Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Aug;85(16):5769–5773. doi: 10.1073/pnas.85.16.5769

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

J Doehmer 1, S Dogra 1, T Friedberg 1, S Monier 1, M Adesnik 1, H Glatt 1, F Oesch 1
PMCID: PMC281846  PMID: 3137560

Abstract

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme.

Full text

PDF
5771

Images in this article

5772Image
on p.5772
5772Image
on p.5772
5772Image
on p.5772

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adesnik M., Atchison M. Genes for cytochrome P-450 and their regulation. CRC Crit Rev Biochem. 1986;19(3):247–305. doi: 10.3109/10409238609084657. [DOI] [PubMed] [Google Scholar]
  2. Battula N., Sagara J., Gelboin H. V. Expression of P1-450 and P3-450 DNA coding sequences as enzymatically active cytochromes P-450 in mammalian cells. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4073–4077. doi: 10.1073/pnas.84.12.4073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradley M. O., Bhuyan B., Francis M. C., Langenbach R., Peterson A., Huberman E. Mutagenesis by chemical agents in V79 chinese hamster cells: a review and analysis of the literature. A report of the Gene-Tox Program. Mutat Res. 1981 Sep;87(2):81–142. doi: 10.1016/0165-1110(81)90029-4. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  6. Friedberg T., Waxman D. J., Atchison M., Kumar A., Haaparanta T., Raphael C., Adesnik M. Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1. Biochemistry. 1986 Dec 2;25(24):7975–7983. doi: 10.1021/bi00372a028. [DOI] [PubMed] [Google Scholar]
  7. Fujii-Kuriyama Y., Mizukami Y., Kawajiri K., Sogawa K., Muramatsu M. Primary structure of a cytochrome P-450: coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 cDNA from rat liver. Proc Natl Acad Sci U S A. 1982 May;79(9):2793–2797. doi: 10.1073/pnas.79.9.2793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  9. Ishii K., Maeda K., Kamataki T., Kato R. Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450. Mutat Res. 1986 Jun;174(2):85–88. doi: 10.1016/0165-7992(86)90095-3. [DOI] [PubMed] [Google Scholar]
  10. Jefferson D. M., Clayton D. F., Darnell J. E., Jr, Reid L. M. Posttranscriptional modulation of gene expression in cultured rat hepatocytes. Mol Cell Biol. 1984 Sep;4(9):1929–1934. doi: 10.1128/mcb.4.9.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kimura S., Smith H. H., Hankinson O., Nebert D. W. Analysis of two benzo[a]pyrene-resistant mutants of the mouse hepatoma Hepa-1 P(1)450 gene via cDNA expression in yeast. EMBO J. 1987 Jul;6(7):1929–1933. doi: 10.1002/j.1460-2075.1987.tb02453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Law M. F., Byrne J. C., Howley P. M. A stable bovine papillomavirus hybrid plasmid that expresses a dominant selective trait. Mol Cell Biol. 1983 Nov;3(11):2110–2115. doi: 10.1128/mcb.3.11.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lubet R. A., Nims R. W., Mayer R. T., Cameron J. W., Schechtman L. M. Measurement of cytochrome P-450 dependent dealkylation of alkoxyphenoxazones in hepatic S9s and hepatocyte homogenates: effects of dicumarol. Mutat Res. 1985 Mar;142(3):127–131. doi: 10.1016/0165-7992(85)90052-1. [DOI] [PubMed] [Google Scholar]
  15. Marx J. L. The cytochrome P450's and their genes. Science. 1985 May 24;228(4702):975–976. doi: 10.1126/science.4001932. [DOI] [PubMed] [Google Scholar]
  16. Mizukami Y., Sogawa K., Suwa Y., Muramatsu M., Fujii-Kuriyama Y. Gene structure of a phenobarbital-inducible cytochrome P-450 in rat liver. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3958–3962. doi: 10.1073/pnas.80.13.3958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Montisano D. F., Hankinson O. Transfection by genomic DNA of cytochrome P1-450 enzymatic activity and inducibility. Mol Cell Biol. 1985 Apr;5(4):698–704. doi: 10.1128/mcb.5.4.698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  19. Nebert D. W., Adesnik M., Coon M. J., Estabrook R. W., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F., Kemper B., Levin W. The P450 gene superfamily: recommended nomenclature. DNA. 1987 Feb;6(1):1–11. doi: 10.1089/dna.1987.6.1. [DOI] [PubMed] [Google Scholar]
  20. Nebert D. W., Gelboin H. V. Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J Biol Chem. 1968 Dec 10;243(23):6242–6249. [PubMed] [Google Scholar]
  21. Nebert D. W., Gonzalez F. J. P450 genes: structure, evolution, and regulation. Annu Rev Biochem. 1987;56:945–993. doi: 10.1146/annurev.bi.56.070187.004501. [DOI] [PubMed] [Google Scholar]
  22. Pelkonen O., Nebert D. W. Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol Rev. 1982 Jun;34(2):189–222. [PubMed] [Google Scholar]
  23. Platt K. L., Utesch D., Gemperlein-Mertes I., Glatt H. R., Oesch F. Metabolizing systems in short-term in vitro tests for carcinogenicity. Food Chem Toxicol. 1986 Jun-Jul;24(6-7):721–729. doi: 10.1016/0278-6915(86)90171-7. [DOI] [PubMed] [Google Scholar]
  24. Rizzolo L. J., Finidori J., Gonzalez A., Arpin M., Ivanov I. E., Adesnik M., Sabatini D. D. Biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids. J Cell Biol. 1985 Oct;101(4):1351–1362. doi: 10.1083/jcb.101.4.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Russell W. C., Newman C., Williamson D. H. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature. 1975 Feb 6;253(5491):461–462. doi: 10.1038/253461a0. [DOI] [PubMed] [Google Scholar]
  26. Seglen P. O. Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion. Exp Cell Res. 1973 Dec;82(2):391–398. doi: 10.1016/0014-4827(73)90357-1. [DOI] [PubMed] [Google Scholar]
  27. Shimada T., Nakamura S., Imaoka S., Funae Y. Genotoxic and mutagenic activation of aflatoxin B1 by constitutive forms of cytochrome P-450 in rat liver microsomes. Toxicol Appl Pharmacol. 1987 Oct;91(1):13–21. doi: 10.1016/0041-008x(87)90189-x. [DOI] [PubMed] [Google Scholar]
  28. Shimizu T., Sogawa K., Fujii-Kuriyama Y., Takahashi M., Ogoma Y., Hatano M. Expression of cytochrome P-450d by Saccharomyces cerevisiae. FEBS Lett. 1986 Oct 27;207(2):217–221. doi: 10.1016/0014-5793(86)81491-0. [DOI] [PubMed] [Google Scholar]
  29. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  30. Stow N. D., Wilkie N. M. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J Gen Virol. 1976 Dec;33(3):447–458. doi: 10.1099/0022-1317-33-3-447. [DOI] [PubMed] [Google Scholar]
  31. Turchi G., Glatt H. R., Doerjer G., Oesch F. A benzo[a]pyrene metabolite-DNA adduct occurring in the activating cell but not in exogenous DNA. Mutat Res. 1987 Jan;190(1):31–34. doi: 10.1016/0165-7992(87)90078-9. [DOI] [PubMed] [Google Scholar]
  32. Waxman D. J., Walsh C. Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms. J Biol Chem. 1982 Sep 10;257(17):10446–10457. [PubMed] [Google Scholar]
  33. Wiebel F. J., Singh J., Schindler E., Summer K. H. UDP-glucuronosyl-, phenol sulfo-, and glutathione-S-transferase activities of mammalian cells in permanent culture. Toxicology. 1980;17(2):123–126. doi: 10.1016/0300-483x(80)90084-0. [DOI] [PubMed] [Google Scholar]
  34. Zuber M. X., Simpson E. R., Waterman M. R. Expression of bovine 17 alpha-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells. Science. 1986 Dec 5;234(4781):1258–1261. doi: 10.1126/science.3535074. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES