The Construction of Cell Cultures Genetically Engineered for Metabolic Competence towards Xenobiotics

  • Johannes Doehmer
Conference paper
Part of the NATO ASI Series book series (volume 90)


Analytical tools have always played an important role for the study and understanding the complex biotransformation process. It needs technology to develop analytical tools. In this sense, gene technology is the most recent technology being applied in the construction of in vitro system useful for metabolism studies of xenobiotics. This includes cloning of genes encoding xenobiotics metabolizing enzymes, construction of recombinant expression vectors, and genetically engineering of cell cultures.


Human Cytochrome Recombinant Vaccinia Virus Cytochrome P450 Gene Biological Endpoint Packaging Cell Line 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Adesnik M, Bar-Nun S, Maschio F, Zunich M, Lippman A, Bard E, (1981) Mechanism of induction of cytochrome P-450 by phénobarbital. J Biol Chem 256: 10340–10345PubMedGoogle Scholar
  2. Aoyama T, Gonzalez FJ, Gelboin HV, (1989) Mutagen activation by cDNA-expressed Pl–450, P3–450, and P450a. Mol Carcinog 1: 253–259PubMedCrossRefGoogle Scholar
  3. Aoyama T, Yamano S, Guzelian PS, Gelboin HV, Gonzalez FJ, (1990) Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin Bl. Proc Natl Acad Sci USA 87: 4790–4793PubMedCrossRefGoogle Scholar
  4. Barnes HJ, Arlotto MA, Waterman MR, (1991) Expression and enzymatic activity of recombinant cytochrome P450 17α- hydroxylase in Escherichia coli. Proc Natl Acad Sci USA 88: 5597–5601PubMedCrossRefGoogle Scholar
  5. Battula N., (1989) Transduction of cytochrome P3–450 by retroviruses: constitutive expression of enzymatically active microsomal hemoprotein in animal cells. J Biol Chem 264: 2991–2996PubMedGoogle Scholar
  6. Battula N, Sagara J, Gelboin HV, (1987) Expression of Pl–450 and P3–450 DNA coding sequences as enzymatically active cytochromes P-450 in mammalian cells. Proc Natl Acad Sci USA 84: 4073–4077PubMedCrossRefGoogle Scholar
  7. Battula N, Schut HAJ, Thorgeirsson SS, (1991) Cytochrome P450IA2 constitutively expressed from transduced DNA mediates metabolic activation and DNA-adduct formation of aromatic amine carcinogens in NIH3T3 cells. Mol Carcinog 4: 407–414PubMedCrossRefGoogle Scholar
  8. Black SM, Ellard S, Meehan RR, Parry JM, Adesnik M, Beggs JD, Wolf CR (1989) Carcinogenesis 10: 2139PubMedCrossRefGoogle Scholar
  9. Cullin C, Pompon D, (1988) Functional expression of mouse cytochrome P-450 PI and chimeric P-450 P3–1 in yeast saccharomyces cerevisiae. Gene 65: 203–217PubMedCrossRefGoogle Scholar
  10. Crespi CL, Langenbach R, Rudo K, Chen YT, Davies RL, (1989) Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line AHH-1, and use of the recombinant cell line in gene mutation assays. Carcinogenesis 10: 295–301PubMedCrossRefGoogle Scholar
  11. Crespi CL, Penman BW, Leakey JAE, Arlotto MP, Stark A, Parkinson A, Turner T, Steimel DT, Rudo K, Davies RL, Langenbach R, (1990) Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1. Carcinogenesis 11: 1293–1300PubMedCrossRefGoogle Scholar
  12. Davies RL, Crespi CL, Rudo K, Turner TR, Langenbach R, (1989) Development of a human cell line by selection and drug- metabolizing gene transfection with increased capacity to activate promutagens. Carcinogenesis 10: 885–891PubMedGoogle Scholar
  13. Dogra S, Doehmer J, Glatt HR, Mölders H, Siegert P, Friedberg T, Seidel A, Oesch F, (1990) Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing. Mol Pharmacol 37: 608–613PubMedGoogle Scholar
  14. Doehmer J, Barinaga M, Vale W, Rosenfeld MG, Verma IM, Evans R, (1982) Introduction of rat growth hormone gene into mouse fibroblasts via a retroviral DNA vector: expression and regulation. Proc Natl Acad Sci USA 85: 5769–5773CrossRefGoogle Scholar
  15. Doehmer J, Dogra S, Friedberg T, Monier S, Adesnik M, Glatt HR, Oesch F, (1988) Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin Bl. Proc Natl Acad Sci USA 85: 5769–5773PubMedCrossRefGoogle Scholar
  16. Doehmer J, (1993) V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies. Toxicol 82: 105–118CrossRefGoogle Scholar
  17. Eugster HP, Sengstag C, Meyer UA, Hinnen A, Würgler FE, (1990) Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: An alternative enzyme source for in vitro studies. BBRC 172: 737–744Google Scholar
  18. Pompon D (1988) cDNA cloning and expression in yeast 5 cerevisiae of beta-naphtoflavone induced rabbit liver P-450 LM4 and LM6. Eur J Biochem 177: 285-293Google Scholar
  19. Faletto MB, Koser PL, Battula N, Townsend GK, MacCubbin AE, Gelboin HV, Gurtoo HL (1988) Cytochrome P3–450 cDNA encodes aflatoxin Bl-4-hydroxylase. J Biol Chem 2 63: 12187–12189Google Scholar
  20. Fisher CW, Caudle DL, Martin-Wixtrom CA, Quattrochi LC, Tukey RH, Waterman MR, Estabrook RW, (1992) High-level expression of functional human cytochrome P450 1A2 in Escherichia coli. FASEB J 6: 759–764PubMedGoogle Scholar
  21. Fujii-Kuriyama Y, Taniguchi T, Mizukami Y, Saki M, Tashiro Y, Muramatsu M, (1981) Construction and identification of a hybrid plasmid containing a DNA sequence complementary to phenobarbital-inducible cytochrome P-450 messenger RNA from rat liver. J Biochem 89: 1869–1879PubMedGoogle Scholar
  22. Fujita VS, Thiele DJ, Coon MJ, (1990) Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1). DNA Cell Biol 9: 111–118PubMedCrossRefGoogle Scholar
  23. Gonzalez FJ, Kasper CB, (1982) Cloning of DNA complementary to rat liver NADPH-cytochrome c(P450) oxidoreductase and cytochrome P-450b mRNAs. J Biol Chem 257: 5962–5968PubMedGoogle Scholar
  24. Gonzalez FJ, Mackenzie PI, Kimura S, Nebert DW, (1984) Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome Pl–450 and P3–450. Gene 29: 281–292PubMedCrossRefGoogle Scholar
  25. Gonzalez FJ, Aoyama T, Gelboin HV, (1990) Activation of promutagens by human cDNA-expressed cytochrome P450s. Mutation and the Environment, part B, 77–86Google Scholar
  26. Graham FL, van der Eb A, (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. J Virol 52: 456–467CrossRefGoogle Scholar
  27. Hardwick JP, Gonzalez FJ, Kasper CB, (1983) Cloning of DNA complementary to cytochrome P-450 induced pregnenolone 16a- carbonitrile: characterization of its mRNA, gene, and induction response. J Biol Chem 258: 10182–10186PubMedGoogle Scholar
  28. Kempf A, Nef P, Meyer UA, (1991) Bacterial expression of spectrally active rat cytochrome P-450 2G1 (P-450 olf). 3rd International ISXX Meeting, AmsterdamGoogle Scholar
  29. Kimura S, Smith HH, Hankinson N, Nebert DW, (1987) Analysis of two benzo[a]pyrene-resistance mutants of the mouse hepatoma Hepa-1 Pl–450 gene via cDNA expression in yeast. Embo J 6: 1929–1933PubMedGoogle Scholar
  30. Larson JR, Coon MJ, Porter TD, (1991) Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli. J Biol Chem 266: 7321–7324PubMedGoogle Scholar
  31. Negishi M, Swan DC, Enquist LW, Nebert DW, (1981) Isolation and characterization of a cloned DNA sequence associated with the murine Ah locus and 3-methylcholanthrene-induced form of cytochrome P-450. Proc Natl Acad Sci USA 78: 800–804PubMedCrossRefGoogle Scholar
  32. Nouso K, Thorgeirsson SS, Battula N, (1992) Stable expression of cytochrome P450IIE1 in mammalian cells: metabolic activation of nitrosodimethylamine and formation of adducts with cellular DNA. Cancer Res 52: 1796–1800PubMedGoogle Scholar
  33. Oeda K, Sakaki T, Ohkawa H, (1985) Expression of rat liver cytochrome P-4 50 MC cDNA in Saccharomyces cerevisiae. DNA 4: 203–210PubMedCrossRefGoogle Scholar
  34. Pellicer A, Robins D, Wold B, Sweet R, Jackson J, Lowy I, Roberts JM, Sim GK, Silverstein S, Axel R, (1980) Altering genotype and phenotype by DNA-mediated gene transfer. Science 209: 1414–1422PubMedCrossRefGoogle Scholar
  35. Puga A, Raychaudhuri B, Kalman S, Zhang YH, Nebert DW, (1990) Stable expression of mouse CYP1A1 and human CYP1A2 cDNAs transfected into mouse hepatoma cells lacking detectable P450 enzyme activity. DNA and Cell Biol 9: 425–432CrossRefGoogle Scholar
  36. Renaud JP, Cullin C, Pompon D, Beaune P, Mansuy D, (1990) Expression of human liver cytochrome P-450 IIIA4 in yeast: a functional model for the hepatic enzyme. Eur J Biochem 194: 889–896PubMedCrossRefGoogle Scholar
  37. Sakaki T, Akiyoshi-Shibata M, Yabusaki Y, Manabe K, Murakami H, Ohkawa H, (1991) Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochrome P450cl7 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P4 50 oxidoreductase. Pharmacogenetics 1: 86–93PubMedCrossRefGoogle Scholar
  38. Schmalix WA, Maser H, Kiefer F, Reen R, Wiebel FJ, Gonzalez FJ, Seidel A, Glatt HR, Greim H, Doehmer J, (1993) Stable expression of human cytochrome P450 1A1 cDNA in V7 9 Chinese hamster cells and metabolic activation of benzo[a]pyrene. Eur J Pharmacol Env Toxicol Pharmacol 248: 251–261CrossRefGoogle Scholar
  39. Shimizu T, Sogawa K, Fujii-Kuriyama Y, Takahashi M, Ogoma Y, Hatano M, (1986) Expression of cytochrome P-450d by saccharomyces cerevisiae. FEBS 207: 217–221CrossRefGoogle Scholar
  40. States JC, Quan TH, Hines RN, Novak RF, Runge-Morris M, (1993) Expression of human cytochrome P450 IA1 in DNA repair deficient and proficient human fibroblasts stably transformed with an inducible expression vector. Carcinogenesis, in pressGoogle Scholar
  41. Thompson LH, Wu RW, Felton JS, (1991) Introduction of cytochrome P450IA2 metabolic capability into cell lines genetically matched for DNA repair proficiency/deficiency. Proc Natl Acad Sci USA 88: 3827–3831PubMedCrossRefGoogle Scholar
  42. Trinidad AC, Wu RW, Thompson LH, Felton JS, (1991) Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair- proficient CHO cells. Mol Carcinog 4: 510–518PubMedCrossRefGoogle Scholar
  43. Waxman DJ, Lapenson DP, Aoyama T, Gelboin HV, Gonzalez FJ, Korzekwa K (1991) Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s. Arch Biochem Biophysics 290: 160–166CrossRefGoogle Scholar
  44. Wölfel C, Platt KL, Dogra S, Glatt HR, Wächter F, Doehmer J, (1991) Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17b-estradiol and 2-aminofluorene in V79 Chinese hamster cells. Mol Carcinog 4: 489–498PubMedCrossRefGoogle Scholar
  45. Zuber MX, Simpson ER, Waterman MR, (1986) Expression of bovine 17a-hydroxylase cytochrome P-450 cDNA in nonsteroidogeni(COS 1) cells. Science 234: 1258–1261PubMedCrossRefGoogle Scholar
  46. Zurbriggen B, Boehlen E, Sanglard D, Kappeli O, Fiechter A, (1989) Controlled expression of heterologous cytochrome P- 450c cDNA in Saccharomyces cerevisiae I. Construction and expression of a complete rat cytochrome P-450c cDNA. Int Biotechnol 9: 255–272Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1995

Authors and Affiliations

  • Johannes Doehmer
    • 1
  1. 1.Institut für Toxikologie und UmwelthygieneTechnische Universität MünchenMünchenGermany

Personalised recommendations