Molecular characterization of cytochrome P450 1A and 3A and the effects of perfluorooctanoic acid on their mRNA levels in rare minnow (Gobiocypris rarus) gills
Introduction
Perfluorinated compounds (PFCs), such as perfluorooctanoic acid (PFOA), are remarkably stable compounds that do not undergo photolysis, hydrolysis, or biodegradation. Commercial use of PFCs for the past several decades has resulted in a broad distribution of stable precursors/metabolites in wildlife in terrestrial and aquatic environments (Giesy and Kannan, 2001, Prevedouros et al., 2006, Martin et al., 2004, Smithwick et al., 2005, Yeung et al., 2006, Dai et al., 2006). PFOA potentially produces hepatomegaly, induces hepatic peroxisomes, and alters endocrine function in rodents (O’Brien and Wallace, 2004, Seacat et al., 2002, Hanson et al., 2005, Lau et al., 2004). In teleosts, PFOA increases hepatic fatty acyl-CoA oxidase activity, increases oxidative damage, and affects the circulating sex steroid levels (Ankley et al., 2005, Oakes et al., 2004).
Cytochrome P450s (CYPs) are a large superfamily of heme-proteins that play key roles in the biotransformation of xenobiotics and in the synthesis and degradation of physiologically important endogenous substrates (Nelson et al., 1996, Guengerich, 1999). Alteration of the expression of CYPs markedly affects the potential risks and benefits of xenobiotics and, thus, is very important from a toxicological point of view (Williams et al., 1998). On the basis of sequence similarity, CYPs can be classified into various families and subfamilies (Nelson et al., 1993), including CYP1A, CYP3A, and CYP4A. Transcriptional expression of CYP1A and CYP3A are regulated via the aryl hydrocarbon receptor (AhR)-mediated pathway and the pregnane X receptor (PXR) pathway, respectively (Hahn, 2002, Kliewer et al., 2002). Ligand-activated AhR heterodimerizes with aryl hydrocarbon nuclear translocator (ARNT) and activates the transcription of target genes such as CYP1A through xenobiotic response elements (XREs) (Hahn, 2002). Upon activation, PXR, which is a member of the nuclear receptor superfamily, interacts with retinoid X receptor (RXR). This heterodimer binds to specific DNA sequences and regulates transcriptional expression of target genes, including CYP3A (Wang and LeCluyse, 2003, Mikamo et al., 2003).
PFOA is a potent inducer of hepatic CYPs in mice (Permadi et al., 1992). Recent studies using microarray analysis demonstrated that several CYP450 genes (e.g. CYP2B15 and CYP2J4) were induced by PFOA in rat liver (Guruge et al., 2006); however, information on the modulation of CYPs by PFOA in teleosts is relatively limited. Thus, the freshwater teleost rare minnow (Gobiocypris rarus) was used in the PFOA exposure experiments. Rare minnows are suitable for aquatic toxicity experiments due to their sensitivity to chemicals, small size, wide temperature range, ease of laboratory culture, and short embryonic development period (Qun-Fang et al., 2002, Zhong et al., 2005). We focused on the toxic effect of PFOA on the gills mainly because gills are the tissue involved in gas exchange and one of the primary tissues exposed directly to aquatic xenobiotic compounds. Here, in order to probe the xenobiotic compound response in rare minnow gills, CYP1A, and CYP3A cDNA was isolated, and the modulatory effects of PFOA on the CYP1A and CYP3A signaling pathways were further investigated by quantitative polymerase chain reaction (Q-PCR) method.
Section snippets
Fish and PFOA exposure experiments
PFOA (98%) was purchased from Acros Organics (Geel, Belgium). All rare minnows were obtained from a laboratory hatchery. Two hundred and forty mature males and females (about 9 months old with body weight of 1.4 ± 0.4 g and total length of 47.7 ± 3.6 mm) were randomly assigned to eight 20 L glass tanks (30 individuals per tank) and acclimated for a week. Fish were supplied with dechlorinated tap water under continuous flow-through conditions at 25 ± 2 °C and subjected to a photoperiod of 16:8 h
Pathological changes in gills following PFOA exposure
Several histopathological studies of PFOA exposure on rodents (Pastoor et al., 1987, Berthiaume and Wallace, 2002) revealed that the liver was the primary target organ for PFOA. Treatment with this compound initiated a characteristic sequence of morphological and biochemical changes in liver due to increased peroxisomes and beta-oxidation of peroxisomal fatty acid. High doses of PFOA resulted in multifocal coagulation and liquefaction necrosis in the liver (Son et al., 2008). Relatively few
Acknowledgements
This work was funded by the National Basic Research Program of China (2006CB403306) and the National Natural Science Foundation of China (20677060 and 20777074).
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