Effects of 17β-Estradiol and Testosterone on Hepatic mRNA/Protein Levels and Catalytic Activities of CYP2M1, CYP2K1, and CYP3A27 in Rainbow Trout (Oncorhynchus mykiss)

Abstract

There is growing concern that exposure to chemicals in the environment can disrupt the endocrine systems of wildlife and humans, causing reproductive problems or other adverse effects. The expression of many cytochrome P450s (CYPs) is under hormonal control, hence, levels of these enzymes can be affected by exposure to endocrine-disrupting chemicals. Previous research has reported that treatment of fish and other animals with the estrogenic and androgenic hormones 17β-estradiol (E2) and testosterone (T) alters the P450 content or enzyme activities in the treated animals. However, the results of many of these studies are either incomplete or in disagreement and in most cases the effect on specific P450 forms has not been determined. Therefore, to better understand the effects of gonadal hormones on the expression of P450s and their associated enzyme activities, it was of interest to undertake a comprehensive investigation of the transcriptional and translational expression of three constitutive hepatic P450s in the rainbow trout (Oncorhynchus mykiss) following hormone exposure. Accordingly, juvenile trout were injected intraperitoneally with propylene glycol vehicle and the most active estrogenic and androgenic hormones E2 (3 mg/kg) or T (3 mg/kg) on days 1, 4, 7, 13, and 15 and euthanized on day 19. After treatment with E2, hepatic microsomes showed significantly lower levels (percentage of control) in total P450 contents (52%), lauric acid hydroxylase (32%), and 6β-progesterone hydroxylase activities (27%), [3H]aflatoxin–DNA binding (31%), and the protein levels of individual cytochrome P450s (CYPs) LMC1 (CYP2M1), LMC2, (CYP2K1), and LMC5 (CYP3A27) (average for three isoforms a reduction to 29% of control values) with only minor differences between sexes. Treatment with T had either no effect or resulted in small increases in total P450 in males (42%), in lauric acid hydroxylase in females (24%), and in 6β-progesterone hydroxylase activity in males (21%). Biological variabilities among fish were high and a polymorphic or new LMC2-like form was detected at about 52 kDa in some liver microsomal samples after exposure of fish to either hormone. Female liver RNAs were analyzed through Northern blots and an average decrease of 94% in CYP2 M1, CYP2K1, and CYP3A27 mRNA levels occurred in the E2-treated trout. In livers from T-treated trout, the changes of mRNA levels of CYP2M1 and CYP3A27 were negligible, but CYP2K1 mRNA level decreased by about 60%. Additional CYP2K1 cDNA hybridizable mRNAs were seen in some fish as faint bands at about 2.8 kb for both hormone treatments. Results of this study, therefore, indicated that E2 down-regulated while T produced small but variable effects on the hepatic mRNA/protein levels of CYP2K1, CYP2M1, and CYP3A27 in juvenile rainbow trout. This study, therefore, suggests that exposure of fish and other wildlife to environmental endocrine disruptors, especially estrogen mimics, can adversely affect a number of physiological processes through mechanisms involving altered levels of expression of specific P450 isozymes.