Evidence for an enterohepatic circulation of ochratoxin A in mice.

The distribution and elimination of [3H]ochratoxin A (OTA) from stomach content and tissue, intestine content and tissue, liver, bile, serum and urine of Swiss male mice which had received a single low dose of OTA by intubation was followed as a function of time. The profiles of radioactivity do not show a smooth decline after the absorption period, but an oscillating pattern with rapid declines followed by increases which favour the assumption of an enterohepatic circulation. Between 28% and 68% of conjugated OTA together with OTA cleavage products were found in bile giving evidence for biliary excretion of OTA and its metabolites in mice. When given i.m. to mice [3H]OTA is already found after 30 min in bile and intestine contents and its elimination patterns show several peaks confirming the biliary excretion and the enterohepatic circulation. Cholestyramine, which is known to prevent the enterohepatic circulation of drugs and toxins, changes the profile of elimination of OTA which no longer presents the cyclic pattern. This result is also in favour of an enterohepatic circulation of OTA. When phenylalanine is given together with OTA by oral gavage the toxicokinetics of the mycotoxin change completely in the different body fluids, in stomach and intestine content and tissues. Phenylalanine seems to facilitate the gastric absorption of OTA and the gastro-intestinal transit. It increases also its early excretion into urine and bile. However, its elimination pattern no longer shows the oscillating pattern. Thus phenylalanine seems to inhibit the intestinal reabsorption of OTA conjugates.

Transplacental transfer of T2-toxin: pathological effect.

Radioactivity was recovered in fetuses of pregnant rats following administration of radioactive T2-toxin. The transplacental effects of T2-toxin were studied by ip administration or oral feeding of pregnant rats at doses equivalent to natural contaminations and compatible with the maintenance of pregnancy. Body weights of the newborn rats from treated females were similar to the body weights of the control animals but their thymuses were atrophic. This atrophy was reversible in one week. Since the measurement of antibody production for fetuses and newborns is not feasible, the lymphoblastic response to mitogen of the splenic and thymic cells of baby rats from treated and control females was tested. At 4 and 6 days after birth, a good response to PHA for the thymic cells of the mother treated young rats was observed. Histological examination of the thymus showed that one day after birth the cortex was atrophic while the medulla was proliferative; on day six the situation was reversed. For the spleen, both B and T cells were impaired and their responsiveness to PHA and LPS decreased. On days 1 and 6, the periarteriolar sheats, as well as the follicles, appeared atrophic. These results show that T2-toxin easily passes the placental barrier and that T2-toxin injection or feedings at levels (2 ppm) similar to those in naturally contaminated foods, produced an impairment of the immune system of the newborn.

DNA adduct formation in mice treated with ochratoxin A.

Several authors have reported the occurrence of renal and hepatic tumours in mice and rats exposed to ochratoxin A in long-term studies. The compound was not mutagenic, however, in various microbial and mammalian gene mutation assays, either with or without metabolic activation. Contradictory results were obtained for induction of unscheduled DNA synthesis and sister chromatid exchange. We showed previously that ochratoxin A causes DNA damage, manifested as single-strand breaks in mouse spleen cells and in vivo. These findings, which suggest that ochratoxin A is weakly genotoxic to mammalian cells, prompted us to search for DNA adducts using a modified 32P-postlabelling method, the sensitivity of which was improved by treatment with nuclease P1. DNA was isolated from liver, kidney and spleen excised from mice 24, 48 and 72 h after oral treatment with ochratoxin A at 0.6, 1.2 and 2.5 mg/kg body weight. Several adducts were found in the DNA of the three organs, the levels varying greatly. After administration of 2.5 mg/kg body weight, 40 adducts per 10(9) nucleotides were found in kidney DNA and 7 adducts per 10(9) nucleotides in liver after 72 h. The levels of most of the adducts increased from 24 to 72 h, but those of others diminished after 24 or 48 h. Adducts were found in spleen only at 24 and 48 h. These results confirm the genotoxicity of ochratoxin A.

The myocotoxin ochratoxin A is a substrate for phenylalanine hydroxylase in isolated rat hepatocytes and in vivo.

Ochratoxin A (OTA), is a myocotoxin contaminating food and feed stuffs, consisting of a chlorinated dihydroisocoumarin linked through a 7-carboxyl group to L-phenylalanine by an amide bond. When OTA (0.12-1.4 mM) is incubated with freshly isolated rat hepatocytes, it inhibits both the hydroxylation of phenylalanine (0.05 mM) to tyrosine, catalyzed by phenylalanine hydroxylase and the subsequent metabolism of tyrosine as measured by homogentisate oxidation. The IC50 of OTA for phenylalanine hydroxylation is 0.43 mM. OT alpha, (0.5-1.0 mM), the dihydroisocoumarin moiety of OTA, does not inhibit phenylalanine hydroxylase activity under these conditions. During incubations of hepatocytes with uniformly labelled [3H]-OTA and unlabelled phenylalanine, tyrosine-ochratoxin A is formed (up to 6% of the total mycotoxin added), indicating that ochratoxin can act as a substrate for phenylalanine hydroxylase. In vivo tyrosine-OTA is also found in liver of poisoned animals.