a study with a similar protocol, markers of neuritic outgrowth and differentiation (NF68 and 160 kDa, MAP2 and MAP5) were affected at significantly lower concentrations than the markers of cytotoxicity. Although the presentation of the data is unclear, the IC₅₀ values for the most sensitive parameters appeared to be 20–50 nmol/L for the embryonic brain and neural retinal cultures. Binding of ochratoxin A to bovine serum albumin resulted in significantly decreased potency (IC₅₀ values increased by 15–30-fold). Differences were noted between serum-free primary cultures and the cell lines. In these culture systems, phenylalanine did not decrease the effects of ochratoxin A and in contrast appeared to cause a concentration-related decrease in the IC₅₀ [no statistical analysis presented]. The authors concluded that ochratoxin A specifically affected neurite formation and that its toxicity was decreased by protein binding but not by phenylalanine (Bruinink & Sidler, 1997).

These authors also investigated whether the effects of ochratoxin A could be attributed to its isocoumarin structure, by comparing the toxicity of ochratoxin A with that of ochratoxin alpha and ochracin in serum-free embryonic chick brain cultures. Ochratoxin A decreased the end-points at concentrations > 15 nmol/L, with a greater effect on neurite outgrowth (neurofilament 68 kD). Ochratoxin alpha and ochracin had minimal effects at concentrations up to 1 mmol/L. The isocoumarin structure was therefore considered not to be responsible for the toxicity of ochratoxin A in this brain cell culture model (Bruinink et al., 1998).

The regional selectivity of ochratoxin A was investigated in primary cultures of neurons and astrocytes isolated from embryonic or newborn rat brain ventral mesencephalon and cerebellum. The cultures were exposed to ochratoxin A in a medium containing 10% fetal calf serum for 46 h, before measurement of DNA and protein synthesis, lactate dehydrogenase leakage, and lipid peroxidation. Ochratoxin A inhibited protein and DNA synthesis in all cell types, with IC₅₀ values ranging from 14 to 69 µmol/L. Neuronal cells were more sensitive than astrocytes, and the cells of the ventral mesencephalon were more sensitive than those of the cerebellum. Increases in lactate dehydrogenase leakage and lipid peroxidation were also seen, but the sensitivity of the cell types did not mirror that for DNA and protein synthesis. The authors concluded that ochratoxin A is neurotoxic and may affect particular structures of the brain (Bruinink et al., 1998).

(d) Nephrotoxicity

Renal function and morphology are greatly affected at high doses of ochratoxin A, as indicated by increased kidney weight, urine volume, blood urea nitrogen (Hatey & Galtier, 1977), urinary glucose, and proteinuria (Berndt & Hayes, 1979). The last two findings indicate that the site of reabsorption, i.e. the proximal convoluted tubules, is damaged. The NOELs for changes in renal function depend on the species and on the parameter tested. At low doses of ochratoxin A, no increase in blood urea nitrogen, creatinine, or glucose was found in the urine of male or female rats given 210 µg/kg bw per day by gavage for 6–12 months, but a mild to moderate decrease in the ability to concentrate urine was seen. The NOEL for this effect was 70 µg/kg bw per day for male rats and 21 µg/kg bw per day for female rats (National Toxicology Program, 1989).

Various groups of investigators have shown that this specific nephrotoxic effect is due to an ochratoxin A-induced defect of the organic anion transport mechanism located on the brush border of the proximal convoluted tubule cells and basolateral membranes (Endou et al., 1986; Sokol et al., 1988). The organic ion transport system is also the mechanism by which ochratoxin A enters proximal tubular cells (Friis et al., 1988; Sokol et al., 1988).

The middle (S2) and terminal (S3) segments of the proximal tubule of isolated nephron segments were found to be the most sensitive to the toxic effects of ochratoxin A (0.05 mmol/L), as shown by a significant decrease in cellular ATP and a dose-related decrease in mitochondrial ATP content (Jung & Endou, 1989).

Several investigators have measured the effect of ochratoxin A on the release of enzymes from the kidney into the urine. Changes in enzyme and protein patterns can be used to distinguish different types of renal injury (Stonard et al., 1987).

Subcutaneous doses of ochratoxin A at 10 mg/kg bw for 5 days decreased the activity of muramidase and then decreased the activities of lactate dehydrogenase, alkaline phosphatase, glutamate dehydrogenase, and acid phosphatase in the kidney (Ngaha, 1985). The activities of alanine peptidase, leucine amino peptidase, and alkaline phosphatase were decreased by 60%, 50%, and 35%, respectively in isolated kidney tubules in the presence of 0.1 mmol/L ochratoxin A (Endou et al., 1986).

In male rats given ochratoxin A at 0.1–2 mg/kg bw per day orally for 2–5 days, the phosphoenolpyruvate carboxykinase activity decreased by 50–70% at the highest dose (Meisner et al., 1983; Meisner & Krogh, 1986). The minimum effect level was 0.1 mg/kg bw per day (Meisner & Polsinelli, 1986); at 2 mg/kg bw per day, enzymes such as pyruvate carboxylase, malate dehydrogenase, hexokinase, and gamma-glutamyl transpeptidase were not affected (Meisner & Selanik, 1979).

In rats given ochratoxin A by gavage at a dose of 0.14 mg/kg bw every 48 h (equivalent to about 2 mg/kg diet) for 8–12 weeks, the activities of lactate dehydrogenase, alkaline phosphatase, leucine aminopeptidase, and gamma-glutamyl transferase decreased significantly. The last three enzymes are located in the brush border of the proximal convoluted tubules, indicating damage at that site. Concomitantly with the decrease of enzyme activity in the kidney, these enzymes appeared in the urine. A late event was a