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Protocol no. 107
USE OF STABLE CELL LINES EXPRESSING CYTOCHROMES CYP cDNA IN TOXICITY TESTING

The cDNA of different members of the cytochromes CYP family can be inserted into cell lines such as V79 Chinese Hamster cells, which are used for in vitro toxicity testing. This means that the metabolites of xenobiotics are produced in the same cells in which any toxic effect will be observed, thus negating the problems associated with co-cultures and the use of subcellular fractions.

CONTACT

Dr J. Doehmer Institut fur Toxikologie und Umwelthygiene der Technischen Universitęt Munchen Lazarettstrasse 62 D-80636 Munchen Germany Tel: 49(0)89-3187-2973 Fax: 49(0)89-3187-3449

RATIONALE

The monooxygenases of the cytochrome CYP family have a detoxifying effect on some xenobiotics; however they can also act as activating agents that convert many promutagens and procarcinogens into cytotoxic, mutagenic or carcinogenic forms. For this reason, the toxicity testing of xenobiotics should incorporate testing of the metabolites after conversion by CYP. Unfortunately, cell culture lines used in toxicity testing do not express, or express very low levels, of these cytochromes. Therefore, CYP activity must be supplied exogenously to imitate the in vivo level of activity. Previously, this has been achieved through co-culture with rodent hepatocytes, or with a subcellular fraction of the hepatocytes. However, neither the use of intact hepatocytes, nor of subcellular fractions, is an ideal means of supplementing the xenobiotic metabolism in cell culture toxicity testing systems. Gene transfer of CYP cDNA into cell lines used for toxicity testing makes it possible to overcome these problems. This protocol discusses the use of genetically engineered Chinese hamster cells (V79) in mutagenicity testing and mechanistic studies of mutagens. The V79 cell line was chosen as it is the most commonly used cell line in in vitro mutagenicity tests, partly due to its short generation time of 12 hours. The mechanistic investigations described involve the use of genetically engineered cell lines that are metabolically competent, in order to distinguish aneugenic test compounds from clastogenic compounds.

BASIC PROCEDURE

Cytotoxicity and Mutagenicity Testing Cells are cultured for 18 hours and are then exposed to the test compound for 24 hours. The exposure is terminated by changing the medium, the cells are cultured for a further 4 days and then cytotoxicity is determined by neutral red uptake. Mutagenicity may be determined by determining the number of cells resistant to thioguanine. Micronucleus Assay Cells are exposed to the test compound for 24 hours and then incubated for a further 24 hours without the test compound. After treatment with a hypotonic buffer, the cells are fixed in methanol/glacial acetic acid and stained with Hoechst 33258 solution. Micronuclei are counted under a fluorescence microscope.

TEST STATUS

Genetically engineered V79 cells lines are in routine use in many industrial and academic laboratories.

CHEMICALS TESTED

2-Amino anthracene
Benzo[a]pyrene
Benz[a]anthracene-3,4-diol
Benzo[c]chrysene-9,10-diol
Benzo[g]chrysene-11,12-diol
Caffeine
Chrysene-1,2-diol
Chrysene-3,4-diol
Cyclophosphamide
Diethylnitrosamine
9,10-Dimethylanthracene-1,2-diol
7-Dimethylbenz[a]anthracene-3,4-diol
Dimethylnitrosamine
(trans)-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene
1-Hydroxy-BP-7,8-diol
3-Hydroxy-BP-7,8-diol
9-Hydroxychrysene-1,2-diol
7-Methylbenz[a]anthracene
7,12-Methylbenz[a]anthracene-3,4-diol
N-Methyl-N'-nitro-N-nitrosoguanidine
Nicotine
p-Nitrophenol
Phenanthrene-9,10-diol
(-)-Picene-3,4-diol
(+)-Picene-3,4-diol
Sterigmatocystin
Theophylline
Tobacco particulate matter

REFERENCES

  1. Denner K., Vogel R., Schmalix W., Doehmer J., and Bernhardt R. (1994) Cloning and stable expression of the human mitochondrial cytochrome P450 11B1 cDNA in V79 Chinese hamster cells and their application for testing of potential inhibitors. Submitted for publication.
  2. Doehmer J. (1993) V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies. Toxicology 82: 105-118.
  3. Doehmer J., Dogra S., Friedberg T., Monier S., Adesnik M., Glatt H., and Oesch F. (1988) Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1 Proc. Natl. Acad. Sci. USA 85: 5769-5773.
  4. Doehmer J., Glatt H.R., Seidel A., and Oesch F. (1990) Genetically engineered V79 Chinese hamster cells metabolically activate the cytostatic drugs cyclophosphamide and ifosfamide. Environ. Health Perspect. 88: 63-65.
  5. Doehmer J., and Oesch F. (1991) V79 Chinese hamster cells genetically engineered for stable expression of cytochromes P450. in Methods in Enzymology vol. 206 (eds. M. Waterman and E.F. Johnson) Academic Press; San Diego, pp. 117-123.
  6. Dogra S., Doehmer J., Glatt H., Mēlders H., Siegert P., Freidberg T., Seidel A., and Oesch F. (1990) Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing. Molecular Pharmacology 37: 608-613.
  7. Ellard S., Mohammed Y., Dogra S., Wolfel C., Doehmer J., and Parry J. (1991) The use of genetically engineered V79 Chinese hamster cultures expressing rat liver CYP1A1, 1A2 and 2B1 in micronucleus assays. Mutagenesis 6: 461-470.
  8. Fuhr U., Doehmer J., Battula N., Wolfel C., Kudla C, Keita Y., and Staib A. (1992) Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Biochemical Pharmacology 43: 225-235.
  9. Glatt H.R., Pauly K., Wēlfel C., Dogra S., Seidel A., Lee H., Harvey R.G., Oesch F., Doehmer J. (1993) Stable expression of heterologous cytochromes P450 in V79 cells: Mutagenicity studies with polycylic aromatic hydrocarbons. Polycyclic Aromatic Compounds 3: 1167-1174.
  10. Jensen K.G., Loft S., Doehmer J., and Poulsen H.E. (1993) Metabolism of phenacetin in V79 Chinese hamster cell cultures expressing rat liver cytochrome P450 1A2 compared to isolated rat hepatocytes. Biochem. Pharmacol. 45: 1171-1173. Jensen K.G., .nfelt A., Poulsen H.E., Doehmer J. and Loft S. (1993) Effects of benzo[a]pyrene and ()-trans-7,8-dihydrobenzo[a]pyrene on mitosis in Chinese hamster V79 cells with stable expression of rat cytochrome P450 1A1 or 1A2. Carcinogenesis 14: 2115-2118.
  11. Kiefer F., Cumpelik O., Reen R., Doehmer J., and Wiebel F.J. (1994) Arylamines suppress their own activation and that of nitroarenes in V79 Chinese hamster cells by competing for acetyltransferases. Environ. Health Perspect. 102: 1-3.
  12. Kulka U., Doehmer J., Glatt H.R., Bauchinger M. (1993) Cytogenetic effects of promutagens in genetically engineered V79 Chinese hamster cells expressing cytochromes P450. Eur. J. Pharmacol., Sect. Environ. Toxicol. Pharmacol. 228: 299-304.
  13. Schmalix W.A., Męser H., Kiefer F., Reen R., Wiebel F.J., Gonzalez F., Seidel A., Glatt H.R., Greim H., and Doehmer J. (1993) Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene. Eur. J. Pharmacol., Sect. Environ. Toxicol. Pharmacol. 248: 251-261.
  14. Stadler J., Trockfeld J., Schmalix W.A., Brill T., Siewert J.R., Greim H., Doehmer J. (1994) Inhibition of cytochromes P450 by nitric oxide. Proc. Nat. Acad. Sci. USA 91: 3559-3563. Wolfel C., Heinrich-Hirsch B., Schultz-Schalge T., Seidel A., Frank H., Ramp U., Wachter F., Wiebel F., Gonzalez F., Greim F., and Doehmer J. (1992) Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450 1A2. Eur. J. Pharmacol. Sect. Environ. Toxicol. Pharmacol. 228: 95-102.
  15. Wolfel C., Platt K.-L., Dogra S., Glatt H., Wachter F., and Doehmer J. (1991) Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17-estradiol and 2-aminofluorene in V79 hamster cells. Molecular carcinogenesis 4: 489-498.

IP-107 November/94