NOTE Toxicology
Identification and Phylogenetic Analysis of Novel Cytochrome P450 1A Genes from Ungulate Species
Wageh Sobhy DARWISH1,2), Yusuke KAWAI1), Yoshinori IKENAKA1), Hideaki YAMAMOTO3), Tarou MUROYA1) and Mayumi ISHIZUKA1)*
1)Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18, W9, Kita-ku, Sapporo 060–0818, Japan, 2)Food Control Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt and 3)Maruyama Zoo, Miyagaoka 3, Banchi 1, Chuo-ku, Sapporo 064–0959, Japan
(Received 10 January 2010/Accepted 10 April 2010/Published online in J-STAGE 24 April 2010)
ABSTRACT. As part of an ongoing effort to understand the biological response of wild and domestic ungulates to different environmental pollutants such as dioxin-like compounds, cDNAs encoding for CYP1A1 and CYP1A2 were cloned and characterized. Four novel CYP1A cDNA fragments from the livers of four wild ungulates (elephant, hippopotamus, tapir and deer) were identified. Three frag- ments from hippopotamus, tapir and deer were classified as CYP1A2, and the other fragment from elephant was designated as CYP1A1/ 2. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 76 to 97% with other animal CYP1As. The phylogenetic analysis of these fragments showed that both elephant and hippopotamus CYP1As made separate branches, while tapir and deer CYP1As were located beside that of horse and cattle respectively in the phylogenetic tree. Analysis of dN/dS ratio among the identified CYP1As indicated that odd toed ungulate CYP1A2s were exposed to different selection pressure. KEY WORDS: CYP1A, sequence, ungulates. J. Vet. Med. Sci. 72(9): 1237–1241, 2010
Cytochrome P450 (CYP) enzymes are a large group of our knowledge, this is the first report to present the molecu- monooxygenase enzymes located in the endoplasmic reticu- lar cloning of CYP1A genes in wild ungulate species. This lum or mitochondrial inner membrane. An important group report also establishes a molecular phylogeny that covers of cytochrome P450s are the phase I enzymes that insert an the CYP1A genes of different ungulates. oxygen atom into molecules as the first step of excretion, Samples from the elephant and tapir were gifted from the conjugation and/or further transformation of xenobiotics Maruyama Zoo (Sapporo, Hokkaido, Japan) and were [16]. Of particular interest, the CYP1A genes can be upreg- received directly after the death of the animals. Ezo shika ulated by exposure to aryl hydrocarbon receptor (AhR) ago- deer were hunted from wild life (Hokkaido, Japan) in nists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8- November 2003. Hippopotamus samples were collected in TCDD), polycyclic aromatic hydrocarbons, phytochemi- Zambia in October 2007 and samples entered Japan after cals, and endogenous substances [4, 8]. Recent researches agreement of the specific authorities in Zambia and Japan. reported the unique expression pattern and substrate speci- All samples were adult females. The liver samples were fro- ficity of CYP1A in some ungulate species [7, 10]. Since zen in liquid nitrogen immediately after dissection and ungulates are herbivores, the markedly high CYP1A-depen- stored at –80C until use. Total RNA was prepared from dent activity leads us to consider the fact that the differences each liver sample by the single-step method [2], using TRI in the CYP1A characters between species might be due to an reagent from Sigma Chemical Co. (St. Louis, MO, U.S.A.). evolutionary adaptation against numerous plant chemicals. Degenerate primers, sense (5’-TCT TTG GRG CWG GNT However, the CYP1A subfamily has been extensively stud- TTG ACA C-3’) and anti-sense (5’-TGG TTR AYC TGC ied in only laboratory animals such as rats and subsequently CAC TGG TT-3’), were both positioned upstream from the in humans, but not in ungulates. The lack of CYP1A well-described cysteine-containing heme-binding site and sequence information in the ungulate species constructs used to amplify fragments of CYP1A cDNAs, the binding “black box” in the mammalian CYP1A phylogenetic tree. site itself was not chosen for a primer region to increase the In this study, we chose typical ungulate species from each specificity of CYP1A primers [12]. This region is regarded branch of the phylogenetic tree, including rare animal spe- as an unconverted region of CYP1A1 and CYP1A2 in mam- cies. We newly report the identification of novel ungulate mals and is thought to be the appropriate region for analyses CYP1A genes from the livers of elephant (Elephas maximus of the CYP1A1 sequence. The PCR reaction was started indicus), hippopotamus (Hippopotamus amphibius), tapir with a single 4 min cycle at 94C, followed by 40 cycles of (Tapirus indicus) and deer (Cervus nippon yesoensis). To a 15 sec denaturation at 94C, a 45 sec annealing at 54C and a 1 min extension at 72C. The nucleotide sequences *CORRESPONDENCE TO: ISHIZUKA, M., Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate were determined by an automated DNA Sequencer (ABI School of Veterinary Medicine, Hokkaido University, N18, W9, Prism 310 genetic analyzer). The cDNA and amino acid Kita-ku, Sapporo 060–0818, Japan. sequences of the examined ungulates were aligned with e-mail : [email protected] those of other mammals using the ClustalW 1.83 program. 1238 W. S. DARWISH ET AL.
Phylogenetic tree for amino acid sequences was constructed fied to be CYP1A2, while the elephant fragment was by the neighbor-joining method based on Molecular Evolu- classified as CYP1A1/2. The isolated fragment from the tionary Genetic Analysis (MEGA) program [11]. Phyloge- elephant showed the highest homology with cattle and horse netic Analysis of Maximum Likelihood (PAML) [14] was 1A1s (86 and 85%, respectively, Table 1). The hippopota- used to test the differences in selective pressure on mamma- mus fragment showed high homology with the horse lian CYP1A genes. We applied the F3x4 codon model and CYP1A2 (80%). The isolated fragment from the tapir was allowed for estimating synonymous and nonsynonymous regarded as CYP1A2 based on the homology percentage substitution (dN/dS) rate. reported in Table 1. For the deer fragment, the highest sim- In this study, we succeeded in identifying four novel ilarity was recorded to be with cattle CYP1A2 by (96%), CYP1A fragments from the liver of the examined wild suggesting that this clone is CYP1A2. ungulates. Each amplified cDNA fragment was 264 bp In the phylogenetic analysis, it was shown that within the long, excluding the 5’-end and 3’-end PCR primer regions cluster of ungulate CYP1As, the genes were assembled into and encoded a deduced 88 amino acid peptide. The frag- groups of two subfamilies, CYP1A1 and CYP1A2, which ments are positioned at a region corresponding to the 336– resembled the topology of the full length CYP1A phylogeny 423 amino acids in rat CYP1A1 sequence. The accession [6], as seen in Fig. 2. The elephant CYP1A1/2 made a sep- numbers of the obtained nucleotide sequences in the Gene arate cluster in between other ungulates CYP1A1 and bank are AB540169 (Elephas maximus indicus), AB540170 CYP1A2. We considered the name of the elephant clone as (Hippopotamus amphibius), AB540648 (Tapirus indicus), CYP1A1/2, functional characteristics may help to decide AB540168 (Cervus nippon yesoensis), the deduced amino lately. Interestingly, the hippopotamus CYP1A2 gene made acid sequences of those fragments are shown in Fig. 1. The a separate branch in the CYP1A2 cluster, that unique sepa- homology percentage of the isolated fragments ranged ration may be reasonable, as this animal species spends part between 76 and 97% with other mammalian CYP1As. of its life in the water. Yasué et al. [15] analyzed sequences Based on the homology percentage and the phylogenetic of a short interspersed repetitive element (SINE) and analysis with previously reported ungulates, three of the iso- reported that the hippopotamus had separately branched off lated fragments (hippopotamus, tapir and deer) were identi- from other ungulates. The position of the deer CYP1A2
Fig. 1. Alignments of the deduced amino acid sequences of the novel identified CYP1As of different ungulates with other mammals and the rat. Asterisks indicate the regions conserved among all sequences in the alignment. The colons indicate that the strong groups are fully conserved. The predicted amino acid sequences are shown aligned with the rat (Rattus norvegicus) CYP1A1 (NP_036672) sequence of the corresponding region. Underlined is the well-described heme-binding region with the cysteine positioned in the center (arrow). CYP1A SEQUENCES IN UNGULATES 1239
Table 1. Homology of the deduced amino acid sequences from the identified CYP1As with other ungulate CYP1A genes Animal species Accession Elephant Hippopotamus Tapir Deer number CYP1A1 CYP1A2 CYP1A2 CYP1A2 Cattle CYP1A1 Xp_588298 86% 80% 83% 84% Horse CYP1A1 Xp_001493959 85% 79% 82% 81% Dog CYP1A1 XM_544773 84% 80% 76% 79% Ribbon Seal CYP1A1 BAB20377 84% 81% 81% 82% Cattle CYP1A2 Np_001092834 77% 77% 89% 96% Horse CYP1A2 Xp_001493936 83% 80% 97% 88% Dog CYP1A2 NP_001008720 84% 80% 85% 84% Ribbon Seal CYP1A2 BAB20378 81% 78% 89% 86%
Fig. 2. Phylogenetic analysis of mammalian deduced amino acid sequences of CYP1As. This is a rooted tree for amino acid sequences in the partial region constructed by the neighbor- joining method based on Molecular Evolutionary Genetic Analysis (MEGA) program. The numbers on the tree represent local bootstrap values from 100 replicates. Values less than 10 were removed. zebrafish (Danio rerio) (NP_571954) and killifish (Fundulus heteroclitus) (AF026800) CYP1As were used as the out-group species. 1240 W. S. DARWISH ET AL. confirmed our speculation that this clone belonged to the however, these animals are exposed to environmental con- CYP1A2 isoform as shown in Fig. 2. The tapir CYP1A2 taminants and naturally occurring chemicals during their located beside the horse CYP1A2 and this position agrees lifetime [7, 10, 13]. In some cases, cancer in wild popula- with the morphological classification, the tapir is related to tions approaches or even exceeds that in humans, and the other hooved mammals known as Perissodactyla or odd- cause appear to be environmental pollution [5]. Some envi- toed ungulates such as the horse and rhinoceros. ronmental pollutants require metabolic activation by phase I In Fig. 3, we tested the hypothesis that odd toed ungulates enzymes, especially CYP 1A, prior to reaction with DNA, to (Perissodactyla) had a close relationship with carnivores exert its genotoxic effects [1]. For the conservation of such animals as mentioned by Nishihara et al. [9], who proposed ungulates, it is important to know the molecular and bio- an unexpected mammalian clade (Pegasoferae) for the Chi- chemical characteristics of the CYP1As in these animals, roptera, Perissodactyla, Carnivora, and Pholidota based on which may be associated with the toxicokinetics of such an extensive application of retroposon (L1) insertion analy- contaminants and the enzyme induction or deterioration [3, sis to the phylogenetic relationships among almost all mam- 12]. malian orders. By analysis of dN/dS ratio among the In general, although our obtained sequences were only identified CYP1As using PAML, only tapir CYP1A2 (odd- partial, the constructed tree was estimated to bear reliability toed herbivorous animal) branch showed significantly to draw conclusions on the novel gene positions in the phy- higher dN/dS ratio than dog (carnivorous animal) as clear in logenetic tree. In conclusion, we successfully isolated par- Fig. 3. This result indicates that odd toed ungulate tial genes of CYP1A isozymes from some wild ungulates. CYP1A2s were exposed to different selection pressure after We subsequently made a phylogenetic analysis of the novel divergence from carnivorous animal CYP1A2s, which may CYP1As which reflected the possible position of the reflect the effect of diet on this selection. This speculation obtained CYP1As in the tree. Identification of CYP1As in agrees with the proposal of Nishihara et al. [9]. wild and domestic ungulates that are exposed to high risks Odd-toed and cloven-hoofed members of either wild or of environmental pollution is an introductory step which domesticated ungulates are considered endangered species; may provide us with new insights into the metabolic or tox- icological functions of CYP1As in these animals. Based on the results of this study, further approaches to clone and express the full-length sequence of ungulate CYP1As for complete characterization of these isoforms will be of inter- est.
ACKNOWLEDGMENT. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan awarded to M. Ishizuka (No. 19671001).
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