Hepatic xenobiotic biotransformation enzyme levels during feeding and depuration periods in Atlantic salmon (Salmo salar) fed graded levels of the phenolic antioxidant butylated hydroxytoulene (BHT)

Synthetic antioxidants are used by the Norwegian aquaculture industry as preservative in fish feed and fish feed ingredients against the auto-oxidation of unsaturated lipids. The growth of Atlantic salmon in seawater increases with increasing lipid levels in the diet up to approximately 40%. This has lead to increase of lipid content in fish feed during the last 30 years and has led to the increased use of antioxidants such as BHT, which is usually added to fish oil. This study was performed to investigate the toxicological aspects of BHT in Atlantic salmon during a 12-week feeding period and 2-week depuration period with 15 mg (low), 150 mg (medium) and 1500 mg/kg feed (high) using genomic and catalytic assay methods. In addition, levels of parent BHT accumulated or retained in fish fillet were measured during feeding and depuration periods using a HPLC method. During the feeding period, liver samples were collected at 3, 7, 14 and 84 days, and in the depuration period at 3, 7 and 12 days. Total RNA were isolated from these liver samples, transcribed to cDNA and analyzed for gene (AhR isotypes, UGT, GST, CYP1A1 and CYP3A) expression patterns using real-time PCR. Enzyme activities for EROD (CYP1A1), GST and UGT were analysed using flourimetric and spectrophotometer methods with specific substrates. Our data showed a linear relationship between doses of parent BHT in feed and that retained in fish fillet during feeding and depuration periods. Comparison of feeding and depuration periods showed that BHT was highly retained in fish fillet as only 17-29% of fed BHT was eliminated during the 2-week depuration period. For the biotransformation system, our data show that consumption of dietary BHT produced an exposure and timespecific gene expression pattern for AhRa, AhRb. CYP1A1, CYP3A, GST and UGT during feeding and depuration periods. These unique patterns of expression can be described as increase in transcriptional expression at feeding days 3 and 7, and thereafter decrease at feeding days 14 and 84. During the depuration period the patterns could generally be described as increase in transcriptional expression at depuration day 3, and thereafter decrease at day 7 and 12. The expression of biotransformation enzymes reflects the subtle differences in the retention of dietary BHT suggesting the involvement of phase I and/or phase II biotransformation enzymes whose identity is subject of continued study in our laboratory. The food safety implications of the findings in the present study will be discussed.