In VivoAflatoxin B1Metabolism and Hepatic DNA Adduction in Zebrafish (Danio rerio)

Abstract

The zebrafish (Danio rerio) is assuming prominence in developmental genetics research. By comparison, little is known of tumorigenesis and nothing is known of carcinogen metabolism in this species. This study evaluated the ability of zebrafish to metabolize a well-characterized human carcinogen, aflatoxin B1(AFB1), to phase I and phase II metabolites and assessed hepatic AFB1–DNA adductionin vivo.Fish ip injected with 50–400 μg [3H]AFB1/kg body wt displayed a linear dose response for hepatic DNA binding at 24 hr. AFB1–DNA adduct levels among treatments showed no statistical difference over the period from 1 to 21 days after injection, suggesting poor adduct repair in this species. DNA binding in female fish was 1.7-fold higher than that in males (p< 0.01). Anin vitroAFB1metabolism assay verified that zebrafish liver extracts oxidize AFB1to the 8,9-epoxide proximate electrophile (Km= 79.0 ± 16.4 μm,Vmax= 11.7 ± 1.4 pmol/min/mg protein at 28°C). The excretion of AFB1and its metabolites was also examined by HPLC. As is typical of other fish studied, major metabolites excreted were aflatoxicol (AFL) and aflatoxicol–glucuronide (AFL-g), followed by unreacted AFB1. AFL appeared as early as 5 min after injection, whereas AFL-g was a significant metabolite after 18 hr. This study shows thatin vivoadministration of AFB1to zebrafish results in moderate adduction of the carcinogen to liver DNA and that zebrafish have the capacity for both phase I and phase II metabolism of AFB1. The approximate fourfold difference between rainbow trout and zebrafish AFB1–DNA covalent binding index appears insufficient to explain the relative resistance of zebrafish to dietary AFB1hepatocarcinogenicity.