In vitro DNA and dGMP adducts formation caused by ochratoxin A.
Ochratoxin A (OTA), a nephrotoxic and nephrocarcinogenic mycotoxin, leads to the formation of DNA adducts after administration to animals. This could be due to an epigenetic effect. In vitro assays can exclude an indirect effect, where the xenobiotic can generate, in vivo, endogenous reactive compounds which give adducts on DNA. Microsomes prepared from mice or rabbit kidney and liver, used as metabolic activators, were incubated in the presence of commercial salmon testes DNA and OTA, with NADPH or arachidonic acid used as cofactors. Upto 126 DNA adducts for 10(9) nucleotides were detected using the 32P postlabeling method after incubation with the mouse kidney system. Similar results were obtained with rabbit kidney microsomes. Using liver microsomes, the number of DNA adducts detected was much lower. When NADPH was used as a cosubstrate (to explore the cytochrome P450 metabolic pathways), with mice kidney microsomes, the adduct level was only 44% of the one obtained with arachidonic acid. These results lend support to the hypothesis of the preferential activation of OTA by the peroxidase activity of prostaglandin synthases and/or lipoxygenases to direct genotoxic metabolites, and are in agreement with the previously obtained results after in vivo treatment of mice. In order to identify the nucleotides of DNA modified by the OTA metabolites, dAMP, dGMP, dTMP and dCMP were used as substrates under the same conditions as with DNA. The adducts were found only on dGMP. The total adduct level was of 344 adducts per 10(9) nucleotides with the appearance of three major adducts in the presence of arachidonic acid. With NADPH, 271 adducts were obtained per 10(9) nucleotides, with again three major adducts, but only two of them were similar to two adducts obtained in the presence of arachidonic acid. Desferal (desferrioxamine B methanesulphonate), at a 50 microM concentration, did not reduce the adduct level. Adducts were also obtained when polydG, polydC and dG-p-dG were used as alternative substrates, whereas no adducts were obtained with polydA, polydT and polydC. The major adduct obtained after incubation of DNA with OTA, comigrated with the major adduct obtained with dGMP, in two chromatographic solvents. These results show that OTA is metabolized to genotoxic metabolite(s) which interact with the guanine residues of DNA.
Obrecht-Pflumio
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Laboratoire de Biologie végétale appliquée, IUT Louis Pasteur, allée d'Athènes, F-67300, Schiltigheim, France.
Dirheimer
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G
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002845:Chromatography; D018736:DNA Adducts / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D005260:Female; D007668:Kidney / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D009183:Mycotoxins / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D009793:Ochratoxins / Q000494:pharmacology*; D009195:Peroxidase / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry
D002273:Carcinogens; D002614:Chelating Agents; D018736:DNA Adducts; D003848:Deoxyguanine Nucleotides; D009183:Mycotoxins; D009711:Nucleotides; D009793:Ochratoxins; C025589:ochratoxin A; D016718:Arachidonic Acid; D009249:NADP; C007257:2'-deoxyguanosine 5'-phosphate; D009195:Peroxidase; D003676:Deferoxamine