David J. Doolittle

Evaluation of eight in vitro assays for assessing the cytotoxicity of cigarette smoke condensate

The accurate assessment of cytotoxicity is important for evaluating the potential of a test agent to induce pathologies that result from cell killing and to determine appropriate doses for other endpoints, such as genetic toxicology studies. The objective of this work was to determine the most sensitive assays for assessing cytotoxicity in Chinese hamster ovary (CHO) cells following short-term (1 h) and long-term (24 h) exposure to cigarette smoke condensate (CSC). Eight in vitro cytotoxicity assays with different endpoints were used to evaluate the cytotoxicity of Kentucky reference 1R4F (K1R4F) CSC in CHO cells. The assays used for this study were neutral red uptake, LDH release, kenacid blue binding, MTT formation, XTT formation, acid phosphatase activity, sulforhodamine B binding and resazurin binding. Four of the more widely used cytotoxicity assays (neutral red, MTT, kenacid blue and LDH) were also evaluated at 3-, 6-, 12- and 18-h time points. At the 1-h exposure time, LDH was the most sensitive with toxicity observed beginning at 100 microg/ml. None of the other assays demonstrated a concentration-dependent increase in toxicity after 1-h exposures even at the maximum concentration of 150 microg/ml of CSC. Following 24 h of exposure, neutral red and kenacid blue were the most sensitive. The results of our study indicate the assay that measured membrane integrity was the most sensitive for short exposure times, whereas the neutral red and kenacid blue assays that measured total cell number were more sensitive for longer exposure times.

Comparative QSAR evidence for a free-radical mechanism of phenol-induced toxicity

Phenol and 14 substituted-phenols were tested for their ability to impair epithelial cell membrane integrity in WB rat liver cells as determined by an increase in lactate dehydrogenase release. Two quantitative structure-activity relationship (QSAR) regression equations were developed which showed that separate mechanisms of phenolic cytotoxicity are important - nonspecific toxicity due to hydrophobicity and formation of phenoxyl radicals. The equations most predictive of phenol toxicity are denoted as log1/C=-0. 98sigma(+)+0.77logP+0.23 or log1/C=-0.11BDE+0.76logP+0.21, respectively, where C is the minimum concentration of substituted-phenol required for a toxic response. P is the octanol-water partition coefficient, sigma(+) is the electronic Hammett parameter and BDE is the OH homolytic bond dissociation energy. In the literature, phenol toxicity correlated to sigma(+) is rare, but there is strong evidence that phenols possessing electron-releasing groups may be converted to toxic phenoxyl radicals. A common feature in a variety of cells is generation of elevated amounts of reactive oxygen species (ROS) associated with a rapid growth rate. The slightly elevated cancer risk associated with the use of Premarin may be due to phenoxyl-type radicals derived from one or more of its components.

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter

A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market. In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.

Chemical and biological studies of a newCigarette that primarily heats tobacco. Part 2. In vitro toxicology of mainstreamsmoke condensate

Abstract The genotoxic and cytotoxic potential of mainstream cigarette smoke condensate (CSC) from a new cigarette that primarily heats tobacco (TOB-HT) was compared with that of CSC from a Kentucky reference low “tar” cigarette (1R4F) representative of the current US cigarette market, and Kentucky Reference 1R5F, representative of ultra-low “tar” cigarettes on the US market. TOB-HT was evaluated at concentrations which induced concentration-dependent positive responses with 1R4F and 1R5F in an in vitro toxicology test battery which included sister chromatid exchange, chromosome aberration, and neutral red cytotoxicity assays in CHO cells, and the Ames bacterial mutagenicity assay. CSC from 1R4F and 1R5F was positive in the Ames assay with Salmonella typhimurium strains TA98, TA100, TA1538 and TA1537, and negative with TA1535, while CSC from TOB-HT was negative in all five strains. CSC from 1R4F and 1R5F cigarettes was positive in sister chromatid exchange (SCE), chromosome aberration (CA) and neutral red cytotoxicity assays, while CSC from the TOB-HT cigarette yielded negative results in all the above endpoints. These data indicate that in these assays the genotoxic and cytotoxic potential of CSC from the new cigarette that primarily heats tobacco is significantly less than CSC from Kentucky reference 1R4F and 1R5F cigarettes, which are representative of cigarettes currently sold in the US.

A comparison of the mutagenicity of mainstream cigarette smoke condensates from a representative sample of the U.S. cigarette market with a Kentucky reference cigarette (K1R4F)

The Salmonella mutagenicity assay has been used to investigate the mutagenicity of cigarette smoke and cigarette smoke condensate. The Kentucky reference (K1R4F) cigarette is designed to be representative of full-flavor, low-tar cigarettes sold in the U.S. and to serve as a reference standard for comparative studies on the chemistry and biological activities of cigarette smoke and condensate. The objective of this study was to determine if the mutagenicity of mainstream smoke condensate from the K1R4F, as measured by the Salmonella mutagenicity assay, is representative of the mutagenic activity of U.S. cigarettes. Mainstream smoke condensates prepared in dimethyl sulfoxide from the K1R4F and 73 brand styles (representing greater than 70% of the total U.S. cigarette market) were assayed using Salmonella typhimurium TA98 and TA100 (+S9) at concentrations of 0, 25, 50, 75, 100, 125 and 250 micrograms/plate. Revertants/mg condensate were determined by calculating the slopes of the dose-response curves using linear and nonlinear regression models. Revertants/cigarette were determined by multiplying the revertants/mg condensate by the mg condensate/cigarette. No significant differences (p > 0.05) were observed between the mean mutagenicity of U.S. market and K1R4F mainstream smoke condensates in terms of revertants/mg condensate or revertants/cigarette. Increased variability in mutagenicity was observed among the U.S. brands versus that of the K1R4F. This is not surprising since variability among the U.S. brands would be expected to have both measurement error and brand style variability while the K1R4F variability contains only the measurement error portion. These results demonstrate that the K1R4F is a representative model for the U.S. cigarette market in comparative Salmonella mutagenicity studies using mainstream smoke condensates.

Assessment of Mitochondrial Membrane Potential as an Indicator of Cytotoxicity

Mechanistically based short-term in vitro tests to evaluate the relative cytotoxicity of of chemicals will complement in vitro genotoxicity testing during the initial phases of toxicity evaluation as well as provide information on the cellular site of action for chemicals found to be toxic in animals. The objective of this study was to characterize a procedure for evaluating mitochondrial membrane potential, an integral component of cellular energy homeostasis and normal cellular function, as an in vitro indicator of chemically induced cytotoxicity. Rhodamine 123, a cationic fluorescent dye whose mitochondrial fluorescence intensity decreases quantitatively in response to dissipation of mitochondrial transmembrane potential, was used to evaluate disturbances in mitochondrial membrane potential. Cultured rat liver epithelial cells (WB cell line) or human skin fibroblasts (MSU-2 cell line) treated with the oxidative phosphorylation uncoupler 2,4-dinitrophenol (DNP) or the cytochrome oxidase inhibitor sodium azide were used to characterize the system. In addition, acetaldehyde, which has been reported to damage the plasma membrane, but not the mitochondrial membrane, was used to demonstrate the specificity of this assay system. Mitochondrial membrane potential was not significantly affected by the cell culture density, as long as the cells were in the logarithmic phase of growth. The stage of the cell cycle influenced the mitochondrial membrane potential in human skin fibroblasts (highest in late G1-early S) but not in rat liver cells. DNP and sodium azide significantly (p less than 0.01) reduced the mitochondrial membrane potential in both cell lines compared to untreated cells, while acetaldehyde did not reduce the mitochondrial membrane potential in either cell line. This assay provides a tool for evaluating the effect of chemical treatments on mitochondrial membrane potential, as well as an indicator of cytotoxicity which does not require the use of animals.

Effect of cooking methods on the mutagenicity of food and on urinary mutagenicity of human consumers

The effects of cooking methods on the in vitro mutagenicity of individual foods, the in vitro mutagenicity of meals containing those foods, and the mutagenic exposure of human volunteers following consumption of the meals were examined using Ames bacterial strain TA98 with S-9 metabolic activation. Three methods of food preparation--boiling, baking and frying/flame-broiling--were compared. With meats, frying or broiling resulted in higher in vitro mutagenicity (10- to 50-fold) than did baking or boiling, whereas for carbohydrates, eggs or vegetables mutagenicity did not vary markedly with cooking method. The observed (experimental) mutagenic activity of the meals was quite similar to their calculated (predicted) mutagenicity, obtained by summing the mutagenicity of the individual foods in the meal. The close agreement between experimental and predicted mutagenicity indicated that components of the meal did not interact in either a synergistic or inhibitory manner. The mutagenicity of fried flame-broiled meals was approximately 10-fold greater than the mutagenicity of baked or broiled meals, which were similar in mutagenicity. The mutagenicity of human urine following consumption of the meals was related to the in vitro mutagenicity of the meals themselves. The in vitro mutagenicity of meals is markedly affected by the cooking method used to prepare them and the mutagenicity of the diet may be reflected in the mutagenicity of body fluids.

Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents

Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher that that of the N-nitrosamines. The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N(1)-nitrosonornicotine (NNN). These nitrosamines require metabolic activations by cytochrome P-450s for the expression of mutagenicity. Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported, In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9). All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner. The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine. None of the observed reductions in mutagenicity could be explained by cytotoxicity. These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines.

Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.

Ninety-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette : DNA adducts and alveolar macrophage cytogenetics

The chemical constituents of cigarette smoke are greatly diluted in environmental tobacco smoke (ETS). In the typical indoor environment where cigarettes are smoked, the mean value of respirable suspended particles is approximately 0.1 mg/m3. In this study, we used aged and diluted sidestream smoke (ADSS) of 1R4F University of Kentucky research cigarettes as a surrogate for ETS and exposed Sprague-Dawley rats nose-only to 0, 0.1, 1.0, and 10 mg wet total particulate matter (WTPM)/m3 for 6 hr per day for 14 consecutive days. DNA from lung, heart, larynx, and liver was tested for adduct formation after 7 and 14 days of exposure and after 14 days of recovery. In addition, alveolar macrophages from animals exposed for 7 days were examined for chromosomal aberrations. Exposure-related DNA adducts were not observed in any of the animals at 0.1 or 1.0 mg WTPM/m3, which represent ambient and 10-fold exaggerated ETS concentrations, respectively. Slight diagonal radioactive zones, characteristic of adducts observed in human smokers and in animals exposed to mainstream smoke, were observed, but only in lung and heart DNA of animals exposed to the highest concentration of ADSS (10 mg WTPM/m3), a 100-fold exaggeration of typical field measurements of ETS. The mean relative adduct labeling values (+/- SE) were 8.7 (+/- 0.2) adducts per 10(9) nucleotides for lung DNA and 5.7 (+/- 0.7) adducts per 10(9) nucleotides for heart DNA after 14 days of exposure. No elevation in chromosomal aberrations was observed in alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)