Aflatoxin B1 carcinogenesis and its relation to DNA adduct formation and adduct persistence in sensitive and resistant salmonid fish

Rainbow trout (Salmo gairdneri)and coho salmon (Oncorhyn-chuskisutch) were exposed to aflatoxin B1(AFB1) either by passiveembryo uptake or by dietary treatment after hatching and feedingonset. Trout exposed as embryos to an aqueous solution of 0.5p.p.m. AFB1 for 15 min showed a 62% tumor incidence 12 monthslater, whereas coho salmon exposed to a similar solution for30 min showed only a 9% incidence. The difference between salmonand trout response was even greater by dietary AFB1 treatment.Trout exposed for 4 weeks to 20 p.p.b. dietary AFB1 had a 62%tumor response 12 months later, whereas salmon exposed to 40p.p.b. dietary AFB1 for 4 weeks failed to develop tumors. A5% tumor incidence was observed in salmon 12 months after 3weeks exposure to 5000 p.p.b. dietary AFB1, a lethal dose fortrout. In addition to a lower tumor incidence when comparedto trout, the neoplastic response of salmon to AFB1 is to producebenign hepatic adenomas in contrast to the malignant hepatocellularcarcinomas seen in trout. AFB1 metabolism, DNA adduct formation,adduct persistence in vivo and in vitro and cytochrome P-450isozyme composition were compared in livers of trout and salmonto understand the role of metabolism and initiation in thisspecies difference. AFB1-DNA binding was 7–56 times greaterin trout than salmon liver at various times after AFB1 injection,20 times greater in embryos or in freshly isolated trout hepatocytepreparations after a 1 h incubation with aflatoxin Bl, and 18times greater in trout liver after a three week dietary (80p.p.b.) exposure. The major AFB1-DNA adduct was 8, 9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB1 in both species. Persistence of AFB1-DNA adducts in vivoin liver was high compared to mamalian systems, implying thatactive enzymatic removal of bulky DNA adducts is low in bothspecies and probably not a factor in their differential responseto aflatoxin. Species differences in other phase I and phaseII metabolism pathways and in AFB1 elimination were, overall,much less striking than those previously observed for troutfed inhibitors of aflatoxin carcinogenesis. Rates of bileeliminationof AFB1 detoxication products, and total excretion of aflatoxinsinto water after AFB1 exposure, were not significantly differentbetween trout and salmon. Since detoxication differences werenot observed, the species difference in AFB1-DNA binding appearsto reflect less efficient cytochrome P-450 metabolism of aflatoxinto the reactive 8, 9-epoxide in salmon, compared to trout. Insupport of this hypothesis, trout liver microsomes displayeda K_m (7.5 µM)for AFB1-DNA adduction in vitro that was7-fold lower than salmon (52 µM). Furthermore, immunoquantitationof various P-450 isozymes suggest that salmon liver microsomeshave much lower amounts of an isozyme immunochemically relatedto trout P-450 LM₂ which has previously been shown to be themajor isozyme catalyzing AFB1 8, 9-epoxidation. Other, post-initiationdifferences were not ruled out by these studies and may contributeto the differential response of rainbow trout and coho salmonto AFB1 hepatocarcino-genesis.