Molecular Cloning and Expression of Two Novel Avian Cytochrome P450 1A Enzymes Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Abstract

Transcriptional regulation by the aryl hydrocarbon receptor, for which the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent ligand, leads in mammalian liver to the induction of genes for two distinct cytochrome P450 (CYP)1A enzymes, CYP1A1 and -1A2. Fish seem to have only one CYP1A enzyme. CYP1A enzymes have been regarded as injurious largely because of their ability to activate chemical carcinogens. We report here the cloning and sequencing of cDNAs for two catalytically distinct TCDD-induced CYP enzymes in chick embryo liver. One mediates classic CYP1A1 activities. The other has some -1A2-like activities and is also responsible for TCDD-induced arachidonic acid epoxygenation, a much more conspicuous effect in liver of chicks than of mammalian species. Amino acid sequence analysis shows that although each chick enzyme can be classified in the CYP1A family, both are more like CYP1A1 than -1A2, and neither can be said to be directly orthologous to CYP1A1 or -1A2. Phylogenetic analysis shows that the two chick enzymes form a separate branch in the CYP1A family tree distinct from mammalian CYP1A1 and -1A2 and from fish CYP1A enzymes. The findings suggest that CYP1A progenitors split into two CYP enzymes with some parallel functions independently in two evolutionary lines, evidence for convergent evolution in the CYP1A family. Northern analysis shows that the chick enzymes have a different tissue distribution from CYP1A1 and -1A2. Polymerase chain reaction and in situ hybridization data show that both chick enzymes are expressed in response to TCDD even before organ morphogenesis. The findings further suggest that beyond their role in activating carcinogens, CYP1A enzymes have conferred evolutionary and developmental advantages, perhaps as defenses in maintaining homeostatic responses to toxic chemicals. Activation of the aryl hydrocarbon (Ah)1 receptor by toxic polyhalogenated aromatic hydrocarbons like TCDD leads to the transcriptional induction of genes in the CYP1 family and to a toxicity syndrome that includes immunologic, hormonal, and cardiac dysfunction and tumor promotion (1, 2). There are large unexplained species differences in sensitivity to TCDD toxicity that cannot be attributed to differences in Ah receptor concentration or binding affinity and seem to involve factors downstream of the Ah receptor (1, 2). Ligand activation of the Ah receptor in mammalian liver induces expression of CYP1A1 and -1A2 and the more recently discovered CYP1B1 (3). These enzymes exhibit differences in substrate specificity, regulation, and tissue distribution. Because single amino acid differences can change CYP catalytic activity (4), species differences in CYP1 structure and function may contribute to differences in sensitivity to TCDD toxicity. The chicken and chick embryo close to hatching are among the more sensitive species to TCDD toxicity. They have long served as models for the study of Ah receptor-mediated toxicity and changes in heme and hemoprotein synthesis (2), but the chick TCD...

Identification of the Aryl Hydrocarbon Receptor Target Gene TiPARP as a Mediator of Suppression of Hepatic Gluconeogenesis by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and of Nicotinamide as a Corrective Agent for This Effect

“…1B). Concurrently, TCDD increased expression of major AHR target genes, CYP1A4 and CYP1A5, chick orthologs of mammalian CYP1A1 and 1A2, respectively (25). Effects in ovo were seen as early as 12 h after treatment (not shown) and were sustained for at least 96 h (Fig.…”

A Revised Evolutionary History of the CYP1A Subfamily: Gene Duplication, Gene Conversion, and Positive Selection

“…The fact that this orthology is not reflected in traditional phylogenetic analyses (e.g. Gilday et al, 1996a) may be attributed to concerted evolution via inter-paralog gene conversion.…”

“…Phylogenetic analyses of nucleic acid and amino acid sequences show that chicken CYP1A4 and CYP1A5 cluster together, separately from the mammalian CYP1A1/1A2 clade (Gilday et al, 1996b). This has been interpreted to mean that CYP1A4 and CYP1A5 resulted from an independent gene duplication that occurred subsequent to the divergence of the avian and mammalian lineages.…”

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

“…Previous work from this laboratory showed that TCDD-induced CYP1A4 is entirely responsible for the increased 7-ethoxyresorufin deethylase (EROD) activity and CYP1A5 for the increased epoxygenation of the endogenous membrane fatty acid, arachidonic acid (aa), in chick embryo (CE) liver microsomes [9][10][11]. Further, TCDD treatment suppressed formation of another P450 aa metabolite, 20-HETE, in both mammalian and CE liver microsomes [12,13].…”