Molecular Cloning and Expression of Two Novel Avian Cytochrome P450 1A Enzymes Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin
1996; Elsevier BV; Volume: 271; Issue: 51 Linguagem: Inglês
10.1074/jbc.271.51.33054
ISSN1083-351X
AutoresDeidre Gilday, Maureen Gannon, Katherine E. Yutzey, David M. Bader, Arleen B. Rifkind,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoTranscriptional 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. 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. INTRODUCTIONActivation of the aryl hydrocarbon (Ah) 1The abbreviations used are: Aharyl hydrocarbonAAarachidonic acidAHHaryl hydrocarbon hydroxylaseCYPcytochrome P450PCRpolymerase chain reactionRACErapid amplification of cDNA endsRTreverse transcriptaseTCDD2,3,7,8-tetrachlorodibenzo-p-dioxinUTRuntranslated regionbpbase pair(s)kbkilobase(s). 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 (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar). 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 (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar). Ligand activation of the Ah receptor in mammalian liver induces expression of CYP1A1 and −1A2 and the more recently discovered CYP1B1 (3Sutter T.R. Tang Y.M. Hayes C.L. Wo Y.-Y.P. Jabs E.W. Li X. Yin H. Cody C.W. Greenlee W.F. J. Biol. Chem. 1994; 269: 13092-13099Abstract Full Text PDF PubMed Google Scholar). These enzymes exhibit differences in substrate specificity, regulation, and tissue distribution. Because single amino acid differences can change CYP catalytic activity (4Lindberg R.L.P. Negishi M. Nature. 1989; 339: 632-634Crossref PubMed Scopus (366) Google Scholar), 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 (2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar), but the chick TCDD-induced CYP enzymes have only been partially characterized. We have identified and purified two catalytically distinct TCDD-induced chick embryo liver CYP enzymes with nearly identical molecular weights (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar). One, designated TCDDAHH, was selectively active in classic CYP1A1-mediated mixed function oxidations, aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin deethylase. The other, TCDDAA, was active in epoxygenation of the endogenous fatty acid arachidonic acid (AA) and lacked AHH or 7-ethoxyresorufin deethylase activity. As TCDDAA was also preferentially active in some CYP1A2-selective activities, i.e. uroporphyrinogen decarboxylation 2Sinclair, P. R., Gorman, N., Walton, H. S., Sinclair, J. F., Bentivegna, C. S., Hamilton, J. W., Lee, C., and Rifkind, A. B. (1994) Proceedings of the 10th International Conference on Microsomes and Drug Oxidations, p. 533, University of Toronto, Toronto. and estradiol 2-hydroxylation,3 and shared immunologic epitope(s) with rat CYP1A2 (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar), it seemed that TCDDAA and TCDDAHH might be orthologous to mammalian CYP1A2 and −1A1, respectively.RESULTS AND DISCUSSIONImmunoscreening identified three partial clones as TCDDAA and two as TCDDAHH (Fig. 1). Rescreening of the library (6Strauss W.J. Ausubel F. Brent R. Kingston D. Moore J.G. Seidman J. Smith S. Struhl K. Current Protocols in Molecular Biology. Vol. 1. John Wiley & Sons, Inc., NY1994: 6.3.1-6.3.3Google Scholar) with a 812-bp probe (full clone 8) and a 265-bp EcoRI/SphI fragment from clone 4 produced two 1.9-kb clones (clones 10 and 2B). Both contained the N-terminal amino acid sequence found for the purified TCDDAA protein (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar). The full coding sequence of TCDDAHH was obtained by RT-PCR and 5′-RACE. It contained the N-terminal amino acids found for the purified protein (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar) plus the initial methionine.The TCDDAA cDNA (Fig. 2) contained a 5′-UTR of 147 bp, an open reading frame of 1584 bp coding for 528 amino acids, and a 3′-UTR of 107 bp. The TCDDAHH cDNA (Fig. 3) contained 71 bp of 5′-UTR sequence, 1590 bp coding for 530 amino acids, and a 3′-UTR of 425 bp. Both enzymes contained the characteristic heme binding motif (15Black S.D. Coon M.J. Meister A. Advances in Enzymology. vol. 60. John Wiley & Sons, Inc., NY1987: 35-87Google Scholar) underlined in Fig. 2, Fig. 3. Their distributions of basic, acidic, and hydrophobic amino acids were consistent with the distribution in other CYP enzymes (15Black S.D. Coon M.J. Meister A. Advances in Enzymology. vol. 60. John Wiley & Sons, Inc., NY1987: 35-87Google Scholar) (64, 63, and 182, respectively, for TCDDAA and 60, 56, and 188 for TCDDAHH). The N-terminal amino acid sequences for both contained the typical hydrophobic signal sequence that anchors P450 in the endoplasmic reticulum membrane, followed by a halt-transfer signal containing several positively charged amino acids (15Black S.D. Coon M.J. Meister A. Advances in Enzymology. vol. 60. John Wiley & Sons, Inc., NY1987: 35-87Google Scholar). The N-terminal sequences extending to the halt-transfer signals were longer for the chick than for the mammalian or fish CYP1 enzymes, suggesting that the chick enzymes have a longer segment inserted in the membrane or a part that is free in the lumen of the endoplasmic reticulum. The two cDNAs have dissimilar 3′- and 5′-UTRs and 80% DNA sequence and 78% amino acid identities in their coding regions. A partial sequence reported by Murti et al. (16Murti J.R. Adiga P.R. Padmanaban G. Biochem. Biophys. Res. Commun. 1991; 175: 928-935Crossref PubMed Scopus (13) Google Scholar) resembles TCDDAA but lacks the canonical heme binding region and several other CYP1A consensus sites.Fig. 3cDNA and predicted amino acid sequence for chick TCDDAHH. The same features are highlighted as described in the legend to Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TCDDAA was found to have several potential secondary modification sites not present in TCDDAHH or the other CYP1 proteins listed in Table I. These include an N-linked glycosylation site (NFSI, amino acids 153-156 (all numbering is based on the sequence for TCDDAA in Fig. 4)), a cAMP/cGMP protein kinase phosphorylation site (KRFS, amino acids 520-523), a tyrosine kinase phosphorylation site (KQSDPYRY, amino acids 190-198), a protein kinase C phosphorylation site (SMK, amino acids 523-525), three casein II phosphorylation sites (SVLE, amino acids 60-63; TAVE, amino acids 273-276; and SIQD, amino acids 491-494), and an amidation site (DGKK, amino acids 494-497). TCDDAHH had a protein kinase C phosphorylation site (SFK, amino acids 435-437) and a casein II phosphorylation site (SRTE, amino acids 450-453) not present in TCDDAA. These findings suggest that TCDDAA and to a lesser extent TCDDAHH have the potential to undergo several posttranslational modifications distinct from each other. Protein kinase C and casein II phosphorylation sites, amidation sites, and N-myristylation sites were also found that were common to both TCDDAA and TCDDAHH. Some but not all of those were also present in other CYP1 enzymes. Both chick enzymes, but not the mammalian or fish CYP1A enzymes, also contained a 10-amino acid sequence identified as a prokaryotic membrane lipoprotein lipid attachment site (IAASPTASSSC, amino acids 156-166).Table IPercent amino acid identities between TCDDAA and TCDDAHH and other CYP enzymesPercent identityTCDDAATCDDAHHMammalian CYP1A1Human6159Mouse6359Hamster6058Rabbit6159Rat6260Guinea pig6158Mammalian CYP1A2Human6155Mouse5753Hamster5753Rabbit5853Rat5753Guinea pig5956Fish CYP1APlaice5553Scup5855Toadfish5555Trout5655Mammalian CYP1BHuman3433Rat3534Mammalian CYP2Rat 2A12527Rat 2B12829Rabbit 2C32525Rat 2E12526ChickenCYP2H12527CYP2H22527CYP172928 Open table in a new tab Fig. 4Multiple sequence alignments for TCDDAA and TCDDAHH, human, rat, and rabbit CYP1A1 and −1A2, and two fish CYP1A enzymes. Amino acids common to six or more sequences are shaded; TCDDAA numbering is used as the reference.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of the amino acid sequences for the chick enzymes and other CYP enzymes (Fig. 4 and Table I) showed identities of both chick enzymes to CYP1A1 ranging from 58-63%, to CYP1A2 ranging from 53-61%, and to fish CYP1A enzymes ranging from 53-58%. Those values meet the criteria for inclusion in the CYP1A family (46% or greater similarity for amino acid sequences of different species to be included in the same family) (17Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2633) Google Scholar). However, as shown in Table I, both chick enzymes were more similar to CYP1A1 than CYP1A2. Moreover, even though TCDDAA is enzymatically and immunologically more like −1A2 and TCDDAHH is enzymatically more like CYP1A1, the amino acid sequence of TCDDAA is more like that of CYP1A1 than is the sequence of TCDDAHH. Thus, neither TCDDAHH nor TCDDAA can be said to be directly orthologous to CYP1A1 or −1A2.There is other evidence for differences between chick TCDDAA and TCDDAHH and mammalian CYP1A2 and −1A1. 1) Whereas CYP1A2 and −1A1 iron are found in high and low spin states, respectively, both TCDDAA and TCDDAHH are low spin (18Nakai K. Ward A.M. Gannon M. Rifkind A.B. J. Biol. Chem. 1992; 267: 19503-19512Abstract Full Text PDF PubMed Google Scholar). 2) Furafylline, at concentrations selectively inhibitory for human and rat CYP1A2 (19Sesardic D. Boobis A.R. Murray B.P. Murray S. Segura J. De la Torre R. Davies D.S. Br. J. Clin. Pharmacol. 1990; 29: 651-663Crossref PubMed Scopus (285) Google Scholar), did not inhibit chick liver TCDD-induced arachidonic acid metabolism. 5C. A. Lee, K. Kunze, and A. B. Rifkind, unpublished observations. 3) Isosafrole, a selective CYP1A2 inducer (20Ryan D.E. Thomas P.E. Levin W. J. Biol. Chem. 1980; 255: 7941-7955Abstract Full Text PDF PubMed Google Scholar), did not increase TCDDAA or TCDDAHH in chick embryo liver. 6D. Gilday and A. B. Rifkind, unpublished observations. 4) CYP1A2 has been found only in liver (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar), but TCDDAA is also present in kidney. 5) The amino acid sequence of TCDDAHH is less like that of CYP1A1 than is the sequence of TCDDAA. 6) TCDDAHH is expressed in myocardium, 3K. Hirada, C. A. Lee, and A. B. Rifkind, unpublished observations. whereas fish CYP1A was found in endocardium and not in myocardium (22Morrison H.G. Oleksiak M.F. Cornell N.W. Sogin M.L. Stegeman J.J. Biochem. J. 1995; 308: 97-104Crossref PubMed Scopus (100) Google Scholar). For these reasons, and because one of the two trout enzymes has been designated "CYP1A3" (17Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2633) Google Scholar), we suggest naming chicken TCDDAHH, CYP1A4 and TCDDAA, 1A5.Phylogenetic analysis (Fig. 5) shows that neither chick enzyme clustered with the mammalian CYP1A1 or −1A2 sequences or with the fish 1A sequences. Rather, the two chick enzymes formed a separate branch between the fish and mammalian CYP1A sequences. Identification of the chick sequences, therefore, adds a new branch to the CYP1A phylogenetic tree (22Morrison H.G. Oleksiak M.F. Cornell N.W. Sogin M.L. Stegeman J.J. Biochem. J. 1995; 308: 97-104Crossref PubMed Scopus (100) Google Scholar) and demonstrates that the chick CYP1A enzymes are a separate lineage.Fig. 5Phylogenetic tree for the CYP1A family enzymes. The scale represents the number of amino acid residue substitutions from the base of the tree to the furthest branch. The same relationships were found when parsimony analysis was done using Phylogenetic Analysis Using Parsimony (D. L. Swofford, Version 3.0 s).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Data from Morrison et al. (22Morrison H.G. Oleksiak M.F. Cornell N.W. Sogin M.L. Stegeman J.J. Biochem. J. 1995; 308: 97-104Crossref PubMed Scopus (100) Google Scholar) that fish have only one CYP1A enzyme (with the possible exception of trout, which have two nearly identical CYP1A enzymes (23Berndtson A.K. Chen T.T. Arch. Biochem. Biophys. 1994; 310: 187-195Crossref PubMed Scopus (140) Google Scholar)) indicate that CYP1A1 and −1A2 split after land animals diverged from fish about 400 million years ago (24Carroll R.L. Vertebrate Paleontology and Evolution. W. H. Freeman and Co., NY1988Google Scholar). Our data further imply that 1) the earliest land animals in the common ancient reptilian/mammal/bird line, did not contain a CYP1A2 or a TCDDAA precursor distinct from CYP1A1, and 2) mammalian CYP1A1 and −1A2 and chick TCDDAHH and TCDDAA each split after precursors of modern mammals branched from that line about 300 million years ago (24Carroll R.L. Vertebrate Paleontology and Evolution. W. H. Freeman and Co., NY1988Google Scholar). Thus, these findings advance the time of the split of mammalian −1A1 and −1A2 by about 100 million years. More precise timing will require data on CYP1A enzymes in earlier evolving mammals and other avian orders. The evidence that amino acid identities are lower for CYP1A1 and −1A2 (66%, mean for the six species compared in Table I) than for TCDDAA and TCDDAHH (78%) suggests that the two TCDD-induced mammalian enzymes are more divergent than the two chick enzymes.The amino acid sites that differ for CYP1A1 and −1A2 are principally contained in a sequence of about 127 amino acids (amino acids 197-323 in Fig. 4). That region contains 13 sites where fish and chick CYP1As are conserved with mammalian CYP1A1 but none with −1A2, suggesting that the ancestral CYP1A progenitors were more −1A1- than −1A2-like (see also Ref. 22Morrison H.G. Oleksiak M.F. Cornell N.W. Sogin M.L. Stegeman J.J. Biochem. J. 1995; 308: 97-104Crossref PubMed Scopus (100) Google Scholar). That is supported by evidence that fish CYP1A (21Stegeman J.J. Smolowitz R.M. Hahn M.E. Toxicol. Appl. Pharmacol. 1991; 110: 486-504Crossref PubMed Scopus (91) Google Scholar), mammalian CYP1A1 (25Dees J.H. Masters B.S.S. Muller-Eberhard U. Johnson E.F. Cancer Res. 1982; 42: 1423-1432PubMed Google Scholar) and TCDDAHH4 but not mammalian −1A2 or TCDDAA are all expressed in vascular endothelial cells as well as liver. The 127-amino acid region also includes 11 sites selectively shared by TCDDAA and −1A2 but only three by TCDDAHH and −1A2. One or more of the former may contribute to the −1A2-like activity of TCDDAA. TCDDAA, however, lacks the CYP1A2-specific immunologic epitope (amino acids 291-298 in Fig. 4) (26Edwards R.J. Murray B.P. Singleton A.M. Murray S. Davies D.S. Boobis A.R. Biochem. Pharmacol. 1993; 46: 213-220Crossref PubMed Scopus (37) Google Scholar). Thus, another region must account for the selective immunologic cross-reactivity of immunopurified anti-TCDDAA serum to TCDDAA and rat CYP1A2 (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar).On Northern analysis (Fig. 6A) mRNA from TCDD-treated but not control embryos contained single transcripts of approximately 1.9 kb for TCDDAA in liver and kidney but not heart and single transcripts of 2.3 kb for TCDDAHH in liver, kidney, and heart, consistent with the tissue-specific expression of the CYP enzyme proteins (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar). The findings demonstrate definitively that TCDD induces two distinct chick CYP1A genes. They also provide additional evidence for differences between the chick enzymes and CYP1A1 and −1A2 because −1A2 is expressed only in liver and is found constitutively as well as in the induced state.Fig. 6A, Northern analysis of TCDDAA, TCDDAHH, and actin mRNA in liver, heart, and kidney of 17-day-old chick embryos 16 h after treatment with 1 nmol of TCDD (+) or solvent alone (−) (0.005 ml of dioxane). Lanes 1 and 2, liver; Lanes 3 and 4, heart; Lanes 5 and 6, kidney (10 μg total RNA for each). B, RT-PCR analysis of TCDDAA and TCDDAHH expression. Amplification of TCDDAA (AA), TCDDAHH (AHH), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from livers of control (−) and TCDD-treated (+) embryos using gene specific primers. Top panel, livers of 17-day-old chick embryos. Lower three panels, whole embryos treated with TCDD at 24 h of development. The time (h) after TCDD treatment is shown at the top of each panel. TCDDAHH, upper middle panel; TCDDAA, lower middle panel; glyceraldehyde-3-phosphate dehydrogenase, lower panel. C, localization by in situ hybridization of TC-DDAHH and TCDDAA in 72-h embryos treatedwith TCDD at 24 h. Top left panel, control embryo probed with TCDDAHH antisense probe; top middle and bottom left panels, TCDD-treated embryo probed with TCDDAHH antisense probe; right panels, TCDD-treated embryo probed with TCDDAA antisense probe. g, gut endoderm; da, dorsal aorta; e, endocardium; md, mesonephric duct. Anterior, top; dorsal, left. Magnification: upper panels, ×25; lower panels, same areas at ×50.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PCR analysis performed on whole 28-72-h-old embryos treated with TCDD at 24 h (middle panels of Fig. 6B) and for comparison on livers of 17-day-old TCDD-treated embryos (top panel) amplified bands of the same size (400 bp for TCDDAHH and 200 bp for TCDDAA) in both the younger embryos and the embryos close to hatching. From the top panel it is also seen that liver glyceraldehyde-3-phosphate dehydrogenase was increased by TCDD treatment in livers of the 17-day-old chick embryos, as has been reported in human keratinocytes exposed to TCDD (27McNulty S.E. Toscano Jr., W.A. Biochem. Biophys. Res. Commun. 1995; 212: 165-171Crossref PubMed Scopus (39) Google Scholar). TCDDAHH (upper middle panel) was induced in 48-h-old embryos 24 h after TCDD treatment, and the induction was increased in the 72-h-old embryos. TCDDAA (lower middle panel) was expressed in the controls at the earliest time examined (28 h of development), suggesting that there is constitutive expression of this gene in the early embryo. The expression of TCDDAA was increased by TCDD in the 36-h-old embryos 12 h after treatment and further increased at 48-72 h of development. AHH induction has been reported in 5-day-old chick embryos (28Hamilton J.W. Denison M.S. Bloom S.E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3372-3376Crossref PubMed Scopus (103) Google Scholar). Our findings indicate that there is a functional Ah receptor in the developing chicken 3-4 days earlier than previously recognized.In situ hybridization studies in 72-h-old embryos 48 h after TCDD treatment (Fig. 6C) showed that mRNAs for both enzymes are expressed in the areas of gut epithelium that later develop into liver. Sense probes did not hybridize (data not shown). Both antisense probes were negative for the solvent-treated controls and positive for the TCDD-treated embryos. TCDDAHH but not TCDDAA was expressed in the dorsal aorta and other vascular endothelial cells and in endocardium. TCDDAA but not TCDDAHH was expressed in the mesonephric duct, which gives rise to the kidney. The localization of TCDDAHH in vascular endothelium further suggests that TCDD may have widespread effects via its expression in blood vessels.Our evidence that mammalian and chick CYP1A enzymes branched into two functionally analogous pairs of enzymes independently suggests that the split in both cases may have responded to similar evolutionary pressures. That, together with the evidence that both chick enzymes are expressed very early in development, suggests that CYP1A enzymes have functions beyond their harmful actions as activators of carcinogens, perhaps providing a defense against environmental pollutants. For example, with respect to known activities of these enzymes, increased estradiol-2-hydroxylation may help to maintain tissue estrogen levels. Increased arachidonic acid epoxygenase activity (which occurs for TCDDAA and some but not all CYP1A2 enzymes (29Rifkind A.B. Lee C. Chang T.K.H. Waxman D.J. Arch. Biochem. Biophys. 1995; 320: 380-389Crossref PubMed Scopus (212) Google Scholar)) may help to maintain calcium, fluid, and hormonal homeostasis (30Capdevila J.H. Falck J.R. Dishman E. Karara A. Methods Enzymol. 1990; 187: 385-394Crossref PubMed Scopus (89) Google Scholar). High activity of CYP1A1 for metabolism of growth-modulating retinoids has recently been shown (31Raner G.M. Vaz D.N. Coon M.J. Mol. Pharmacol. 1996; 49: 515-522PubMed Google Scholar).The common view of CYP1A1 and −1A2 as injurious has not succeeded in explaining the role of these enzymes in TCDD toxicity. Thus, the carcinogenic effect of TCDD does not involve metabolic activation of TCDD by CYP1A enzymes (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar) and there is little CYP1A induction in the liver of TCDD-treated guinea pig, the species most sensitive to TCDD toxicity (2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar). Although CYP1A enzymes may have some potentially carcinogenic effects (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar, 32Park J.-Y.K. Shigenaga M.K. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2322-2327Crossref PubMed Scopus (240) Google Scholar), our results suggest that investigation of their advantageous, possibly protective actions and of their effects on endogenous metabolism may prove productive. INTRODUCTIONActivation of the aryl hydrocarbon (Ah) 1The abbreviations used are: Aharyl hydrocarbonAAarachidonic acidAHHaryl hydrocarbon hydroxylaseCYPcytochrome P450PCRpolymerase chain reactionRACErapid amplification of cDNA endsRTreverse transcriptaseTCDD2,3,7,8-tetrachlorodibenzo-p-dioxinUTRuntranslated regionbpbase pair(s)kbkilobase(s). 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 (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar). 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 (1Okey A.B. Riddick D.S. Harper P.A. Trends Pharmacol. Sci. 1994; 15: 226-232Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar). Ligand activation of the Ah receptor in mammalian liver induces expression of CYP1A1 and −1A2 and the more recently discovered CYP1B1 (3Sutter T.R. Tang Y.M. Hayes C.L. Wo Y.-Y.P. Jabs E.W. Li X. Yin H. Cody C.W. Greenlee W.F. J. Biol. Chem. 1994; 269: 13092-13099Abstract Full Text PDF PubMed Google Scholar). These enzymes exhibit differences in substrate specificity, regulation, and tissue distribution. Because single amino acid differences can change CYP catalytic activity (4Lindberg R.L.P. Negishi M. Nature. 1989; 339: 632-634Crossref PubMed Scopus (366) Google Scholar), 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 (2Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2319) Google Scholar), but the chick TCDD-induced CYP enzymes have only been partially characterized. We have identified and purified two catalytically distinct TCDD-induced chick embryo liver CYP enzymes with nearly identical molecular weights (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar). One, designated TCDDAHH, was selectively active in classic CYP1A1-mediated mixed function oxidations, aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin deethylase. The other, TCDDAA, was active in epoxygenation of the endogenous fatty acid arachidonic acid (AA) and lacked AHH or 7-ethoxyresorufin deethylase activity. As TCDDAA was also preferentially active in some CYP1A2-selective activities, i.e. uroporphyrinogen decarboxylation 2Sinclair, P. R., Gorman, N., Walton, H. S., Sinclair, J. F., Bentivegna, C. S., Hamilton, J. W., Lee, C., and Rifkind, A. B. (1994) Proceedings of the 10th International Conference on Microsomes and Drug Oxidations, p. 533, University of Toronto, Toronto. and estradiol 2-hydroxylation,3 and shared immunologic epitope(s) with rat CYP1A2 (5Rifkind A.B. Kanetoshi A. Orlinick J. Capdevila J.H. Lee C. J. Biol. Chem. 1994; 269: 3387-3396Abstract Full Text PDF PubMed Google Scholar), it seemed that TCDDAA and TCDDAHH might be orthologous to mammalian CYP1A2 and −1A1, respectively.
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