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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.

Heating tobacco, rather than burning it, reduces tobacco combustion and pyrolysis products. This study tested the hypothesis that the simplified smoke chemistry of a cigarette which primarily heats tobacco (TOB-HT) significantly reduces the potential to alter the structure or function of cellular plasma membranes relative to low "tar" 1R4F and ultra low "tar" lR5F Kentucky reference cigarettes which burn tobacco. Gap junction intercellular communication (GJIC) and lactate dehydrogenase release (LDH) were used to quantify functional and structural changes to the plasma membrane, respectively. Cigarette smoke condensate (CSC) from the mainstream smoke of TOB-HT, lR4F and 1R5F cigarettes were compared in the GJIC and LDH release assays following a 1-hr exposure in vitro. Human bronchial/tracheal epithelial cells, coronary artery endothelial cells, coronary artery smooth muscle cells, foreskin keratinocytes and the WB-344 rat liver epithelial cell line were studied. TOB-HT did not inhibit GJIC in any of the human cell types tested (P0.05) at concentrations where 1R4F and lR5F did inhibit GJIC (P<0.05). TOB-HT did not elevate LDH release (P0.05) when tested at concentrations where lR4F and lR5F did elevate LDH release (P<0.05). Our results suggest that CSC from TOB-HT cigarettes is less damaging to the structure or function of the cellular plasma membranes of a variety of human cell lines than CSC from 1R4F and 1R5F tobacco burning reference cigarettes.

The neutral red assay, a rapid and accurate method for estimating the cytotoxicity of chemicals, has been used to assess the cytotoxicity of cigarette smoke condensate (CSC), a complex chemical mixture containing over 3000 identified compounds. The first objective was to optimize the neutral red assay for evaluation of CSCs. This study also assessed and compared the cytotoxicity of smoke condensates from three reference cigarettes which differ in 'tar' content; cigarettes of different tobacco type composition; an ultra-low tar cigarette (R1); and an RJR test cigarette which heats but does not burn tobacco. Finally, this study investigated the cytotoxicity of a specific CSC component, nicotine, and its metabolite, cotinine. Exposure times of 24 hours or longer using CHO cells provided optimal conditions for evaluation of CSC cytotoxicity. The cytotoxicity of CSCs from reference cigarettes was similar. CSC from cigarettes comprised of flue-cured tobacco exhibited greater cytotoxicity than CSC from cigarettes comprised of burley tobacco. CSC from the R1 cigarette exhibited similar cytotoxicity compared with 1R4F CSC. The CSC from a cigarette that heated but did not burn tobacco (RJR test cigarette) demonstrated no cytotoxicity in CHO cells. Finally, nicotine and cotinine were not cytotoxic to CHO cells. The neutral red assay has been proved useful for quantifying differences in cytotoxicity of smoke condensates from cigarettes which vary in 'tar' yield and for assessing specific smoke constituents.