The chemical structures of I
The chemical structures of I3C, 3MOI, and 3MI and the Valproic acid responsible for 3MI metabolism in rainbow trout and carp are shown in Fig. 4.
Discussion To determine the individual CYP450 isoforms involved in 3MI metabolism, we used specific inhibitors for CYP1A, CYP3A and CYP2E1. Our results suggest that CYP1A and CYP3A contribute to the metabolism of 3MI. CYP1A is well studied in fish microsomal enzymes and is responsible for the biotransformation of many xenobiotics (Hansson et al., 2006, Celander, 2011). This enzyme in fish is of interest because of its involvement in the metabolism of various environmental pollutants, including polycyclic aromatic hydrocarbons. CYP3A is another enzyme that is involved in xenobiotic metabolism in fish and therefore has been widely studied (Celander et al., 1989, Hegelund et al., 2004, Hasselberg et al., 2008, Zlabek and Zamaratskaia, 2012). The substrate specificity of both CYP1A and CYP3A is very broad. It is accordingly not surprising that 3MI is also metabolised by CYP1A and CYP3A. The metabolism of 3MI by mammalian CYP450 has been well documented (Yan et al., 2007, Zamaratskaia and Squires, 2009), and it was suggested that mammalian CYP1A and CYP3A might be involved in 3MI hepatic metabolism, although their role is not major. CYP2E1 has been repeatedly reported to be involved in 3MI metabolism in pigs (Zamaratskaia and Squires, 2009). Our inhibition study showed that DAS, an inhibitor of mammalian CYP2E1, did not decrease the formation of 3MI metabolites, suggesting that CYP2E1-like protein is not involved in 3MI metabolism in fish, and providing evidence for inter-species differences in in vitro 3MI metabolism. A possible limitation of our study was the use of inhibitors of mammalian CYP450 isoforms to investigate hepatic metabolism of 3MI by fish microsomes. However, these inhibitors have already been evaluated in fish microsomes. Thus, ellipticine strongly inhibited EROD activity in fish gill filaments (Beijer et al., 2010) and in fish hepatic S9 fractions (Rotchell et al., 2000), and ketoconazole inhibited both EROD and BFCOD activities (Zlabek and Zamaratskaia, 2012). Studies of the effects of DAS on fish CYP450 have been limited. Other investigations previously showed that DAS inhibited CYP2E1-like protein activity in microsomes from Atlantic salmon (Zamaratskaia and Zlabek, 2011) and medaka (Geter et al., 2003), but it was suggested that DAS is a considerably stronger inhibitor of PNPH in mammals than in fish. Additionally, there are indications that DAS can also inhibit fish CYP1A activity (Zamaratskaia and Zlabek, 2011). Nevertheless, DAS did not inhibit 3MI metabolite formation in the present study, indicating that CYP2E1-like protein is not involved in 3MI metabolism by fish hepatic microsomes.
Conflicts of interest
Acknowledgements This work was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic – projects “CENAKVA” (No. CZ.1.05/2.1.00/01.0024) and “CENAKVA II” (No. LO1205 under the NPU I program), by the Grant Agency of the University of South Bohemia in Ceske Budejovice (No. 012/2016/Z), by the Czech Science Foundation (No. 15-04258S), and by a research grant from C.F. Lundströms Stiftelse, Sweden. The authors thank Dr. Carl Brunius for the synthesis of 3-hydroxy-3-metyloxindole. We would like to thank American Manuscript Editors, an affiliate of Grammar Labs, LLC, for English language editing.
Introduction Over the past decade, a wide range of pharmaceutical compounds for human use, agriculture, and aquaculture have been detected in the aquatic environment (Beausse, 2004, Fent et al., 2006). Pharmaceutical compounds and their derivatives have effects on non-target aquatic organisms, including fish, even at the low concentrations typical of aquatic systems (Schwaiger et al., 2004, Corcoran et al., 2010, Hirakawa et al., 2012). The majority of detoxification occurs in the liver via a multitude of enzyme systems. Phase I, usually reactions of oxidation, reduction, or hydrolysis and phase II conjugation reactions biochemically transform toxic substances into more water-soluble, and therefore excretable, substances (Liska et al., 2006). Phase I enzymes is primarily cytochrome P450 (CYP450), which is a superfamily of heme-containing proteins involved in the biotransformation of many endogenous compounds and exogenous substrates. The proteins belonging to the first three families of CYP450: CYP1, CYP2, and CYP3 are closely associated with drug activation and metabolism (Uno et al., 2012).