Effect of Cytochrome P-450 induction on the metabolism and toxicity of Ochratoxin A

Omar, Rabeea Fahmy (1994) Effect of Cytochrome P-450 induction on the metabolism and toxicity of Ochratoxin A. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The role of cytochrome P-450 in the stimulation of lipid peroxidation induced by the mycotoxin ochratoxin A (OTA) has been investigated. Purified cytochrome P-450 (IIB1) could effectively replace EDTA in stimulating lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe³⁺, EDTA and NADPH, suggesting that it could mediate the transfer of electrons from NADPH to Fe³⁺. Microsomes isolated from livers of cobalt protoporphyrin IX-treated rats (in which cytochrome P-450 was depleted) underwent OTA-dependent lipid peroxidation much more slowly than control microsomes. -- The role of cytochrome P-450 in OTA metabolism was also investigated. To determine which cytochrome P-450 isoforms are involved in the metabolism of OTA, we used different cytochrome P-450 inducers to induce the major isoforms of cytochrome P-450 in the rat liver. Microsomes from these livers were used to investigate their effect on OTA metabolism. Pretreatment of rats with pregnenolone -16α-carbonitrile (PCN), phenobarbital (PB), 3-methyl cholanthrene (3MC), and isosafrole (ISF) greatly induced 4(R)-4-OH-OTA formation; 4(S)-4-OII-OTA formation was also induced after pretreatment with PB, PCN, 3MC and ISF. INH pretreatment primarily induced the 4(S) isomer formation. The formation of the 4(R) and 4(S) isomers showed significant differences with respect to pH optima, effect of antioxidants and iron chelators. The 4(R) isomer formation showed a pH optimum of 6.0 using microsomes from rats treated with 3MC and ISF, and 6.5 using microsomes from rats treated with PB and PCN and was not inhibited by antioxidants or iron chelators. In contrast, both the 4(S) isomer formation and lipid peroxidation showed a pH optimum of 7.0 - 7.5 and both activities were highly sensitive to inhibition by antioxidants and iron chelators. Lipid peroxides were not involved in the 4(S) isomer formation since addition of linoleic acid hydroperoxide to microsomes did not give rise to the 4(S) isomer. Cytochrome P-450 appeared to be essential since other hemoproteins such as horseradish peroxidase and hemoglobin were ineffective in metabolizing OTA. Microsomes from rats pretreated with Co-protoporphyrin IX resulted in no metabolism of ochratoxin A. 7-Ethoxy- and 7-pentoxyresorufin assays showed specificity towards cytochromes P-450 induced by 3MC (IA1/IA2) and PB (IIB1) respectively. Also, metyrapone (inhibitor of cytochrome P-450 IIB1) preferentially inhibited OTA metabolism by microsomes from rats treated with PB, and α-naphthoflavone (inhibitor of cytochrome P-450 IA1/IA2) preferentially inhibited OTA metabolism by microsomes from 3MC and ISF treated rats. Monoclonal antibodies (MAbs) 1-7-1 (against P-450 IA1/IA2) and 2-66-3 (against P-450 IIB1) showed preferential inhibition of OTA metabolism by microsomes from 3MC and PB treated rats respectively. -- Excretion of renal enzymes in urine is a sensitive non-invasive index of renal damage. Therefore, we examined the effect of cytochrome P-450 induction on the excretion of alkaline phosphatase and ɤ-glutamyl transferase (ɤ-GT) in a PB-treated rat group and a control rat group (both groups received OTA). At the fourth day of OTA treatment higher enzyme levels were found in the control group compared to the PB group. The results provide evidence for the stimulatory role of cytochrome P-450 in OTA-induced lipid peroxidation. Also, our data suggest that 4(R)-4-OH-OTA is formed by normal mixed function oxidation but that 4(S)-4-OH-OTA formation may involve free iron. Also, our results show that the major cytochrome P-450 isoforms catalyzing OTA hydroxylation are IA1/IA2 (3MC and ISF), IIB1 (PB) and IIIA1/IIIA2 (PCN). The results from in vivo experiments suggest a protective effect of PB against OTA toxicity (through inducing cytochrome P-450, thus metabolizing more OTA and excreting it faster from the body compared to controls). This was also evident from in vivo experiments of [³H]OTA distribution. Therefore, OTA hydroxylation is more likely to be a detoxification pathway.

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