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  • The hepatic cytochrome P CYP superfamily


    The hepatic cytochrome P450 (CYP450) superfamily is essential for metabolising foreign chemicals, fatty acids, vitamins, hormones and other compounds and consists of 18 subfamilies (Uno et al., 2012). The number of discovered CYP genes is increasing as a result of intense work with CYP450 gene structures (Kubota et al., 2013). The first three subfamilies (CYP1, CYP2 and CYP3) are mainly responsible for metabolising xenobiotics, and variations in their expression and activity can be used as indicators of exposure to environmental contaminants. CYP1A is a catalyst of environmental pollutants, including human pharmaceuticals (Laville et al., 2004, Navas et al., 2004, Smith et al., 2012); therefore it is critical in finding the pathways leading to detoxification. This enzyme is highly conservative among vertebrates. Mammals, melanin inhibitor and some fish species (eel and rainbow trout) possess genes for two CYP1A isoforms (Berndtson and Chen, 1994, Rifkind et al., 1994, Gorman et al., 1998, Mahata et al., 2003). A widely used assay for CYP1A is the measurement of EROD activity, which is routinely used as a biomarker to determine the presence of organic pollutants (Mandal, 2005). The majority of CYP450s are substrate-inducible via mechanisms often including ligand activation of transcription factors such as the aryl hydrocarbon receptor (AhR), pregnane X receptor, constitutive androstane receptor and others. The mechanism of CYP1A induction in fish is well known; numerous bioactive compounds induce CYP1A via binding to AhR and subsequent initiation of transcription. The less-studied mechanism of CYP450 regulation involves stabilization of mRNA and changes in protein turnover, leading to an increase in CYP450 activity, which largely depends on biotransformation of environmental pollutants in aquatic organisms. Factors that alter this activity might also alter the toxicity of CYP450 substrates. Natural and synthetic glucocorticoids perform their biological action in the organism through receptor-dependent mechanisms (e.g., by binding to the glucocorticoid receptors). It has been determined that glucocorticoid receptors cross-talk with other nuclear receptors, including AhR (Celander et al., 1996b, Dvorak et al., 2008, Vrzal et al., 2009). Dexamethasone is a typical inducer of mammalian CYP3A (Donato et al., 1995, McCune et al., 2000, Pascussi et al., 2000a, Pascussi et al., 2000b) and CYP2E1 activity (Tamasi et al., 2001). In fish, DEX increased protein expression and CYP1A catalytic activity in hepatocellular carcinoma cells from Poeciliopsis lucida (Celander et al., 1996a). In this study, we investigated the in vivo effect of DEX on the catalytic activity of selected hepatic CYP450s in rainbow trout. Because DEX concentrations vary greatly in environmental waters, two environmentally relevant DEX concentrations (3 and 30ngL−1) and two higher concentrations (300 and 3000ngL−1) were included. Activities of the following CYP450s were measured: CYP1A (7-ethoxyresorufin O-deethylase [EROD]), CYP2E1-like (p-nitrophenol hydroxylase [PNPH]) and CYP3A-like (7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase [BFCOD] and 7-benzyloxyquinoline O-debenzylase [BQOD]). Moreover, total CYP450 content as well as CYP1A and CYP3A-like proteins were determined.
    Materials and methods
    Discussion The induction of CYP450 in fish has been evaluated as a sensitive early warning signal of organic xenobiotics in the aquatic environment (van der Oost et al., 2003). In this sub-chronic in vivo experiment, we observed that DEX at concentrations ranging from 3 to 300ngL−1 can modify CYP450 activity when expressed as amount of product formed per min per nmol total CYP450, and it can affect total CYP450 content. Several recent studies focused on the modulation of CYP450 activity by numerous pharmaceuticals including DEX (Smith and Wilson, 2010, Wassmur et al., 2010). In general, no consistent response of fish CYP450 to DEX was found across published studies. In one study, increased metabolism of a number of CYP450 substrates was found after treating rainbow trout with DEX at a dose of 2mg/kg body weight (Haasch et al., 1994). Rainbow trout injected with DEX at a dose of 100mg/kg body weight showed significant induction in 3-cyano-7-ethoxycoumarin metabolism (mainly metabolised by CYP1A enzymes), while EROD and 7-methoxyresorufin O-demethylation activities were not altered (Smith and Wilson, 2010). Similarly, injection of DEX at 0.03–3μg/kg into male H. malabaricus did not alter EROD activity (Guiloski et al., 2015). CYP3A activity was induced in grass carp after DEX treatment (Li et al., 2008). This dissimilarity in responses can be associated with many factors, such as the age of the fish, type of exposure and differences in experimental concepts.