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Ecotoxicology

, Volume 24, Issue 7–8, pp 1574–1582 | Cite as

Gene cloning and expression analysis of AhR and CYP4 from Pinctada martensii after exposed to pyrene

  • Junqiao Du
  • Chenghong Liao
  • Hailong Zhou
  • Xiaoping DiaoEmail author
  • Yuhu Li
  • Pengfei Zheng
  • Fuqiang Wang
Article

Abstract

Pyrene, a typical polycyclic aromatic hydrocarbon, is a common pollutant in the marine environment. Polycyclic aromatic hydrocarbons initiate cellular detoxification in an exposed organism via the activation of the aryl hydrocarbon receptor (AhR). Subsequent metabolism of these xenobiotics is mainly by the cytochrome P450 enzymes of the phase I detoxification system. Full-length complementary DNA sequences from the pearl oyster Pinctada martensii (pm) encoding AhR and cytochrome P4 were cloned. The P. martensii AhR complementary DNA sequence constitutes an open reading frame that encodes for 848 amino acids. Sequence analysis indicated PmAhR showed high similarity with its homologues of other bivalve species. The cytochrome P(CYP)4 complementary DNA sequence of P. martensii constitutes an open reading frame that encodes for 489 amino acids. Quantitative real-time analysis detected both PmAhR and PmCYP4 messenger RNA expressions in the mantle, gill, hepatapancreas and adductor muscle of P. martensii exposed to pyrene. The highest transcript-band intensities of PmAhR and PmCYP4 were observed in the gill. Temporal expression of PmAhR and PmCYP4 messenger RNAs induction was observed in gills and increased between 3 and 5 days post exposure; then returned to control level. These results suggest that messenger RNAs of PmAhR and PmCYP4 in pearl oysters might be useful parameters for monitoring marine environment pyrene pollution.

Keywords

Pyrene cDNA cloning AhR CYP4 Pinctada martensii mRNA expression 

Notes

Acknowledgments

This study was supported by National Natural Sciences Foundation of China (31160126). The authors would like to acknowledge all other members at Dr. Xiaoping Diao Laboratory for their help with sampling and taking care of the pearl oysters.

Conflict of interest

The authors declare that there are no conflicts of interest for their work.

Supplementary material

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References

  1. Atchley WR, Fitch WM (1997) A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA 94:5172–5176CrossRefGoogle Scholar
  2. Bo J, Wu SJ, Li YH, Ren HL, Fan DQ, Chen FY, Wang KJ (2010) The effects of Benzo[a]Pyrene (BaP) exposure on the CYP1A1 mRNA and AhR2 mRNA expression of Red Seabream (Pagrus major). Acta Scientiarum Naturalium Universities Sunyatseni 49:93–97Google Scholar
  3. Boscolo Papo M, Maccatrozzo L, Bertotto D, Pascoli F, Negrato E, Poltronieri C, Binato G, Gallina A, Radaelli G (2014) Expression of CYP4 and GSTr genes in Venerupis philippinarum exposed to benzo(a)pyrene. Annals Anat 196:241–246CrossRefGoogle Scholar
  4. Butler RA, Kelley ML, Powell WH, Hahn ME, Van Beneden RJ (2001) An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding. Gene 278:223–234CrossRefGoogle Scholar
  5. Butler RA, Kelley ML, Olberding KE, Gardner GR, Van Beneden RJ (2004) Aryl hydrocarbon receptor (AhR)-independent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on softshell clam (Mya arenaria) reproductive tissue. Comp Biochem Physiol C 138:375–381Google Scholar
  6. Chaty S, Rodius F, Vasseur P (2004) A comparative study of the expression of CYP1A and CYP4 genes in aquatic invertebrate (freshwater mussel, Unio tumidus) and vertebrate (rainbow trout, Oncorhynchus mykiss). Aquat Toxicol 69:81–94CrossRefGoogle Scholar
  7. Danielson PB (2002) The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr Drug Metab 3:561–597CrossRefGoogle Scholar
  8. Denison MS, Heath-Pagliuso S (1998) The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull Environ Contam Toxicol 61:557–568CrossRefGoogle Scholar
  9. Denison MS, Pandini A, Nagy SR, Baldwin EP, Bonati L (2002) Ligand binding and activation of the Ah receptor. Chem Biol Interact 141:3–24CrossRefGoogle Scholar
  10. Dolwick KM, Swanson HI, Bradfield CA (1993) In vitro analysis of Ah receptor domains involved in ligand-activated DNA recognition. Proc Natl Acad Sci 90:8566–8570CrossRefGoogle Scholar
  11. Dong JY, Wang SG, Shang Z (2009) Water environmental health risk assessment of polycyclic aromatic hydrocarbons in the Lanzhou reach of the Yellow River. J Agro-Environ Sci 28:1892–1897Google Scholar
  12. Feng CL, Lei BL, Wang ZJ (2009) Preliminary ecological risk assessment of polycyclic aromatic hydrocarbons in main rivers of China. China Environ Sci 29:583–588Google Scholar
  13. Fukunaga BN, Hankinson O (1996) Identification of a novel domain in the aryl hydrocarbon receptor required for DNA binding. J Biol Chem 271:3743–3749CrossRefGoogle Scholar
  14. Fukunaga BN, Probst MR, Reisz-Porszasz S, Hankinson O (1995) Identification of functional domains of the aryl hydrocarbon receptor. J Biol Chem 270:29270–29278CrossRefGoogle Scholar
  15. Goksøyr A, Förlin L (1992) The cytochrome P-450 system in fish, aquatic toxicology and environmental monitoring. Aquat Toxicol 22:287–311CrossRefGoogle Scholar
  16. Hahn ME (1998) The aryl hydrocarbon receptor: a comparative perspective. Comp Biochem Physiol C 121:23–53Google Scholar
  17. Hankinson O (1995) The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 35:307–340CrossRefGoogle Scholar
  18. He H, Chen A, Davey R, Ivie G (2002) Molecular cloning and nucleotide sequence of a new P450 gene, CYP319A1, from the cattle tick, Boophilus microplus. Insect Biochem Mol Biol 32:303–309CrossRefGoogle Scholar
  19. Ikuta T, Eguchi H, Tachibana T, Yoneda Y, Kawajiri K (1998) Nuclear localization and export signals of the human aryl hydrocarbon receptor. J Biol Chem 273:2895–2904CrossRefGoogle Scholar
  20. Jain S, Dolwick KM, Schmidt JV, Bradfield CA (1994) Potent transactivation domains of the Ah receptor and the Ah receptor nuclear translocator map to their carboxyl termin. J Biol Chem 269:31518–31524Google Scholar
  21. Jørgensen A, Rasmussen LJ, Andersen O (2005) Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity. Biochem Biophys Res Commun 336:890–897CrossRefGoogle Scholar
  22. Kann S, Huang M-y, Estes C, Reichard JF, Sartor MA, Xia Y, Puga A (2005) Arsenite-induced aryl hydrocarbon receptor nuclear translocation results in additive induction of phase I genes and synergistic induction of phase II genes. Mol Pharmacol 68:336–346Google Scholar
  23. Kazlauskas A, Poellinger L, Pongratz I (1999) Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (aryl hydrocarbon) receptor. J Biol Chem 274:13519–13524CrossRefGoogle Scholar
  24. Kim E-Y, Hahn ME, Iwata H, Tanabe S, Miyazaki N (2002) cDNA cloning of an aryl hydrocarbon receptor from Baikal seals (Phoca sibirica). Marine Environ Res 54:285–289CrossRefGoogle Scholar
  25. Kim E-Y, Iwata H, Suda T, Tanabe S, Amano M, Miyazaki N, Petrov EA (2005) Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) expression in Baikal seal (Pusa sibirica) and association with 2,3,7,8-TCDD toxic equivalents and CYP1 expression levels. Comp Biochem Physiol C 141:281–291CrossRefGoogle Scholar
  26. Liu N, Zhang L (2004) CYP4AB1, CYP4AB2, and Gp-9 gene overexpression associated with workers of the red imported fire ant, Solenopsis invicta Buren. Gene 327:81–87CrossRefGoogle Scholar
  27. Liu N, Pan L, Miao J, Xu C, Zhang L (2010) Molecular cloning and sequence analysis and the response of a aryl hydrocarbon receptor homologue gene in the clam Ruditapes philippinarum exposed to benzo(a)pyrene. Comp Biochem Physiol C 152:279–287Google Scholar
  28. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefGoogle Scholar
  29. Meyer BK, Pray-Grant MG, Vanden Heuvel JP, Perdew GH (1998) Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Mol Cell Bio 18:978–988CrossRefGoogle Scholar
  30. Miao J, Pan L, Liu N, Xu C, Zhang L (2011) Molecular cloning of CYP4 and GSTpi homologues in the scallop Chlamys farreri and its expression in response to benzo[a]pyrene exposure. Marine Genomics 4:99–108CrossRefGoogle Scholar
  31. Miller HC, Mills GN, Bembo D, Macdonald JA, Evans CW (1999) Induction of cytochrome P4501A (CYP1A) in Trematomus bernacchii as an indicator of environmental pollution in Antarctica: assessment by quantitative RT-PCR. Aquat Toxicol 44:183–193CrossRefGoogle Scholar
  32. Nelson DR (1998) Metazoan cytochrome P450 evolution. Comp Biochem Physiol C 121:15–22Google Scholar
  33. Ohi H, Fujita Y, Miyao M, Saguchi K-i, Murayama N, Higuchi S (2003) Molecular cloning and expression analysis of the aryl hydrocarbon receptor of Xenopus laevis. Biochem Biophys Res Commun 307:595–599CrossRefGoogle Scholar
  34. Pan L, Liu N, Xu C, Miao J (2011) Identification of a novel P450 gene belonging to the CYP4 family in the clam Ruditapes philippinarum, and analysis of basal- and benzo(a)pyrene-induced mRNA expression levels in selected tissues. Environ Toxicol Pharmacol 32:390–398CrossRefGoogle Scholar
  35. Reddy JK, Rao MS (1986) Peroxisome proliterators and cancer-mechanisms and implications. Trends Pharmacol Sci 7:438–443CrossRefGoogle Scholar
  36. Rewitz K, Kjellerup C, Jørgensen A, Petersen C, Andersen O (2004) Identification of two Nereis virens (Annelida: Polychaeta) cytochromes P450 and induction by xenobiotics. Comp Biochem Physiol C 138:89–96CrossRefGoogle Scholar
  37. Rewitz KF, Styrishave B, Løbner-Olesen A, Andersen O (2006) Marine invertebrate cytochrome P450: emerging insights from vertebrate and insects analogies. Comp Biochem Physiol C 143:363–381Google Scholar
  38. Schmidt JV, Bradfield CA (1996) Ah receptor signaling pathways. Annu Rev Cell Dev Biol 12:55–89CrossRefGoogle Scholar
  39. Scott JG (1999) Cytochromes P450 and insecticide resistance. Insect Biochem Mol Biol 29:757–777CrossRefGoogle Scholar
  40. Scott JA, Collins FH, Feyereisen R (1994) Diversty of cytochrome P450 genes in the mosquito, Anopheles albimanus. Biochem Biophys Res Commun 205:1452–1459CrossRefGoogle Scholar
  41. Simpson AE (1997) The cytochrome P450 4 (CYP4) family. Gen Pharmacol 28:351–359CrossRefGoogle Scholar
  42. Snyder MJ (1998) Cytochrome P450 enzymes belonging to the CYP4 family from marine invertebrates. Biochem Biophys Res Commun 249:187–190CrossRefGoogle Scholar
  43. Swanson HI, Yang J (1996) Mapping the protein/DNA contact sites of the Ah receptor and Ah receptor nuclear translocator. J Biol Chem 271:31657–31665CrossRefGoogle Scholar
  44. Taysse L, Chambras C, Marionnet D, Bosgiraud C, Deschaux P (1998) Basal level and induction of cytochrome P450, EROD, UDPGT, and GST activities in carp (Cyprinus carpio) immune organs (spleen and head kidney). Bull Environ Contam Toxicol 60:300–305CrossRefGoogle Scholar
  45. Tian S, Pan L, Zhang H (2014) Identification of a CYP3A-like gene and CYPs mRNA expression modulation following exposure to benzo[a]pyrene in the bivalve mollusk Chlamys farreri. Marine Environ Res 94:7–15CrossRefGoogle Scholar
  46. Vrzal R, Stejskalova L, Monostory K, Maurel P, Bachleda P, Pavek P, Dvorak Z (2009) Dexamethasone controls aryl hydrocarbon receptor (AhR)-mediated CYP1A1 and CYP1A2 expression and activity in primary cultures of human hepatocytes. Chem Biol Interact 179:288–296CrossRefGoogle Scholar
  47. Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1:1–9CrossRefGoogle Scholar
  48. Whitelaw ML, Göttlicher M, Gustafsson J, Poellinger L (1993) Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor. EMBO J 12:4169–4179Google Scholar
  49. Wu YY, Wu QH, Luo H, Zhang HJ, Wu YH, Zhang RZ, Xu ZC (2013) Ecological risk assessment for polycyclic aromatic hydrocarbons in river sediments. Acta Scientiae Circumstantiae 33:544–556Google Scholar
  50. Yang P, Tanaka H, Kuwano E, Suzuki K (2008) A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause. J Insect Physiol 54:636–643CrossRefGoogle Scholar
  51. Zhou C, Li C, Zhang W, Jia X (2010) CYP4 gene cloning and expression level analysis of Perna viridis. J Trop Oceanogr 29:82–88Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Junqiao Du
    • 1
    • 3
    • 4
  • Chenghong Liao
    • 2
    • 3
  • Hailong Zhou
    • 2
    • 3
  • Xiaoping Diao
    • 2
    • 3
    Email author
  • Yuhu Li
    • 2
    • 3
  • Pengfei Zheng
    • 2
    • 3
  • Fuqiang Wang
    • 1
    • 3
  1. 1.College of Environment and Plant ProtectionHainan UniversityHaikouChina
  2. 2.College of AgricultureHainan UniversityHaikouChina
  3. 3.Haikou Key Laboratory of Environment ToxicologyHainan UniversityHaikouChina
  4. 4.Key Laboratory of Tropical Biological Resources, Ministry of EducationHainan UniversityHaikouChina

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