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  • At ngL DEX did not affect

    2019-08-13

    At 3000ngL−1, DEX did not affect our investigated parameters, suggesting that DEX is regulated by CYP450 in a biphasic way, a response to xenobiotics that is not uncommon (Heinrichs et al., 1994). These data might also suggest that fish adapted to DEX exposure. It was previously shown that, depending on concentration, DEX might act as either a permissive or suppressive factor in enzyme induction (Ringel et al., 2002). We found that CYP1A1, CYP2E1-like and CYP3A-like enzyme activities increased in the presence of environmentally relevant concentrations of DEX, but not in the presence of the highest tested concentration (3000ngL−1) when activities were calculated per nmol total CYP450 content. No effect was observed when the activities were calculated relative to total protein content. Differences in the CYP450 responses to DEX depended on the calculation, either relative to total protein or to total CYP450 content. This is an important observation. Activities of CYP450-mediated reactions expressed relative to total protein content more closely reflected an in vivo situation, suggesting that overall DEX did not alter measured CYP450 activities. Nevertheless, an increase in the activities of CYP1A, CYP3A and CYP2E1-like proteins, expressed relative to total CYP450 content, suggest an eventual increase in the protein content of individual CYP450 isoforms. In studies using mammals, significant differences in enzyme catalytic activities were also found only after calculation of enzyme catalytic activity per total CYP450 (Wandel et al., 1998, Anzenbacherova et al., 2008). This means that the estimation of enzyme activity, expressed relative to total CYP450, is another way to illustrate a complete picture of the effects of various pharmaceutical drugs other than by calculation per milligram of protein. To further test this concept, protein expressions of CYP1A and CYP3A were measured in the same sample. Samples from fish exposed to DEX for 21d were used because no effect was observed after 42d. Previous studies showed diverse evidence in the regulation of CYP1A and CYP3A ghrelin receptor in fish and fish cell cultures. For example, DEX down regulated CYP1A1 in trout hepatocytes (Dasmahapatra and Lee, 1993), but did not affect CYP3A in rainbow trout (Lee et al., 1993). Moreover, in combination with beta-naphthoflavone (a known AhR activator), DEX increased CYP1A1 protein levels in the P. lucida hepatocellular carcinoma cell line (Celander et al., 1997). Following DEX exposure, the levels of CYP1A1 protein and activity remained unchanged, as seen in our previous in vitro study (Burkina et al., 2013). Dasmahapatra and Lee (1993) reported that 3.9μgL−1 DEX, similar to our highest tested concentration, did not change CYP1A1 protein content. An increase in CYP3A protein content can be related to an adaptive mechanism of fish to DEX exposure. Immunoblotting data did not support the CYP450 activity data. CYP3A-like protein level was increased only at 3ngL−1 DEX, but not for other doses. This study showed that treatment with the same dose of DEX did not cause a change in the activity of BFCOD or BQOD (Fig. 2). In this study, the CYP2E1-like protein was not detected using several antibodies against human and rat epitopes; however the CYP2E1 protein level was successfully determined and responded to carbon tetrachloride treatment in common carp liver (Jia et al., 2014). In future studies, it could be desirable to develop CYP2E1 antibodies against fish CYP2E1. A number of in vitro data have been reported for the effects of DEX on mammalian and fish CYP450 systems, but data from in vivo studies remains limited. It is important to investigate the effects of DEX at concentrations that commonly occur in the aquatic environment to mimic its possible interaction with fish-specific protein targets. Here, we tested the effects of low levels of residual DEX on specific pharmacological targets in fish and found that DEX can modify CYP450 activity in rainbow trout. We also clearly showed that DEX induces hepatic CYP3A-like proteins. This observation is important because there may be further interactions of environmentally relevant DEX concentrations with cellular recovery mechanisms and non-investigated xenobiotic-metabolizing proteins in fish. However, treatment of juvenile rainbow trout with DEX failed to demonstrate a clear and significant effect at high concentrations. In actual conditions, combinations of DEX with other glucocorticoids can raise synergistic actions of this key xenobiotic detoxification pathway.