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Protocol no. 49
H-4-II-E RAT HEPATOMA CELL BIOASSAY

This bioassay utilises cultured H-4-II-E rat hepatoma cells to assess the aryl hydrocarbon hydroxylase (AHH) inducing potencies of planar aromatic hydrocarbons and/or contaminated environmental samples. The response of the cells to pure test chemicals or extracts of mixtures is compared with their response to the standard 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

CONTACT

S Dr. Thomas W. Sawyer Dr. June A. Bradlaw
Biomedical Defence Section In Vitro Toxicology, HFF-162 Defence Research Establishment Suffield Division of Toxicological Studies Box 4000 Food and Drug Administration Medicine Hat, Alberta 200 C Street S.W. Canada T1A 8K6 Washington DC USA 20204 Tel: Canada - 403 544 4708 Tel: USA - 202 245 1080

RATIONALE

The rat hepatoma cell line H-4-II-E has low constitutive activities of the cytochrome P-450 1A1-dependent monooxygenases, aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase. Both enzymes are, however, highly inducible by planar aromatic hydrocarbons (PAH) in this cell line. Thus, when a test sample is incubated with the cells, the level of enzyme activity measured is a quantitative indicator of its AHH-/EROD-inducing potency.

BASIC PROCEDURE

Rat hepatoma H-4-II-E cells are incubated with test samples for 72 hours. The cells are then harvested and whole cell suspensions are assayed fluorimetrically for induction of the cytochrome P-450 1A1-dependent monooxygenases: aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD).

CRITICAL ASSESSMENT

The polychlorinated biphenyls (PCBs), dibenzofurans (PCDFs), and dibenzo-p-dioxins (PCDDs) are structurally related planar aromatic hydrocarbons (PAHs) which have been detected in every component of the global ecosystem, including terrestrial and aquatic food chains. These compounds have been found to be distributed in the tissues of fish, domestic and wild fowl, domestic livestock, and humans, and have demonstrated a marked persistence and toxicity in a variety of animal species. They thus represent a substantial threat to human health. A very strong correlation exists between the in vivo toxicity of the individual PAHs and their potencies as inducers of AHH and EROD. The rat hepatoma cell line H-4-II-E is superior to other cell types for in vitro AHH induction studies because it has low constitutive activities of both AHH and EROD, is highly inducible by the PAHs, has excellent growth characteristics, and is extremely sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, the prototypic AHH inducer). Several classes of compounds have been tested in this system, and in every case their potencies as inducers of AHH and EROD have paralleled their relative rankings with respect to in vivo toxicity. As with other cell types, however, no toxic effects in vitro are evident, even at very high concentrations of PAH. Maximum enzyme induction has been shown to occur 60-72 hours after exposure of cell cultures to the test sample and, although maximally induced enzyme-specific activity can vary from experiment to experiment with a given test sample, the reproducibility of the median effective concentration (EC50) or median effective dose (ED50) is high, even among different laboratories. Solvent effects do appear to be significant in this system, and the solvent of choice (isooctane or DMSO) should be determined experimentally prior to screening of test samples. For example, polychlorinated dibenzo-dioxins seem to be detected with greater sensitivity when isooctane is used as the solvent, while polychlorinated biphenyls are better detected with DMSO. In general, however, the strange properties exhibited by DMSO in vitro and in vivo, and the lower solvent volume per culture of isooctane (2.5% maximum) would tend to make the latter the solvent of choice. The H-4-II-E bioassay can employ two highly sensitive fluorescent enzyme assays to measure the enzyme inducing potencies of PAHs. These include the fluorescent AHH assay, which measures the enzymatic conversion of benzo[a]pyrene to 3-hydroxybenzo[a]pyrene, and the EROD enzyme assay, which measures the deethylation of 7-ethoxyresorufin to resorufin. Both assays yield very similar results, however, the EROD assay is quicker, more economical, easier to perform, and does not include the use of chemical carcinogens. Thus, although elevation of the enzyme AHH has historically been used as the indicator of cytochrome P-450 1A1 induction, the trend more recently has been to use the EROD assay. The sensitivity of the enzyme assays used, coupled with the highly inducible nature of the H-4-II-E cultures, enables the H-4-II-E bioassay to be a sensitive bioanalytical tool for quantifying the AHH inducing potencies of planar aromatic hydrocarbons. The biochemical potencies of test samples in this bioassay correlate very highly with their in vivo toxicities. Thus, this test is a useful tool for predicting the potential in vivo toxicity of pure compounds or contaminated environmental samples.

CHEMICALS TESTED

PCBs, PCDDs, PCDFs and their derivatives. Commercial PCB mixtures (Aroclors, Kaneclors, etc.) Fly ash samples Herbicides Water samples Food extracts

TEST STATUS

Although this assay system has not been subjected to a formal validation study, it has been found to be very reproducible both within and between laboratories. Consequently, it is being used in many laboratories worldwide.

REFERENCES

  1. Bradlaw, J.A. & Casterline, Jr., J.L. (1979) Induction of enzyme activity in cell culture: A rapid screen for detection of planar polychlorinated organic compounds. J. Assoc. Off. Anal. Chem. 62, 904-916.
  2. Bradlaw, J.A., Garthoff, L.H., Hurley, N.E. & Firestone, D. (1980) Comparative induction of aryl hydrocarbon hydroxylase activity in vitro by analogues of dibenzodioxins. Fd. Cosmet. Toxicol. 18, 627-635.
  3. Burke, M.D. & Mayer, R.T. (1974) Ethoxyresorufin: Direct fluorometric assay of a microsomal O-deethylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Disposition 2, 583-588.
  4. Burke, M.D. & Mayer, R.T. (1975) Inherent specificities of purified cytochromes P-450 and P-448 toward biphenyl hydroxylation and ethoxyresorufin deethylation. Drug Metab. Disposition 3, 245-253.
  5. Denomme, M.A., Homonoko, K., Fujita, T., Sawyer, T. & Safe, S. (1985) Effects of substituents on the cytosolic receptor-binding avidities and aryl hydrocarbon hydroxylase induction potencies of 7-substituted 2,3-dichlorodibenzo-p-dioxins. A quantitative structure-activity relationship analysis. Molec. Pharmacol. 27, 655-661.
  6. Nebert, D.W. & Gelboin, H.V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J. Biol. Chem. 243, 6242-6249.
  7. Pohl, R.J. & Fouts, J.R. (1980) A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions. Anal. Biochem. 107, 150-155.
  8. Sawyer, T., Bandiera, S. & Safe, S. (1983) Bioanalysis of polychlorinated dibenzofuran and dibenzo-p-dioxin mixtures in fly ash. Chemosphere 12, 529-535.
  9. Sawyer, T. & Safe, S. (1982) Isomers and congeners: Induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase enzyme activities in rat hepatoma cells. Toxicol. Lett. 13, 87-94.

Tillit, D.E., Giesy, J.P. & Ankley, G.T. (1991) Rat hepatoma cell bioassay as a tool for assessing toxic potency of planar halogenated hydrocarbons (PHHs) in environmental samples. Environ. Sci. Technol. 25, 87-92.

IP-49 © June 1992