In vivo aflatoxin B1 metabolism and hepatic DNA adduction in zebrafish (Danio rerio)
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C M Troxel1, A P Reddy, P E O'Neal
1Department of Food Science and Technology, Oregon State University, Corvallis 97331, USA.
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 adduction in vivo. Fish i.p. injected with 50-400 micrograms [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). An in vitro AFB1 metabolism assay verified that zebrafish liver extracts oxidize AFB1 to the 8,9-epoxide proximate electrophile (Km = 79.0 +/- 16.4 microM, Vmax = 11.7 +/- 1.4 pmol/min/mg protein at 28 degrees C). The excretion of AFB1 and 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 that in vivo administration of AFB1 to 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 AFB1 hepatocarcinogenicity.
Insights
Zebrafish metabolize aflatoxin B1 (AFB1) and form DNA adducts, but show poor repair. This study investigates AFB1 metabolism and DNA binding in zebrafish, revealing moderate adduction and limited repair capacity.
Area of Science:
- Toxicology
- Genetics
- Comparative Metabolism
Background:
- Zebrafish (Danio rerio) are increasingly used in developmental genetics.
- Little is known about zebrafish tumorigenesis and carcinogen metabolism.
- Aflatoxin B1 (AFB1) is a well-characterized human carcinogen.
Purpose of the Study:
- To evaluate zebrafish's ability to metabolize AFB1 into phase I and phase II metabolites.
- To assess hepatic AFB1-DNA adduction in vivo.
- To compare AFB1 metabolism and DNA binding in zebrafish to other species.
Main Methods:
- Zebrafish were injected with [3H]AFB1, and hepatic DNA binding was measured.
- In vitro assays assessed AFB1 metabolism by zebrafish liver extracts.
- HPLC was used to examine the excretion of AFB1 and its metabolites.
Main Results:
- A linear dose response for hepatic DNA binding was observed.
- AFB1-DNA adduct levels remained stable from 1 to 21 days post-injection, indicating poor repair.
- DNA binding was significantly higher in female than male zebrafish.
- Zebrafish liver extracts oxidized AFB1 to its epoxide, and major excreted metabolites included aflatoxicol (AFL) and aflatoxicol-glucuronide (AFL-g).
Conclusions:
- Zebrafish can metabolize AFB1 via phase I and phase II pathways.
- In vivo AFB1 administration leads to moderate liver DNA adduction with limited repair.
- Observed AFB1-DNA binding in zebrafish is insufficient to explain their resistance to AFB1-induced hepatocarcinogenicity compared to rainbow trout.