Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 10 Apr 2010 J-STAGE Advance Published Date: 24 Apr 2010 [Note]

Toxicology

Identification and phylogenetic analysis of novel cytochrome P450 1A genes from species

Wageh Sobhy DARWISHa,b, Yusuke KAWAIa, Yoshinori IKENAKAa, Hideaki YAMAMOTOc,

Tarou MUROYAa and Mayumi ISHIZUKAa,*

aLaboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18, W9, Kita-ku, Sapporo 060-0818, Japan bFood Control Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519,

Egypt cMaruyama Zoo, Miyagaoka 3, Banchi 1, Chuo-ku, Sapporo 064-0959, Japan

*Correspondence: Mayumi Ishizuka

Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18, W9, Kita-ku, Sapporo 060-0818, Japan

Tel: +81-11-706-6949; Fax: +81-11-706-5105

E-mail address: [email protected] (M. Ishizuka)

Running Head: CYP1A SEQUENCES IN

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 fragments 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 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 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

Cytochrome P450 (CYP) enzymes are a large group of monooxygenase enzymes located in the endoplasmic reticulum or mitochondrial inner membrane. An important group of cytochrome

P450s are the phase I enzymes that insert an oxygen atom into molecules as the first step of excretion, conjugation and/or further transformation of xenobiotics [16]. Of particular interest, the CYP1A genes can be upregulated by exposure to aryl hydrocarbon receptor (AhR) agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), polycyclic aromatic hydrocarbons, phytochemicals, and endogenous substances [4, 8]. Recent researches reported the unique expression pattern and substrate specificity of CYP1A in some ungulate species [7, 10]. Since ungulates are herbivores, the markedly high CYP1A-dependent activity leads us to consider the fact that the differences in the CYP1A characters between species might be due to an evolutionary adaptation against numerous plant chemicals. However, the CYP1A subfamily has been extensively studied in only laboratory such as rats and subsequently in humans, but not in ungulates. The lack of CYP1A sequence information in the ungulate species constructs

“black box” in the mammalian CYP1A phylogenetic tree.

In this study, we chose typical ungulate species from each branch of the phylogenetic tree, including rare animal species. We newly report the identification of novel ungulate CYP1A genes from the livers of elephant (Elephas maximus indicus), hippopotamus (Hippopotamus amphibius), tapir (Tapirus indicus) and deer (Cervus nippon yesoensis). To our knowledge, this is the first report to present the molecular cloning of CYP1A genes in wild ungulate species. This report also establishes a molecular phylogeny that covers the CYP1A genes of different ungulates.

Samples from the elephant and tapir were gifted from the Maruyama Zoo (Sapporo, Hokkaido,

Japan) and were received directly after the death of the animals. Ezo shika deer were hunted

from wild life (Hokkaido, Japan) in November 2003. Hippopotamus samples were collected in

Zambia in October 2007 and samples entered Japan after agreement of the specific authorities in

Zambia and Japan. All samples were adult females. The liver samples were frozen in liquid nitrogen immediately after dissection and stored at -80ºC until use. Total RNA was prepared from each liver sample by the single-step method [2], using TRI reagent from Sigma Chemical

Co. (St. Louis, MO, USA). Degenerate primers, sense (5´-TCT TTG GRG CWG GNT TTG

ACA C-3´) and anti-sense (5´-TGG TTR AYC TGC CAC TGG TT-3´), were both positioned upstream from the well-described cysteine-containing heme-binding site and used to amplify fragments of CYP1A cDNAs, the binding site itself was not chosen for a primer region to increase the specificity of CYP1A primers [12]. This region is regarded as an unconverted region of CYP1A1 and CYP1A2 in and is thought to be the appropriate region for analyses of the CYP1A1 sequence. The PCR reaction was started with a single 4 min cycle at 94°C, followed by 40 cycles of a 15 sec denaturation at 94°C, a 45 sec annealing at 54°C and a 1 min extension at 72°C. The nucleotide sequences were determined by an automated DNA Sequencer

(ABI Prism 310 genetic analyzer). The cDNA and amino acid sequences of the examined ungulates were aligned with those of other mammals using the ClustalW 1.83 program.

Phylogenetic tree for amino acid sequences was constructed by the neighbor-joining method based on Molecular Evolutionary Genetic Analysis (MEGA) program [11]. Phylogenetic

Analysis of Maximum Likelihood (PAML) [14] was used to test the differences in selective pressure on mammalian CYP1A genes. We applied the F3x4 codon model and allowed for estimating synonymous and nonsynonymous substitution (dN/dS) rate.

In this study, we succeeded in identifying four novel CYP1A fragments from the liver of the examined wild ungulates. Each amplified cDNA fragment was 264 bp long, excluding the 5'-end

and 3'-end PCR primer regions and encoded a deduced 88 amino acid peptide. The fragments are positioned at a region corresponding to the 336-423 amino acids in rat CYP1A1 sequence. The accession numbers of the obtained nucleotide sequences in the Gene bank are AB540169

(Elephas maximus indicus), AB540170 (Hippopotamus amphibius), AB540648 (Tapirus indicus), AB540168 (Cervus nippon yesoensis), the deduced amino acid sequences of those fragments are shown in Figure 1. The homology percentage of the isolated fragments ranged between 76 and 97% with other mammalian CYP1As. Based on the homology percentage and the phylogenetic analysis with previously reported ungulates, three of the isolated fragments

(hippopotamus, tapir and deer) were identified to be CYP1A2, while the elephant fragment was classified as CYP1A1/2. The isolated fragment from the elephant showed the highest homology with cattle and horse 1A1s (86 and 85%, respectively, Table 1). The hippopotamus fragment showed high homology with the horse CYP1A2 (80%). The isolated fragment from the tapir was regarded as CYP1A2 based on the homology percentage reported in Table 1. For the deer fragment, the highest similarity was recorded to be with cattle CYP1A2 by (96%), suggesting that this clone is CYP1A2.

In the phylogenetic analysis, it was shown that within the cluster of ungulate CYP1As, the genes were assembled into groups of two subfamilies, CYP1A1 and CYP1A2, which resembled the topology of the full length CYP1A phylogeny [6], as seen in Figure 2. The elephant CYP1A1/2 made a separate cluster in between other ungulates CYP1A1 and CYP1A2. We considered the name of the elephant clone as CYP1A1/2, functional characteristics may help to decide lately.

Interestingly, the hippopotamus CYP1A2 gene made a separate branch in the CYP1A2 cluster, that unique separation may be reasonable, as this animal species spends part of its life in the water. Yasué et al. [15] analyzed sequences of a short interspersed repetitive element (SINE) and

reported that the hippopotamus had separately branched off from other ungulates. The position of the deer CYP1A2 confirmed our speculation that this clone belonged to the CYP1A2 isoform as shown in Figure 2. The tapir CYP1A2 located beside the horse CYP1A2 and this position agrees with the morphological classification, the tapir is related to other hooved mammals known as

Perissodactyla or odd-toed ungulates such as the horse and rhinoceros.

In figure 3, we tested the hypothesis that odd toed ungulates (Perissodactyla) had a close relationship with carnivores animals as mentioned by Nishihara et al. [9], who proposed an unexpected mammalian clade (Pegasoferae) for the Chiroptera, Perissodactyla, , and

Pholidota based on an extensive application of retroposon (L1) insertion analysis to the phylogenetic relationships among almost all mammalian orders. By analysis of dN/dS ratio among the identified CYP1As using PAML, only tapir CYP1A2 (odd-toed herbivorous animal) branch showed significantly higher dN/dS ratio than dog (carnivorous animal) as clear in Figure

3. This result indicates that odd toed ungulate CYP1A2s were exposed to different selection pressure after divergence from carnivorous animal CYP1A2s, which may reflect the effect of diet on this selection. This speculation agrees with the proposal of Nishihara et al. [9].

Odd-toed and cloven-hoofed members of either wild or domesticated ungulates are considered endangered species; however, these animals are exposed to environmental contaminants and naturally occurring chemicals during their lifetime [7, 10, 13]. In some cases, cancer in wild populations approaches or even exceeds that in humans, and the cause appear to be environmental pollution [5]. Some environmental pollutants require metabolic activation by phase I enzymes, especially CYP 1A, prior to reaction with DNA, to exert its genotoxic effects

[1]. For the conservation of such ungulates, it is important to know the molecular and

biochemical characteristics of the CYP1As in these animals, which may be associated with the toxicokinetics of such contaminants and the enzyme induction or deterioration [3, 12].

In general, although our obtained sequences were only partial, the constructed tree was estimated to bear reliability to draw conclusions on the novel gene positions in the phylogenetic tree. In conclusion, we successfully isolated partial genes of CYP1A isozymes from some wild ungulates.

We subsequently made a phylogenetic analysis of the novel CYP1As which reflected the possible position of the obtained CYP1As in the tree. Identification of CYP1As in wild and domestic ungulates that are exposed to high risks of environmental pollution is an introductory step which may provide us with new insights into the metabolic or toxicological 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 interest.

Acknowledgements

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).

References

1- Arlt, V.M., Stiborová, M., Henderson, C.J., Thiemann, M., Frei, E., Aimová, D., Singh, R.,

Gamboa da Costa, G., Schmitz, O.J., Farmer, P.B., Wolf, C.R. and Phillips, D.H. 2008.

Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with

detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice.

Carcinogenesis 29: 656-665.

2- Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid

guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159.

3- Hestermann, E.V., Stegeman, J.J. and Hahn, M.E. 2000. Relative contributions of affinity

and intrinsic efficacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl. Pharmacol.

168: 160-172.

4- Kawajiri, K. and Fujii-Kuriyama, Y. 2007. Cytochrome P450 gene regulation and

physiological functions mediated by the aryl hydrocarbon receptor. Arch. Biochem. Biophys.

464: 207-212.

5- Letcher, R.J., Norstrom, R.J., Muir, D.C.G., Sandau, C.D., Koczanski, K., Michaud, R., De

Guise, S. and Béland, P. 2000. Methylsulfone polychlorinated biphenyl and 2,2-

bis(chlorophenyl)-1,1-dichloroethylene metabolites in beluga whale (Delphinapterus leucas)

from the St. Lawrence River Estuary and Western Hudson Bay, Canada. Environ. Toxicol.

Chem. 19: 1378–1388..

6- Morrison, H.G., Weil, E.J., Karchner, S.I., Sogin, M.L. and Stegeman, J.J. 1998. Molecular

cloning of CYP1A from the estuarine fish Fundulus heteroclitus and phylogenetic analysis of

CYP1 genes: update with new sequences. Comp. Biochem. Physiol. C 121: 231-240.

7- Nebbia, C., Dacasto, M., Rossetto Giaccherino, A., Giuliano Albo, A. and Carletti, M. 2003.

Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse

and in other agricultural and laboratory species. Vet. J. 165: 53-64.

8- Nelson, 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. and Nebert, D.W.

1996. P450 superfamily: update on new sequences, gene mapping, accession numbers and

nomenclature. Pharmacogenetics 6: 1-42.

9- Nishihara, H., Hasegawa, M. and Okada, N. 2006. Pegasoferae, an unexpected mammalian

clade revealed by tracking ancient retroposon insertions. Proc. Natl. Acad. Sci. USA. 103:

9929-9934.

10- Sivapathasundaram, S., Magnisali, P., Coldham, N.G., Howells, L.C., Sauer, M.J. and

Ioannides, C. 2003. Cytochrome P450 expression and testosterone metabolism in the liver of

deer. Toxicology 187: 49-65.

11- Tamura, K., Dudley, J., Nei, M. and Kumar, S. 2007. MEGA4: Molecular Evolutionary

Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599.

12- Teramitsu, I., Yamamoto, Y., Chiba, I., Iwata, H., Tanabe, S., Fujise, Y., Kazusaka, A.,

Akahori, F. and Fujita, S. 2000. Identification of novel cytochrome P450 1A genes from five

marine species. Aquat. Toxicol. 51: 145-153.

13- Teraoka, H., Dong, W., Tsujimoto, Y., Iwasa, H., Endoh, D., Ueno, N., Stegeman, J.J.,

Peterson, R.E. and Hiraga, T. 2003. Induction of cytochrome P450 1A is required for

circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.Biochem.

Biophys. Res. Commun. 304: 223-228.

14- Yang, Z. 1997. PAML: a program package for phylogenetic analysis by maximum

likelihood. Comput. Appl. Biosci. 13: 555-556.

15- Yasué, M., Quinn, L.J. and Cresswell, W. 2003. Multiple effects of weather on the starvation

and predation risk trade-off in choice of feeding location in Redshanks. Funct. Ecol. 17: 727-

736.

16- Zhang, X., Moore, J.N., Newsted, J.L., Hecker, M., Zwiernik, M.J., Jones, P.D., Bursian, S.J.

and Giesy, J.P. 2009. Sequencing and characterization of mixed function monooxygenase

genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like

compounds. Toxicol. Appl. Pharmacol. 234: 306-313.

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%

FIGURE LEGENDS

Figure 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).

Figure 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.

Figure 3. Phylogenetic Analysis of Maximum Likelihood (PAML) among mammalian CYP1As.

Phylogenetic Analysis of Maximum Likelihood (PAML) was used to test the differences in selective pressure on mammalian CYP1A genes. We applied the F3x4 codon model and allowed for estimating synonymous and nonsynonymous substitution rate (dN/dS) ratio.

We used 7 vertebrate cDNA sequences, our four novel sequences, dog sequence for comparing with odd toed ungulates and zebrafish (Danio rerio) (NP_571954) and killifish (Fundulus heteroclitus) (AF026800) CYP1As were used as the out-group species.

Figure 1 Deer CYP1A2 99

Cattle CYP1A2 59

Tapir CYP1A2

46 99 Horse CYP1A2

Dog CYP1A2 47

61 Ribbon seal CYP1A2

99 Hippopotamus CYP1A2

Cattle CYP1A1

Horse CYP1A1 67

64 Ribbon seal CYP1A1

41 Dog CYP1A1

Elephant CYP1A1

Zebrafish CYP1A

100 Killifish CYP1A

0.02 Figure 2 Killifish CYP1A

Zebrafish CYP1A

Elephant CYP1A1

Deer CYP1A2

Hippopotamus CYP1A2

dN/dS=0.668 Tapir CYP1A2

Dog CYP1A2 dN/dS=0.112

Figure 3