Abstract
Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.
Highlights
Cytochromes P450 (CYPs) comprise a large gene superfamily encoding a diverse group of heme-thiolate monooxygenase enzymes that catalyze the oxidation of organic substances [1,2,3]
The full-length croaker cytochrome P450 1A (CYP1A) cDNA consists of 2581 bp nucleotides, which contains a 59-untranslated region (59-UTR, 174 bp), an open reading frame (1563 bp) encoding a polypeptide of 521 amino acid residues with a predicted molecular weight of the protein of,59 kDa, and a 39-UTR (844 bp)
The six substrate recognition sites (SRSs) and a heme-binding region (HBR) were determined in croaker CYP1A cDNA based of the SRSs and HBR data reported by Gotoh [55] and Poulos et al [56], respectively
Summary
Cytochromes P450 (CYPs) comprise a large gene superfamily encoding a diverse group of heme-thiolate monooxygenase enzymes that catalyze the oxidation of organic substances [1,2,3]. In the CYPs superfamily, the CYP1 family enzymes are of broad interest because they play a major role in the biotransformation of a variety of endogenous substances such as lipids, steroids, and vitamins [1,2,4] and environmental toxicants, in particular halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs) [5,6]. Some regions of the trout CYP1A2 cDNA are identical to mammalian CYP1A1 but differ from CYP1A2 cDNA. These homology patterns suggest that a single CYP1A gene exists in teleost fishes [4,5]
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