Sources of and technical approaches for the abatement of tobacco specific nitrosamine formation in moist smokeless tobacco products.

The presence of TSNA has been suggested as a potentially important cancer risk factor for moist smokeless tobacco (MST) products. We describe studies of the impact of tobacco agronomic and production practices which influence TSNA formation. TSNA were measured at points in the MST production chain from the farm to the finished product at the end of shelf life. Analyses were conducted to define points at which TSNA may occur, the factors related to the magnitude of occurrence, and actions which may be taken to mitigate such occurrence. Weather conditions during the curing season can have a dramatic impact on TSNA levels in tobacco, with wet seasons markedly increasing TSNA levels in cured tobacco. TSNA levels in MST do not increase beyond levels in cured tobacco when production practices limit the presence of nitrate reducing bacteria. Therefore, TSNA in such products are a function of the agronomic practices and conditions under which tobacco is produced at the farm level. Regional and annual variation in TSNA levels results from the stochastic nature of agronomic factors related to TSNA formation during tobacco growing and curing.

Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection by-products.

Cytotoxicity and genotoxicity assays were used to analyze drinking water disinfection by-products (DBPs) in Chinese hamster ovary (CHO) AS52 cells. The DBPs were chosen because they are common in drinking water, resulting from conventional disinfection using chlorination and chloramination. Data were also available to compare these results with cytotoxicity and mutagenicity studies in Salmonella typhimurium. The rank order in decreasing chronic cytotoxicity measured in a microplate-based assay was bromoacetic acid (BA) >> 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > KBrO(3) > tribromoacetic acid (TBA) > EMS (ethylmethanesulfonate, positive control) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA). The induction of DNA strand breaks by these agents was measured by alkaline single-cell gel electrophoresis (SCGE, comet assay) and the rank order in decreasing genotoxicity was BA >> MX > CA > DBA > TBA > EMS > KBrO(3), while DCA and TCA were refractory. BA was more cytotoxic (31x) and genotoxic (14x) than MX in CHO cells. BA was over 400x more genotoxic than potassium bromate. The brominated haloacetic acids (HAAs) were more cytotoxic and genotoxic than their chlorinated analogs. The HAAs expressed a statistically significant inverse relationship in CHO cell cytotoxicity and genotoxicity as a function of increased numbers of halogen atoms per molecule. A quantitative comparison was conducted with results from a previous study with cytotoxicity and mutagenicity in S. typhimurium. There was no correlation between chronic CHO cell and bacterial cell cytotoxicity. DBP-induced CHO cell cytotoxicity was not related to mutagenic potency in S. typhimurium. Cytotoxicity in CHO cells was statistically significant and highly correlated to CHO cell genotoxicity. Finally, we determined that the DBP genotoxic potency in CHO cells and the mutagenic potency in S. typhimurium were not related. This suggests that toxicity data in S. typhimurium did not quantitatively predict the toxic effects of DBPs in mammalian cell systems. The microplate CHO cell cytotoxicity and genotoxicity assays were well suited for the analysis of DBPs, especially when the quantity of test material is limited.

Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium.

We analyzed the cytotoxicity and mutagenicity of the drinking water disinfection by-products (DBPs) bromoform (BF), bromoacetic acid (BA), dibromoacetic acid (DBA), tribromoacetic acid (TCA), chloroform (CF), chloroacetic acid (CA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), and potassium bromate (KBrO3) in Salmonella typhimurium strains TA98, TA100, and RSJ100 +/- S9. Solvent controls of DMSO and ethanol and a positive control of ethylmethanesulfonate (EMS) were also analyzed. We developed a rapid microplate-based method to determine the cytotoxicity of the DBPs and we determined their mutagenic potencies. The distributions of the rank order for the cytotoxicity and mutagenicity of these DBPs were compared and the structure-function relationships were identified. TA100 -S9 was the most sensitive strain for these DBPs. The rank order of the mutagenic potency adjusted with a cytoxicity factor was MX > BA > EMS > DBA > DCA > CA with TBA, TCA, BF, and CF not mutagenic. From a structure-function perspective, the brominated acetic acids were more cytotoxic and mutagenic than their chlorinated analogs. BA was 150x more mutagenic than CA. The mutagenic potency of the haloacetic acids was inversely related to the number of halogen atoms of the molecule. BA was 36x more mutagenic than DBA. The differential cytotoxicity expressed by the DBPs indicated that a cytotoxicity analysis enhanced the sensitivity of the mutagenicity data, which resulted in an enhanced precision for comparing their relative mutagenic strengths. This information is critical when conducting quantitative structure-function analysis of these hazardous agents.