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  • Boc-D-Asp(OtBu)-OH.DCHA receptor MI has been detected in was


    3MI has been detected in wastewater at concentrations of 640–700 μg L−1 (Hwang et al., 1995) and up to 20 mg L−1 in decaying algal water environments (Peller et al., 2014). It is likely that 3MI accumulation in fish might be due to the degradation of endogenous amino acids. The presence of this chemical in aquatic environments is of concern because of its eventual mutagenic properties (Thornton-Manning et al., 1991). To exert mutagenic effects, metabolic activation of 3MI by CYP450 is required. Numerous xenobiotic compounds that might negatively affect physiological processes in fish include substrates of cytochrome P450 (CYP450). The metabolism of 3MI has been predominantly studied in mammalian species, particularly in pigs due to its negative effects on meat quality (Zamaratskaia and Squires, 2009), and in ruminants due to pneumotoxicity (Yost, 1989). Hepatic skatole metabolites formed during phase-I metabolism in mammals include 2-aminoacetophenone (2-AAP), 3-methyloxindole (3MOI), 5-hydroxyskatole, 6-hydroxyskatole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole (HMOI) and indole-3-carbinol (I3C) (Diaz et al., 1999). Currently, no information is available about the metabolism of 3MI by fish. However, the induction of hepatic CYP1A by dietary I3C was previously studied in rainbow trout (Takahashi et al., 1995). It is essential to fully understand the metabolic pathways of 3MI to evaluate its eventual toxicity to fish. Common carp and rainbow trout are economically important fish species. Cytochrome P450 Boc-D-Asp(OtBu)-OH.DCHA receptor involved in xenobiotic metabolism are well characterised in these species (Rabergh et al., 2000, Whyte et al., 2000, Lee et al., 2001, Fisher et al., 2006, Uno et al., 2012). Additionally, both common carp and rainbow trout were used as test organisms in toxicology tests, environmental monitoring and nutrition studies (Gaillard et al., 2014, Salze et al., 2014). In the present study, we investigated 3MI metabolism by fish hepatic microsomes. For this purpose, we used hepatic microsomes from two fish species, carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss), at two ages.
    Materials and methods
    Results Two metabolites of 3MI were detected in the microsomes from rainbow trout and identified as I3C and 3MOI. In the microsomes from carp, the formation of only one metabolite, 3MOI, was detected (Fig. 1). The rate of 3MOI production was similar in both species at both ages. No statistically significant differences in kinetic parameters were observed (p = 0.799 for Vmax, and p = 0.809 for Km). Production of I3C was detected only in the microsomes from rainbow trout. Km values were similar in juvenile and adult fish (p = 0.957 for Km); Vmax was higher in juvenile rainbow trout compared with adults (p = 0.044). The rates of 3MOI and I3C formation from 3MI (38 μM) in the presence of inhibitors are shown in Fig. 2, Fig. 3. The strongest inhibitions were observed in the presence of ellipticine and ketoconazole. These inhibitors reduced the formation of 3MOI in both species (Fig. 2), although the reduction was somewhat higher in the microsomes from rainbow trout (100 μM of ellipticine: from 40.5 to 53.2% in rainbow trout, and from 64.8 to 81.9% in carp; 100 μM of ketoconazole: from 22.3 to 65.8% in rainbow trout, and from 39.9 to 91.3% in carp). Additionally, the presence of ellipticine and ketoconazole reduced I3C formation (100 μM of ellipticine: from 41.0 to 53.7%; 100 μM of ketoconazole: from 28.6 to 51.5%) in the microsomes from rainbow trout (Fig. 2). The inhibitor of CYP2E1 DAS did not significantly reduce the rates of either 3MOI or I3C formation. The activities of CYP1A, CYP3A and CYP2E1-like protein were higher in rainbow trout compared with carp (Table 1). Moreover, in rainbow trout the activity of CYP2E1-like protein decreased by 39% and the activity of CYP1A increased by 72 % with age (p < 0.001 for both); CYP3A activity was similar in juvenile and adult fish (p < 0.001). No age-related differences in any of investigated isoforms were observed in carp (p > 0.05 for all).