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Protocol no. 112
CYP1A1-INDUCING POTENCY AND CYTOTOXICITY TEST IN THE HEPA-1 MOUSE HEPATOMA CELL LINE

This bioassay utilizes cultured Hepa-lclc7 (Hepa-1) mouse hepatoma cells to assess the CYPlA1-inducing potency or cytotoxicity of pure test chemicals or environmental samples. In the Hepa-l induction test , the CYPlA1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities. In the Hepa-l cytotoxicity test , the effect of the sample on cell viability is measured.

CONTACT

Dr. Sirpa Kärenlampi
Department of Biochemistry and Biotechnology
University of Kuopio
P.O.Box 1627
FIN-70211
KUOPIO FINLAND
Fax: 358-71-2811510
e-mail: Sirpa.Karenlampi@uku.fi
Dr. Riitta Törrönen, Dr. Päivi Kopponen
Department of Physiology
University of Kuopio
P.O.Box 1627
FIN-70211
KUOPIO FINLAND
Fax: 358-71-163112
e-mail: Riitta.Torronen@uku.fi
Paivi.Kopponen@uku.fi

RATIONALE

Hepa-1 is a continuous cell line derived from a mouse hepatoma. It has a highly inducible cytochrome P450IA1 (CYPlA1). The enzymatic activity of CYPlA1 can be detected as AHH and EROD activities. Since the CYPlA1-inducing potency of a compound is considered to be an indicator of its toxicity, the Hepa-1 induction test can be used to assess the potential toxicity of individual chemicals or complex mixtures. In the Hepa-1 cytotoxicity test, the effects of the test samples on the viability of the cells are studied. The combination of these tests provides a rapid screening procedure for chemicals, pollutants, contaminants etc. which may represent a health or environmental hazard.

BASIC PROCEDURE

Hepa-1 induction test The cell culture is exposed to several concentrations of the test sample for 24 hours, after which AHH and/or EROD activities in the cultures are assayed. If CYPlA1 inducers are present in the sample, AHH and EROD activities will be higher than in cultures exposed to the control sample (solvent only). The response of the cells to the sample is compared with their response to a reference compound, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepa-1 cytotoxicity test Cells on a multiwell culture plate are exposed to a 1 : 1 dilution series of the sample for 72 hours. Survival of the cells is estimated by assaying total protein content in the cultures using a dye-binding method. If cytotoxic compounds are present in the sample, less protein is detected in the cultures exposed to the sample than in the cultures exposed to the control sample (solvent only).

TEST STATUS

The Hepa-1 cytotoxicity test has been under evaluation since 1989 in the international evaluation programme MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) organized by the Scandinavian Society for Cell Toxicity. So far, 31 laboratories have submitted cytotoxicity results from 68 methods for the first 30 reference chemicals (Clemedson C. et al., MEIC evaluation of acute toxicity for the first 30 reference chemicals; Part II. In vitro results from 68 methods and a comparative cytotoxicity analysis. To be published in a special supplement to ATLA). The results show that the Hepa-1 cytotoxicity test has given satisfactory values for practically all chemicals tested and that mouse cells are, in general, better indicators of human acute toxicity than are rat cells.

CHEMICALS AND MIXTURES TESTED

Dioxin and dioxin-like compounds Kärenlampi S., Törrönen R. (1990) Induction of cytochrome P450IA1 in mouse hepatoma cells as a short-term bioassay. ATLA 17:158-162.
Kopponen P., Mannila E., Kärenlampi S. (1992) Induction of aryl hydrocarbon hydroxylase AHH by two previously uncharacterized pentachlorinated biphenyls in a mouse and a rat hepatoma cell line. Chemosphere 24: 201-210.
Kopponen P., Sinkkonen S., Poso A., Gynther J., Kärenlampi S. (1994a) Sulfur analogues of polychlorinated dibenzo-p-dioxins, dibenzofurans and diphenyl ethers as inducers of CYPlA1 in mouse hepatoma cell culture and structure-activity relationships. Environ. Toxicol. Chem. 13: 1543-1548.
Fly ash from different combustion processes Kopponen P., Törrönen R., Tarhanen J., Ruuskanen J., Kärenlampi S. (1991) Cytochrome P450IA1 induction in mouse hepatoma cell culture as an indicator of polycyclic organic compounds in fly ash. Chemosphere 22: 895-904.
Kopponen P., Törrönen R., Ruuskanen J., Tarhanen J., Vartiainen T., Kärenlampi S. (1992) Comparison of cytochrome P450IA1 induction with the chemical composition of fly ash from combustion of chlorine containing material. Chemosphere 24: 391-401.
Kopponen P., Tarhanen J., Ruuskanen J., Törrönen R., Kärenlampi S. (1993) Peat induces cytochrome P450IA1 in Hepa-1 cell line. Comparison with fly ashes from combustion of peat, coal, heavy fuel oil and hazardous waste. Chemosphere 26: 1499-1506.
Kopponen P., Välttilä O., Talka E., Törrönen R., Tarhanen J., Ruuskanen J., Kärenlampi S. (1994b) Chemical and biological 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents in fly ash from combustion of bleached kraft pulp mill sludge. Environ. Toxicol. Chem. 13: 143-148.
Kopponen P., Törrönen R., Mäki-Paakkanen J., von Wright A., Kärenlampi S. (1994c) Comparison of CYPlA1 induction and genotoxicity in vitro as indicators of potentially harmful effects of environmental samples. Arch. Toxicol. 68: 167-173.
Paper products Kärenlampi S., Törrönen R. (1990) Induction of cytochrome P450IAl in mouse hepatoma cells as a short-term bioassay. ATLA 17:158-162.
Laboratory animal beddings and feeds Törrönen R., Pelkonen K., Kärenlampi S. (1989) Enzyme-inducing and cytotoxic effects of wood-based materials used as bedding for laboratory animals. Comparison by a cell culture study. Life Sci. 45: 559-565.
Törrönen R., Kärenlampi S., Pelkonen K. (1991) Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study. Life Sci. 48: 1945-1951.

REFERENCES

  1. Bernhard H.P., Darlington G.J., and Ruddle F.H. (1973) Expression of liver phenotypes in cultured mouse hepatoma cells: synthesis and secretion of serum albumin. Dev. Biol. 35: 83-96.
  2. Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
  3. Burke M.D., and Mayer R.T. (1974) Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug. Metab. Dispos. 2: 583-588.
  4. Hankinson O. (1979) Single-step selection of clones of a mouse hepatoma line deficient in aryl hydrocarbon hydroxylase. Proc. Natl. Acad. Sci. USA 76: 373-376. Hankinson O., Brooks B.A., Weir-Brown K.I., Hoffman E.C., Johnson B.S., Nanthur J., Reyes H., and Watson A.J. (1991) Genetic and molecular analysis of the Ah receptor and of Cyp1a1 gene expression. Biochimie 73: 61-66.
  5. Kärenlampi S. (1987) Mechanism of cytotoxicity of aflatoxin B1: role of cytochrome P1-450. Biochem. Biophys. Res. Commun. 145: 854-860.
  6. Nebert D.W., and Gelboin H.V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. J. Biol. Chem. 243: 6242-6249.
  7. Lake B.G. (1987) Preparation and characterisation of microsomal fractions for studies on xenobiotic metabolism. In: Biochemical toxicology, a practical approach, (K. Snell and B. Mullock (eds)), IRL Press, Oxford, UK, pp. 183-215.
  8. Lubet R.A., Mayer R.T., Cameron J.W., Nims R.W., Burke M.D., Wolff T., and Guengerich F.P. (1985a) Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat. Arch. Biochem. Biophys. 238: 43-48. Lubet R.A., Nims R.W., Mayer R.T., Cameron J.W., and Schechtman L.M. (1985b) Measurement of cytochrome P-450 dependent dealkylation of alkoxyphenoxazones in hepatic S9s and hepatocyte homogenates: effects of dicumarol. Mutat. Res. 142: 127-131.
  9. Safe S. (1993) Development of bioassays and approaches for the risk assessment of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Environ. Health Persp. 101 (Suppl. 3): 317-325.
  10. Sawyer T.W., Vatcher A.D., and Safe S. (1984) Comparative aryl hydrocarbon hydroxylase induction activities of commercial PCBs in Wistar rats and rat hepatoma H-4-II E cells in culture. Chemosphere 13: 695-701.

IP-112 October1995