In vitro investigation of toxaphene genotoxicity in S. typhimurium and Chinese hamster V79 lung fibroblasts.

The polychlorinated pesticide toxaphene has been identified as a persistent environmental contaminant and is of particular concern in the Great Lakes and Arctic regions of Canada. Inconsistencies in published in vitro genotoxicology studies have hindered risk assessments of toxaphene exposure. When toxaphene mutagenicity was re-evaluated in the Ames Salmonella/microsome assay at 10-10,000 microg/plate, a dose-dependent increase in His revertants occurred in all five strains of S. typhimurium tested (TA97, TA98, TA100, TA102 and TA104) with higher mutation frequencies observed in the absence of S9 metabolic activation. However, the mutagenic potential of toxaphene was relatively low with concentrations greater than 500 microg/plate required to induce mutation. Toxaphene genotoxicity was also examined in a mammalian system using Chinese hamster V79 lung fibroblasts with metabolic activation provided by human HepG2 hepatoma cells. Genotoxicity of 1-10 microg/ml toxaphene was examined by measuring the frequency of sister chromatid exchange (SCE) and mutation induction at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene locus. Although small increases in SCE were observed at toxic concentrations of toxaphene approaching the LD50 (10 microg/ml), they were not found to be statistically significant relative to control. Toxaphene was also unable to induce HGPRT mutagenesis at the concentrations tested. These results show that while toxaphene is a weak, direct-acting mutagen in the Ames Salmonella Test, convincing evidence of dose-dependent SCE induction and mutagenicity at the HGPRT gene locus could not be demonstrated in V79 cells.

Evaluation of cytotoxicity and genotoxicity of okadaic acid, a non-phorbol ester type tumour promoter, in V79 Chinese hamster lung cells.

Okadaic acid, a specific phosphatase inhibitor and non-phorbol ester type tumour promoter, was examined for cytotoxic and genotoxic properties in a hepatocyte-mediated assay with V79 Chinese hamster lung fibroblasts. Genetic endpoints measured were: mutation to 6-thioguanine resistance at the hgprt gene locus and induction of sister chromatid exchange (SCE). The cytotoxicity of okadaic acid to V79 cells was determined in the absence of hepatocytes at concentrations from 0 to 30 ng/ml medium. Okadaic acid decreased colony size at 20 ng/ml, reduced colony formation by 50% (LC(50)) at 24 ng/ml, and was lethal to 100% of the cells at concentrations above 30 ng/ml. Exposure of V79 cells to okadaic acid for as little as 24 hr without hepatocytes invoked morphological changes that were concentration related. By comparison, okadaic acid was less able to invoke changes in morphology of V79 cells when co-cultivated with rat hepatocytes, but did alter hepatocyte morphology. In genotoxicity tests, mutation frequencies were increased signficantly by okadaic acid only in the absence of hepatocytes and only at the highest tested concentration (20 ng/ml medium). In contrast, SCE frequencies were not affected when okadaic acid was tested alone or with hepatocyte activation at concentrations as high as 20 ng/ml. It was concluded that, within the limits of the test system used, okadaic acid was strongly cytotoxic and may function as a direct-acting mutagen, but was otherwise without cytogenetic activity towards V79 cells.

Toxicity of structurally related anthraquinones and anthrones to mammalian cells in vitro.

Anthraquinones and structurally related compounds were cytotoxic to mammalian cell lines using cloning efficiency and MTT reduction as endpoints. In V79 cells, the concentration of chemical causing 50% inhibition ranged from 0.21 to 21.6 mug/ml for cloning efficiency and from 0.86 to 14.6 mug/ml for MTT reduction. The anthrones anthralin and chrysarobin were 4.1 and 3.2 times more toxic, respectively, in the cloning efficiency assay than in the MTT assay. In contrast, the anthraquinones danthron and emodin were 2.8 and 2.1 times more toxic, respectively, in the MTT assay than in the cloning efficiency assay. Among the four mammalian cell lines tested using the MTT assay, the human leukaemia cell line (K562) was the most sensitive to the test chemicals. In contast, anthraquinone toxicity was reduced in rat hepatoma (H4IIE) cultures. In general, structures with carbonyl groups in positions 9 and 10 on the anthracene skeleton (anthraquinones) were less toxic than structures with carbonyl groups in position 9 only (anthrones). Toxicity was also influenced by the position of hydroxy substituents on the tricyclic skeleton. The results suggested that in vitro cytotoxicity assays are useful in elucidating the relationships between structure and biological activity for anthraquinones and related compounds.

A multiple end-point approach to evaluation of cytotoxicity and genotoxicity of erythrosine (FD and C Red No. 3) in a V79 hepatocyte-mediated mutation assay.

V79 Chinese hamster lung cells were used to evaluate in vitro the cytotoxicity and genotoxicity of erythrosine (2', 4', 5', 7'-tetraiodofluorescein disodium salt; FD and C Red No. 3), a color additive used widely in foods, drugs and cosmetics. Erythrosine reduced colony size at 200 micrograms/ml and was lethal to 90% or more of the cells at 400 micrograms/ml. At dose levels of 100, 200 and 300 micrograms/ml of medium, erythrosine was non-mutagenic to V79 cells at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and sodium, potassium ATPase (Na+, K+ -ATPase) gene loci and did not increase the frequency of sister-chromatid exchanges with or without rat hepatocyte-mediated activation. Erythrosine at 300 micrograms/ml, unlike lower dose levels, produced an increase in micronucleus frequency in the absence of hepatocytes. An erythrosine dose-related increase in the mitotic frequency was due to an increase in the number of first mitoses at the expense of later cell divisions. Hepatocytes moderated the effect of erythrosine treatment on micronucleus frequency, mitotic frequency and MII/MI ratio. These results demonstrate the advantage of a multiple end-point approach to the evaluation of cytotoxicity and genotoxicity within a single-assay system.

Lack of induction of sister chromatid exchanges and of mutation to 6-thioguanine resistance in V79 cells by butylated hydroxyanisole with and without activation by rat or hamster hepatocytes.

The antioxidant butylated hydroxyanisole (BHA) and the promutagen/carcinogen 7,12-dimethylbenz[a] anthracene (DMBA) were examined for mutagenicity and the induction of sister chromatid exchanges (SCE) in a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells. Rat and hamster hepatocytes, prepared by in situ collagenase perfusion, were compared in the mutation assay to determine whether there are species differences in the ability to activate BHA and DMBA to ultimate mutagens. At the marginally cytotoxic concentration of 1.0 microM (2.6 micrograms/ml), DMBA induced a significant increase in the frequency of SCE and in the number of mutations to 6-thioguanine resistance (6-TGR) at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus with either rat or hamster hepatocyte-mediated activation, but induced highest mutation frequencies with rat hepatocytes. These findings support the contention that species differences can affect mutational response in hepatocyte-mediated assays with V79 cells. BHA was strongly cytotoxic to V79 cells at dose levels in excess of 0.3 mM (54 micrograms/ml). In contrast to DMBA, BHA showed no evidence of genotoxicity at marginally cytotoxic concentrations up to and including 0.3 mM as shown by the inability of this antioxidant to increase the frequency of sister chromatid exchanges or to induce mutations to 6-thioguanine resistance when activation was provided by rat or hamster hepatocytes.

In vitro short-term testing systems as prescreens for potential carcinogens: simultaneous detection of sister chromatid exchanges and mutation response in a cell-mediated assay with V79 cells.

Sister chromatid exchange (SCE) is recognized as a sensitive indicator of genetic damage, and this has led numerous investigators to suggest that the analysis of SCE can provide a useful step toward the identification of environmental mutagens and/or carcinogens. To explore this approach, we measured SCE induction in V79 Chinese hamster lung cells and the frequency of mutation to 6-thioguanine resistance at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in a cell-mediated mutation assay. Karyotypic analysis of V79 cells showed a stable modal chromosome number of 22 and an XY chromosome complement. When exposed to the procarcinogen 7,12-dimethylbenz[a]anthracene (DMBA) at marginally cytotoxic dose levels of 1.0, 0.5 and 0.25 microM (2.6, 1.3 and 0.65 micrograms/ml), SCE frequencies were highest within the first 24 h of activation with rat or hamster hepatocytes, showed somewhat lower values after 48 h of activation, and, following withdrawal of the chemical, declined to background levels during the period of expression. While this decline may involve several factors, the possibility is not excluded that DNA repair can contribute to the progressive elimination of SCE. The induction of SCE in V79 cells appeared unrelated to the expression of single-point mutation at the HGPRT locus. These findings demonstrate the advantage of multiple endpoint analysis which enabled cytotoxicity, mutagenicity and conditions optimal for the induction of SCE to be determined concurrently in a hepatocyte-mediated assay with V79 cells.

Cytotoxicity and absence of mutagenic activity of vomitoxin (4-deoxynivalenol) in a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells.

Cytotoxicity and mutagenicity of vomitoxin (4-deoxynivalenol), a tricothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium, were determined in vitro with Chinese hamster V79 cells. Cytotoxicity was shown by a reduction in colony size at 1 microgram/ml (ppm); by reduction in the number and size of colonies at 2-3 micrograms/ml or higher; and by lethality to 80-90% of the cells at 10 micrograms/ml. Up to 3 micrograms/ml, vomitoxin was non-mutagenic to V79 cells at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus, with or without hepatocyte-mediated activation; and did not significantly increase the number of 6-thioguanine-resistant mutants at marginally cytotoxic levels of 6 and 8 micrograms/ml (data not shown). These findings, together with previous studies, suggest that vomitoxin, like other 12,13-epoxytricothecenes, may become cytotoxic through inhibition of protein and/or DNA synthesis, and is likely to be non-carcinogenic.

In vitro cytotoxicity of polychlorinated biphenyls (Aroclors 1016, 1242, 1254 and 1260) and their effect on phospholipid and neutral lipid composition of Chinese hamster ovary (CHO-K1) cells.

Cytotoxicity of 4 Aroclors (1016, 1242, 1254 and 1260) was compared in Chinese hamster ovary (CHO-K1) cells in Ham's F-12 medium. When parameters of toxicity were cell numbers or tissue protein, 50% lethality occurred at Aroclor concentrations between 30 and 45 ppm. An in vitro clonal assay with CHO-K1 cells was a sensitive indicator of cytotoxicity of the polychlorinated biphenyls (PCBs). From EC50 values (concentration that allowed 50% survival of formed colonies), cytotoxicity was lower with Aroclor 1016 (32 ppm) and higher with Aroclors 1254 (27 ppm) and 1260 (28 ppm). In cells exposed 24 h to a marginally cytotoxic dose (20 ppm) of each Aroclor, phospholipid (PL) thin-layer chromatography (TLC) showed an increase in phosphatidylcholine (PC) and a decrease in phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Neutral lipid (NL) TLC of cells given Aroclors 1242, 1254 or 1260 showed a 3-4-fold increase in triglyceride (TG) and a similar reduction in cholesteryl esters (CE); in contrast to Aroclor 1016 which produced no change in TG and a smaller (2-fold) reduction in CE. Cholesterol and free fatty acid fractions were unaffected by any of the Aroclors. The TG:PL ratio remained unchanged in cells given Aroclor 1016, but increased 3-4-fold with Aroclors 1242, 1254, or 1260. Compared to total values in the untreated controls, CHO-K1 cells contained less neutral lipid and more phospholipid only with Aroclor 1016. These results support the concept that differences in the behavior of Aroclor 1016 are related to its PCB composition. Changes in membrane PL and NL components, observed at marginally cytotoxic levels of each Aroclor, provided further evidence that the PCBs may affect membrane integrity and associated metabolic functions.