Jerry R. Williams

Evaluation of a human hepatoma cell line as a target cell in genetic toxicology

A cell line derived from a human hepatoblastoma, HepG2, was examined for its ability to activate cyclophosphamide (CY) to a genotoxic form. Metabolism of CY to genotoxic product(s) was determined by the induction of sister-chromatid exchanges (SCE). The dose-dependent response pattern in HepG2 was compared to the patterns obtained by three other mammalian cell lines. HepG2 and a rat hepatoma cell line, H4-II-E, show similar dose-dependent increases of induced SCE, whereas non-hepatic-derived fibroblast lines show little or no CY-induced SCE. Microsomal enzyme activities characteristic of cytochromes P450 and P448 and epoxide hydrolase were examined in the two hepatoma cell lines and compared to levels in rat liver microsomal preparations. Although no cultured cell line can be a universal surrogate for in vivo metabolism, we propose that HepG2 may be useful to determine in a qualitative manner whether human cells possess the ability to activate a chemical to a genetically damaging form.

In vitro assays of in vivo exposure to cyclophosphamide: Induction of sister-chromatid exchanges in peripheral lymphocytes, bone-marrow cells and in cultured cells exposed to plasma

The in vivo genotoxic potential of cyclophosphamide (CY) was assessed by sister-chromatid exchange (SCE) induction after removal and in vitro culture of circulating peripheral lymphocytes and bone marrow from CY exposed male, Fischer 344 rats. Plasma was simultaneously obtained and assessed for genotoxic activity by incubation with 4 cultured mammalian cell lines: HepG2, H4-II-E (H4), V79 and IMR-90. These 4 cell types were used to help discriminate the role of metabolism in generating SCE-inducing factors in plasma. HepG2 and H4 have been shown to metabolize certain xenobiotics while V79 and IMR-90 do not. An in vivo dose response to CY at doses of 0, 5, 10, 20, 30 and 50 mg CY/kg assayed 1 h post-i.p. injection was performed. Phytohemagglutinin (PHA)-stimulated lymphocytes showed a dose-related increase in SCE up to 66.7 SCE/cell at 20 mg/kg (30 mg/kg was cytotoxic). Bone marrow also showed an SCE increase to 34.8 SCE/cell at 10 mg/kg (higher doses were cytotoxic). Plasma induced a dose-dependent SCE increase in the 4 cultured cell lines at all tested doses indicating the presence of direct-acting SCE-inducing metabolites of CY. A time course study using 20 mg CY/kg indicated peak plasma levels of CY genotoxic activity at approximately 0.5-1 h post-injection. By 3 h, the level of genotoxic activity in plasma was considerably reduced. Lymphocytes, however, showed a cumulative increase in SCE to 74.7 SCE/cell after 3 h of exposure. These in vivo exposure--in vitro assay techniques may be useful and facile systems with which to develop an integrative testing system for assessing the in vivo genotoxicity of a chemical.

Induction of sister chromatid exchanges by benzidine in rat and human hepatoma cell lines and inhibition by indomethacin

The genotoxic activity of benzidine was studied in two cell lines derived from rat (H4) and human (HepG2) hepatomas which have been shown to be capable of activating certain promutagens. The responses were compared to results in two lung-derived fibroblast lines (IMR-90 and V79) which appear to have little or no metabolizing capability. Benzidine was found to induce sister chromatid exchanges in the two liver-derived cell lines in a dose-dependent fashion but failed to induce sister chromatid exchanges in the fibroblast lines. Since one proposed pathway for benzidine activation involves prostaglandin-mediated metabolism, we tested the effect of pretreatment with indomethacin, an inhibitor of this metabolic pathway. Indomethacin was highly effective in inhibiting benzidine-induced sister chromatid exchanges in both H4 and HepG2 cells. These results suggest that some DNA damage may occur in the livers of fast acetylating species such as the rat without prior N-acetylation and that some amount of DNA damage may occur in the livers of slow acetylating species, even when the liver is not the target organ for carcinogenesis.

Induction of sister chromatid exchanges in human and rat hepatoma cell lines by cyclophosphamide and phosphoramide mustard and the effects of cytochrome P-450 inhibitors

The activity of the cytochrome P-450-associated metabolic pathway in human (HepG2) and rat (H4-II-E) hepatoma cells was examined. The genotoxic activities of cyclophosphamide and its direct acting metabolite, phosphoramide mustard, were studied in the hepatoma cells as cyclophosphamide is known to be metabolized by phenobarbital-inducible cytochrome P-450-associated metabolic activity. HepG2 and H4-II-E demonstrated the capacity to activate cyclophosphamide to forms capable of inducing sister chromatid exchanges in concentration-dependent fashion. Phosphoramide mustard induced a similar pattern of sister chromatid exchanges at concentrations three orders of magnitude lower than cyclophosphamide. The cytochrome P-450-associated enzyme inhibitors, SKF-525A and metyrapone, were found to reduce the level of cyclophosphamide-induced sister chromatid exchanges in HepG2 and H4-II-E, suggesting that cyclophosphamide was activated by this pathway in both hepatoma lines. Direct evidence for the presence of mRNA transcript coding for a phenobarbital-inducible cytochrome P-450 was demonstrated in HepG2 cells by Northern blot analysis. Comparison of genotoxic responses in human and rat hepatoma cells may allow for an evaluation of responses by different species to potentially mutagenic chemicals.

Ultraviolet-light exposure induces a heritable sensitivity to the induction of SCE by mitomycin-C

The dose-response relationship for mitomycin-C (MMC)-induced sister-chromatid exchange (SCE) has been determined in the progeny of Chinese hamster lung fibroblasts (V79) exposed to 5.0 J/m2 ultraviolet light-C (UVC, 254 nm) and in the progeny of non-UVC-irradiated controls. Progeny of UVC-irradiated cultures exhibited sensitivity to MMC-induced SCE at doses of MMC that were not detectably lethal. This sensitivity was manifest as an increase in SCE per cell in a large proportion of the cells derived from UVC-exposed cultures and thus appears not to result from the expression of a rare event such as mutation.

Utilization of an established rat hepatoma cell line for mutation studies

A selected strain of rat hepatoma cells was evaluated for its utility in the assay of mutation. We demonstrate that these cells have practical and theoretical advantages over most other mammalian cells utilized for the mutation assay at the HGPRT locus. Characteristics of the H4IIE cell line which enhance the ability of the HGPRT assay to detect mutation include: intrinsic metabolic capabilities; small cell size, and lack of mobility; colony-forming efficiency of 85-95%; a background mutation level between 0.9 and 3 mutants per 10(5) viable cells; and the ability to proliferate in medium containing 5% fetal bovine serum and 5% horse serum. The optimal expression period for the maximum frequency of mutants was investigated using UVC as the mutagenic agent, and found to plateau after 8-10 days. The metabolic capacity of this cell line was demonstrated using 2-aminoanthracene and cyclophosphamide, two types of mutagens requiring biotransformation for activity. We conclude that the use of the H4/HGPRT system could prove valuable in the mutagenic screening of suspect environmental agents.

A time-lapse, image digitization videomicroscope system based on a mini computer with large peripheral memory

We describe a time-lapse image digitization videomicroscope system that uses a mini computer as the main processing unit in conjunction with a large peripheral memory for storing multiple digitized images. The advantage of the system lies in its ability to facilitate acquisition and analysis of data relative to size, location and optical density of cells and cellular structure using elementary image processing techniques. This system can also potentially control microscope stage movement, enabling acquisition of larger amounts of data per time-lapse experiment.

Commentary: Science and Politics: The Possible Regulation of Cancer Promoters

To address the problems associated with the regulation of tumor promoters, one must recognize that a regulatory agencys actions in the formulation of policy redound from a matrix of political, social and scientific pressures; each affecting the other, and all influencing the final policy outcome. Scientific understanding of the mechanism(s) of tumor promotion plays a determinant role in such interaction, for the selection of particular biological tests as surrogates for human response to chemical exposure provides the radical upon which such interaction occurs, industry responding to the legal, regulatory instrument that relies in turn on scientific confidence in required tests. These parties operate within the penumbra cast by political expedients, environmental action groups, and special interest lobbies whose concerns involve the cost of testing, the availability of resources, the consideration of particular chemicals of economic or symbolic value, and other such aspects of regulatory policy or its consequence. In examining the complex development of policy for future regulation of tumor promoters, we must attend first to the legal instrument framing such regulations, assessing probable impact on these various scientific, economic, social and political factors. Potential regulation of most tumor promoters would fall within the regulatory ambit of the U.S. Environmental Protection Agency, specifically the Toxic Substances Control Act of 1976 (TSCA). As with other regulatory acts, the making of public policy, in this case concerning the manufacture and use of chemicals, derives from two major concerns: the attempt to achieve technical analyses of risks and benefits as one base for regulation, and a gener-

Dose Patterns of Cellular Effects Induced by Carcinogens: Evidence for the Presence or Absence of Thresholds

The patterns of response of individual cells to carcinogens may be a factor that influences the shape of the dose-response curve for cancer in human populations; thus the existence of a threshold for cellular response may imply a threshold for cancer induction. The patterns of response of cultured mammalian cells to carcinogen treatment is described for four cellular endpoints: cytotoxidty, mutation, sister chromatid exchange, and in vitro transformation. These data, taken in toto, indicate that for some carcinogens a threshold exists for the induction of cellular effects, but for some carcinogens thresholds probably do not exist. Cancer-prone human beings may contain cell populations without thresholds, making them especially sensitive to low doses of environmental carcinogens.