Charles Tong

Definition of conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine—guanine phosphoribosyl transferase (ARL/HGRPT) mutagenesis assay

Conditions for the detection of genotoxic chemicals in the adult rat-liver epithelial cell/hypoxanthine-guanine phosphoribosyl transferase (ARL/HGPRT) mutagenesis assay have been defined. These included (1) a 3-day exposure to activation-dependent carcinogens; (2) a minimum of 14 days for induced mutant expression; (3) seeding density of 1 x 10(4) cells per cm2 for selection of mutants; (4) use of 6-thioguanine and (5) acceptance of genotoxicity of test chemicals if induced mutant incidence is significantly above that of the parallel run control and beyond the 98% confidence limits of the mean of the population spontaneous mutant incidence. With this protocol, the ARL/HGPRT mutagenesis assay has the capacity to activate representative members of the mycotoxin, aminoazo dye, aromatic amine and nitrosamine-types of carcinogens. This assay, offering additional metabolic parameters through intrinsic metabolic capability and providing a reliable end-point of clear biologic significance serves as a useful supplement to the Salmonella/microsome bacterial mutagenesis assay in a battery for the detection of genotoxic chemicals.

Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. I. Genetic and cellular effects.

The cellular effects of the antioxidants butylated hydroxyanisole and butylated hydroxytoluene were studied in a battery of in vitro tests. No evidence of genotoxicity was obtained for either compound in the hepatocyte primary culture/DNA repair test, the Salmonella/microsome mutagenesis test, the adult rat liver epithelial cell/hypoxanthine guanine phosphoribosyl transferase test, or for butylated hydroxyanisole in the Chinese hamster ovary cell/sister chromatid exchange test. Both compounds inhibited intercellular molecular exchange between cultured liver cells, an effect that has been observed for many agents with neoplasm-promoting activity.

Rat Liver Foci and in Vitro Assays to Detect Initiating and Promoting Effects of Chlorinated Ethanes and Ethylenes

Nine chlorinated aliphatics (CAs) were examined in a rat liver foci assay for tumor initiating and promoting activities. In this model, young adult male Osborne Mendel rats were first subjected to a partial hepatectomy, the test chemical was then administered at the maximum tolerated dose in the initiation or promotion phase in conjunction with diethylnitrosamine (DEN; 30 mg/kg b.w.) or phenobarbital (PB; 0.05 percent, w/w, in the diet), and gamma glutamyltranspeptidase (GGT) was used as a putative preneoplastic indicator. When administered in the promotion protocol after initiation with DEN, 1,1-dichloroethane, 1,1,2-trichloroethane (1,1,2-TCE), 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), tetrachloroethylene (TTCY), and hexachloroethane induced significant increases in GGT+-foci above control levels. 1,1,2,2-TTCE, TTCY, and 1,1,2-TCE also induced significant increases in GGT+-foci when administered in the promotion protocol without DEN initiation. Two variants of GGT+-foci were observed: the classical type associated with PB promotion, and the other, which was more diffuse, less intensely stained, resembling foci undergoing redifferentiation and associated with CAs. A number of CAs were also genotoxic in short-term in vitro tests. Taken together, the studies suggest that CAs may be complete carcinogens in vivo with weak initiating activity and stronger promoting activity.

Induction of purine analog-resistant mutants in adult rat liver epithelial lines by metabolic activation-dependent and -independent carcinogens

Abstract Adult rat liver epithelial cultures were found to be useful for mutagenicity studies. 6-Thioguanine was superior to 8-azaguanine in mutant selection. The incidence of purine analog-resistant mutants increased as a result of exposure to either metabolic activation-dependent or activation-independent carcinogens. Expression time studies revealed that an interval of about 10 days between carcinogen exposure and mutant selection was required to recover the maximum incidence of mutants. The parental cell type and its mutants were comparable in sensitivity to the toxic effects of one of the carcinogens. These data on increasing mutant incidence with time following exposure and the lack of specific resistance of the mutants to toxic effects of one of the mutagenic carcinogens indicate that mutant induction rather than selection was the cause of the increased mutation incidence. Thus, the use of these cultures as a self-sufficient screening system for carcinogens/mutagens is feasible.

Absence of mutagenic activity of three forms of asbestos in liver epithelial cells

Abstract The amosite, crocidolite, and chrysotile forms of UICC asbestos were assayed for their ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. Using adult rat liver-derived (ARL-6 and ARL-18) cells in culture, no consistent or significant increase occurred in the number of mutants resistant to the purine analog, 6-thioguanine, after exposure to toxic levels of amosite, crocidolite, or chrysotile. These results support the concept that asbestos is an epigenetic carcinogen that does not alter DNA.

Enhancement of mutagenesis during cell replication of cultured liver epithelial cells.

The susceptibility to mutagenesis of proliferating and non-proliferating mammalian cells was studied in cultured rat liver epithelial cells. Cells brought to growth quiescence by a non-toxic means were stimulated to proliferate and both types of cultures were exposed to methyl methanesulfonate (MMS). Cultures enriched in proliferating cells were more susceptible to both the toxic and mutagenic action of the mutagen than were quiescent cultures with a low level of proliferation.

Sister-chromatid exchange induction by polycyclic aromatic hydrocarbons in an intact cell system of adult rat-liver epithelial cells.

Adult rat-liver epithelial cell lines possess intrinsic metabolic capability for the biotransformation of xenobiotics and thus, are sensitive to a broad spectrum of mutagens/carcinogens in a mutagenesis assay at the hypoxanthine-guanine phosphoribosyl transferase locus. To provide another end-point of biological significance in these lines, we have investigated the application of adult rat-liver epithelial cell line 18 in a sister-chromatid exchange assay. Significant dose-dependent increases in the sister-chromatid exchange frequency occurred when liver cells were exposed to benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene. A weak but positive response was elicited by benz[a]anthracene. The present observations thus confirm the capacity of these cells to generate genotoxic metabolites from activation-dependent mutagens/carcinogens and indicate a relationship between the production of mutations and sister-chromatid exchanges by polycyclic aromatic hydrocarbons.

Differences in responses of 4 adult rat-liver epithelial cell lines to a spectrum of chemical mutagens

An assay for mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in adult rat-liver epithelial cell cultures (ARL) has been developed to take advantage of the capacity of this cell type to metabolically activate promutagens/procarcinogens. A survey of the effect of 5 types of activation-dependent mutagens/carcinogens on 4 ARL lines indicates that the ARL/HGPRT mutagenesis assay with the 4 target cell lines is able to detect a spectrum of activation-dependent carcinogens. Individual ARL lines, however, responded quite differently to a given carcinogen. The ARL/HGPRT mutagenesis assay system thus offers distinct possibilities for the study of the control of chemical biotransformation processes. However, in light of the specificity of the various cell lines to respond to a particular class of mutagens under the current assay condition, this particular assay system cannot be readily applied to routine screening of suspected environmental mutagens of unknown requirements for metabolic activation. Nevertheless, for agents with a structure related to those activated by a specific line, this system can be used to study mutagenesis resulting from intact cellular metabolism.

Rat Hepatocyte-Mediated Mutagenesis of Human Cells by Carcinogenic Polycyclic Aromatic Hydrocarbons but Not Organochlorine Pesticides

Abstract A hepatocyte primary culture-mediated human cell hypoxanthine-guanine phosphoribosyl transferase mutagenesis assay was established. This assay provides an in vitro system that combines a realistic activation capability and an endpoint of clear biological significance in a target cell line relevant to human risk. The genotoxicity of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene were demonstrated in this assay. In the same assay, DDT, chlordane, and mirex were not genotoxic. An epigenetic mechanism for the carcinogenicity of these organochlorine pesticides is suggested.

Polybrominated biphenyls are nongenotoxic and produce an epigenetic membrane effect in cultured liver cells

Polybrominated biphenyls (PBB) were studied for their genotoxic and epigenetic effects in cultured liver cells. PBB did not elicit DNA repair synthesis in rat, mouse, or hamster hepatocytes in primary cultures and did not cause mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in a line of rat liver epithelial cells or in human fibroblasts cocultivated with rat hepatocytes as an activating system. In contrast, PBB inhibited intercellular molecular exchange between rat hepatocytes and liver epithelial cells indicating an epigenetic membrane effect. These data are consistent with the interpretation that PBB act as neoplasm promoters in the production of rodent liver neoplasms.