5402 Most xenobiotics undergo biotransformation before being
Most xenobiotics undergo biotransformation before being excreted. Such biotransformation is classified into two phases. In the first phase, the lipophilic substrate is oxidized by the cytochrome P-450 (CYP450) enzyme system, which introduces a single oxygen 5402 into the molecule (Moutou et al., 1998). In the second phase, the oxidized compound is conjugated with an endogenous molecule such as glucuronic acid, sulfate, glutathione, amino acid, etc. These conjugated products are generally less toxic and readily excreted (Andersson and Förlin, 1992).
The CYP450s constitute a multigene family of hemoproteins responsible for the biotransformation of numerous xenobiotics, including therapeutic drugs, environmental chemicals, dietary constituents, as well as endogenous steroids and bile acids (Quattrochi and Guzelian, 2001, Nelson, 2009). Although CYP450s were originally discovered in mammalian liver microsomal preparations, they have subsequently been found in many organs and tissues of numerous other animals and in some plants, fungi and bacteria. Over 230 individual CYP450s have been characterized according to their protein sequences, and forms of these enzymes appear to be present in every class of biota (Lewis, 1996).
In lower vertebrates such as fish, the ability to metabolize xenobiotics is not restricted to the liver. High levels of such oxidative enzyme activity have also been found in the kidney, gastrointestinal tract and gills (Andersson and Förlin, 1992, Sarasquete and Segner, 2000). However, most information concerning xenobiotic metabolism has been derived from studies on liver, since the highest level of CYP450 activity is found in that organ (Haasch et al., 1994, Moutou et al., 1998, Vaglio and Landriscina, 1999, Buhler et al., 2000, Råbergh et al., 2000). The hepatic CYP450 dependent monooxygenase system is located in the granular endoplasmic reticulum (Lester et al., 1993, Sole et al., 1999). Husøy et al. (1994) showed that induction of hepatic CYP1A in Atlantic cod (Gadus morhua L.) by environmental xenobiotics occurred in hepatocytes, billiary and vascular structures (endothelial cells), while the constitutive CYP3A-like isozyme appeared to be more homogenously distributed in the liver/hepatocytes than the inducible CYP1A1 isozyme. Also, CYP3A was not evident in the billiary epithelium or vascular endothelium in the liver.
Multiple CYP450 isoenzymes have been described in several fish species (Nelson, 2003, Siroka and Drastichova, 2004, Goldstone et al., 2009, Jonsson et al., 2010). The characterization of CYP1A gene multiplicity in fish (Råbergh et al., 2000) represents an ongoing area of research. Attempts were made to find a CYP1A2 form in rainbow trout (Oncorhynchus mykiss) and other fish species, and a possible CYP1A2 ortholog was found (Berndtson and Chen, 1994, Buhler and Wang-Buhler, 1998). The amino-acid sequence for trout CYP1A1 shows similarities to mammalian CYP1A1 and CYP1A2. There is a 57–59% sequence similarity between the trout CYP1A1 and the mammalian representatives of CYP1A1, and a 51–53% similarity between the trout and various mammalian CYP1A2 forms.
Materials and methods
Introduction Residues and metabolites of pharmaceuticals that reach aquatic systems can exert adverse effects on non-target organisms (Kasprzyk-Hordern et al., 2008, Brausch and Rand, 2011, Verlicchi et al., 2012, Baumann et al., 2013). Clotrimazole (CLO), bis-phenyl-2-chlorophenyl-1-imidazolyl methane, is an antifungal agent widely used in human and veterinary medicine for treating fungal, dermatophyte and yeast infections. In the Czech Republic, CLO prescriptions for the treatment of dermatological and genitourinary problems amounted to 3.94 tons in 2013 (SUKL). Clotrimazole is presumably introduced into the aquatic environment mainly through domestic (Thomas and Hilton, 2004, Peschka et al., 2007) and hospital wastewater discharge (Escher et al., 2011, Frédéric and Yves, 2014). It is expected in numerous aquatic environments because of its relative resistance to hydrolysis (at pH 7, its half-life at 4 °C and 25 °C is 242 days and 20 days, respectively) and photolysis (half-life is 3–310 days, depending on sunlight conditions) (OSPAR, 2013). Biotransformation and adsorption to particles are the main processes for its elimination in wastewater treatment plants (WWTPs’) (Peng et al., 2012), and it has been detected in river water at 6–71 ng L−1 (Thomas and Hilton, 2004, Roberts and Thomas, 2006, Huang et al., 2010) and in wastewater at 0.6–111 ng L−1 (Huang et al., 2010, Loos et al., 2013). The Guangzhou (China) WWTP influent, containing between 256 and 1834 ng L−1, had its highest mass load inflow in the winter and its lowest in the fall, ranging between 0.0028 and 0.28 mg person−1 d−1 (Peng et al., 2012). Clotrimazole has recently attracted the interest of the research community as a contaminant of concern (Gyllenhammar et al., 2009, Shi et al., 2012) for its diverse toxicity to aquatic organisms [PNEC = 1 μg L−1 (Peng et al., 2012) to 0.05 pg L−1 (Huang et al., 2010)].