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.     

Toxicological evaluation of an electrically heated cigarette. Part 3: Genotoxicity and cytotoxicity of mainstream smoke

The in vitro toxicity of cigarette mainstream smoke from an electrically heated cigarette (EHC) with controlled combustion was compared with that of the standard University of Kentucky Reference Cigarette 1R4F.In the Salmonella reverse mutation assay, strains TA98, TA100, TA102, TA1535 and TA1537 were used in the absence and presence of a metabolic promutagen activation system (S9) to determine the mutagenic potential of the total particulate matter (TPM), which was collected on a glass-fiber filter. In the neutral red uptake assay, mouse embryo BALB/c 3T3 cells were used to determine the cytotoxic potential of TPM as well as of the water-solubles in the gas/vapor phase trapped in phosphate-buffered saline.The TPM from the electrically heated cigarette was up to 90% lower in mutagenicity than that of the 1R4F calculated on an equal TPM basis. This reduction in mutagenicity is consistent with the significantly lower concentration of nearly all constituents analyzed in EHC smoke. With regard to cytotoxicity when calculated on an equal TPM basis, TPM from the electrically heated cigarette was 40% less active relative to the 1R4F. When calculated on a per cigarette basis, the cytotoxicity of both the TPM fraction and the water-solubles in the gas/vapor phase of smoke from the EHC was ca. 80% lower relative to the 1R4F.     

A comparison of in vitro toxicities of cigarette smoke condensate from Eclipse cigarettes and four commercially available ultra low-"tar" cigarettes.

Eclipse is a cigarette that primarily heats rather than burns tobacco. R.J. Reynolds Tobacco Company (RJRT) has previously reported the results of in vitro toxicity studies comparing Eclipse with University of Kentucky 1R5F and 1R4F reference cigarettes. To characterize the differences between Eclipse and very low yielding/ultra low-"tar" (vULT) tobacco-burning cigarettes, RJRT conducted a comparative evaluation of the genotoxicity and cytotoxicity of mainstream cigarette smoke condensate (CSC) from Eclipse and three vULT tobacco-burning cigarettes (Now 83 Box, Merit Ultima and Carlton Soft Pack) as well as the leading ultra low-"tar" (ULT) brandstyle (Marlboro Ultra Lights) under four smoking regimens: (1) FTC-35 ml puff volume every 60 s for a 2 s duration (35/60/2); (2) 50/30/2, 0% vents blocked; (3) Massachusetts-45/30/2, 50% vents blocked; (4) Canadian-55/30/2, 100% vents blocked. Ames testing indicated that Eclipse CSC was less (P<0.05) mutagenic than CSC from the four cigarettes under all smoking regimens when compared on a revertants per mg Total Particulate Matter (TPM) basis. When mutagenicity was calculated on a revertants per cigarette basis the mutagenicity of Eclipse CSC was lower (P<0.05) than the mutagenicity of Merit Ultima, Carlton Soft Pack, and Marlboro Ultra Lights, regardless of the puffing regimen. On a per cigarette basis, the calculated mutagenicity of Eclipse was higher (P<0.05) than Now 83 Box cigarettes in the FTC and 50/30/2 regimens but lower (P<0.05) in the Massachusetts and Canadian regimens. Eclipse CSC was less (P<0.05) cytotoxic as measured in the neutral red assay (based on EC(50) values-microg TPM/ml media) than the CSC from the four test cigarettes regardless of the regimen used. Collectively, these data demonstrate that the toxicity of CSC from Eclipse is significantly reduced relative to the activity of CSC from the tested vULT cigarettes and the Marlboro Ultra Lights.     

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 reducesthe amount of certain vapor phase constituents of tobacco smokewith greater efficiency than the charcoal filters of cigarettescurrently in the market In vitro indicators of genotoxic andcytotoxic potential were used to compare the cigarette smokecondensate (particulate phase) or whole cigarette smoke (vaporphase and particulate phase) from cigarettes containing thenovel carbon filter with smoke condensate or whole smoke fromcommercial or prototype cigarettes not containing the novelcarbon filter. Ames bacterial mutagenicity, sister chromatidexchange (SCE) in Chinese hamster ovary (CHO) cells, and neutralred cytotoxicity assays in CHO cells were utilized to assessthe genotoxic and cytotoxic potential of the cigarette smokecondensates. SCE and neutral red cytotoxicity assays were utilizedto assess the genotoxic and cytotoxic potential of the wholesmoke. As expected, the novel carbon filter did not significantlyaffect the genotoxic or cytotoxic activity of the smoke condensate,although we did observe that the use of low-nitrogen tobaccoreduced the mutagenicity of the condensate in Salmonella typhimuriumstrain TA98. However, the whole smoke from cigarettes containingthe novel carbon filter demonstrated significant reductionsin genotoxic and cytotoxic potential compared to cigaretteswithout the novel carbon filter. The toxicity of the smoke wascorrelated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCEinduction) to the aggregate mass of several vapor phase components(acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene,ammonia, NO_x, HCN, benzene, isoprene, and formaldehyde) in thesmoke of the cigarettes utilized in this study. In conclusion,this novel carbon filter, which significantly reduced the amountof carbonyls and other volatiles in mainstream cigarette smoke,resulted in significant reductions in the genotoxic and cytotoxicactivity of the smoke as measured by these assays.     

Evaluation of the potential effects of ingredients added to cigarettes. Part 3: in vitro genotoxicity and cytotoxicity.

Cigarette mainstream smoke from blended cigarettes with and without the addition of ingredients was assayed for its cytotoxicity and genotoxicity. In total, 333 ingredients commonly used in cigarette manufacturing were assigned to three different groups. Each group of ingredients was added at a low and a high level to the test cigarettes. The mutagenicity of the particulate phase of the resulting cigarette smoke was assayed in the Salmonella plate incorporation (Ames) assay with tester strains TA98, TA100, TA102, TA1535 and TA1537. The cytotoxicity of the gas/vapor phase and the particulate phase was determined in the neutral red uptake assay with mouse embryo BALB/c 3T3 cells. Within the sensitivity and specificity of the test systems, the in vitro mutagenicity and cytotoxicity of the cigarette smoke were not increased by the addition of the ingredients.     

A Bayesian statistical analysis of mouse dermal tumor promotion assay data for evaluating cigarette smoke condensate

The mouse dermal assay has long been used to assess the dermal tumorigenicity of cigarette smoke condensate (CSC). This mouse skin model has been developed for use in carcinogenicity testing utilizing the SENCAR mouse as the standard strain. Though the model has limitations, it remains as the most relevant method available to study the dermal tumor promoting potential of mainstream cigarette smoke. In the typical SENCAR mouse CSC bioassay, CSC is applied for 29 weeks following the application of a tumor initiator such as 7,12-dimethylbenz[a]anthracene (DMBA). Several endpoints are considered for analysis including: the percentage of animals with at least one mass, latency, and number of masses per animal. In this paper, a relatively straightforward analytic model and procedure is presented for analyzing the time course of the incidence of masses. The procedure considered here takes advantage of Bayesian statistical techniques, which provide powerful methods for model fitting and simulation. Two datasets are analyzed to illustrate how the model fits the data, how well the model may perform in predicting data from such trials, and how the model may be used as a decision tool when comparing the dermal tumorigenicity of cigarette smoke condensate from multiple cigarette types. The analysis presented here was developed as a statistical decision tool for differentiating between two or more prototype products based on the dermal tumorigenicity.     

The relative toxicity of compounds in mainstream cigarette smoke condensate.

Many different in vivo and in vitro tests are currently used to assess the toxicity of chemicals and complex mixtures such as cigarette smoke condensate. In vivo tests include assays in rodents to determine carcinogenicity, tumorigenicity and reproductive effects In vitro tests of mutagenicity are conducted with both bacterial and mammalian cell systems. A first step towards lowering the toxicity of cigarette smoke condensate is the identification of the relevant compound However, changing the concentration of a given smoke component may not linearly alter the biological activity of the complex mixture due to interactive effects. The "effective toxicity" of a chemical constituent is a function of the concentration, the metabolic fate, the potency in in vivo and in vitro assays, and the ability to reach the target tissues. The logarithm of the octanol-water partition coefficient (log P) is an important parameter since it affects metabolism, biological transport properties and intrinsic toxicity. Using concentration data from the International Agency for Cancer Research (IARC), biological activity data from the Registry of Toxic Effects of Chemical Substances (RTECS) database and measured and calculated log P values, we have rank ordered some of the important compounds in cigarette smoke condensate by their measured or potential toxicity. Condensates from different cigarette brands, tar categories and styles vary in their concentrations of these compounds. Chemicals of greater commercial or scientific interest may be toxicity tested more extensively, thereby increasing the probability of positive test results and highlighting the need for consideration of structure-activity relationships.     

Cigarette smoke condensate extracts augment collagen-induced arthritis in mice

Although cigarette smoking is a solid environmental risk factor for rheumatoid arthritis (RA) as revealed by epidemiological studies, the scientific basis has not been provided. Proinflammatory cytokines produced by synoviocytes are implicated in the pathogenesis of RA. As cigarette smoke condensate (CSC) is able to up-regulate the production of proinflammatory cytokines from human fibroblast-like synoviocytes, we studied the effect of CSC on induction of arthritis in the mouse model of collagen type II-induced arthritis (CIA). When mainstream CSC or sidestream CSC was administered into DBA/1J mice at the time of immunization with collagen and complete Freund adjuvant, CSC dose-dependently augmented the induction and clinical development of arthritis at both young and older mice. Peritoneal injected mainstream CSC one day before immunization also exhibited the augmenting effect, suggesting the systemic effect of CSC. These results support the etiological role of cigarette smoking in RA.     

Kinetic analysis of cytokine response to cigarette smoke condensate by human endothelial and monocytic cells

Atherosclerosis is generally considered an inflammatory disease characterized by the accumulation of lipid in large and medium elastic arteries. Individuals who smoke are at increased risk for developing atherosclerosis and the clinical events associated with this disease. Underlying the mechanisms involved in atherosclerotic lesion development exists a complex pattern of signaling, involving molecules (cytokines and chemokines) that mediate the progression of arterial lesions. The unique nature of exposure to tobacco-related toxicants during the process of smoking prompted our investigation of the time-dependent responses of two critical cell types to cigarette smoke condensate exposure. In this study, we examined the kinetic responses, using suspension array technology and RT-PCR of 17 cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17 GM-CSF, G-CSF, INF-gamma, TNF-alpha, MCP-1 and MIP-1beta) in human aortic endothelial cells (HAECs) and THP-1 monocyte macrophages following exposure to cigarette smoke condensate (CSC) for 24h. In HAECs, IL-8 and IL-4 were rapidly stimulated by CSC exposure while, surprisingly, MCP-1 expression was downregulated. In THP-1 macrophages, IL-6, MIP-1beta, MCP-1 and IL-1beta protein expression were suppressed upon CSC exposure. All other measurable cytokines in THP-1 cells exposed to CSC had levels of protein and mRNA similar to controls. Depending on cell type, CSC uniquely influences the expression of cytokines. The complex interplay of these signaling molecules within the framework of atherosclerosis points to the ability of cigarette smoke components to alter such signaling following acute exposure, and by this mechanism may alter the course of both atherogenesis initiation and progression.     

Cigarette smoke condensate and total particulate matter severely disrupts physiological angiogenesis

The cigarette smoke condensate (CSC) and total particulate matter (TPM) in cigarette smoke is extremely toxic and may produce several pathologies. In our study, we used a chicken chorioallantoic membrane assay (CAM) to study the toxicological effects of CSC and TPM on different aspects of angiogenesis. CSC and TPM from four different commercial filtered cigarettes were applied to the CAMs on day 6 of incubation. Macroscopic vascular transformations were observed among all treated CAMs. The application of CSC disks caused obliteration of main blood vessels, while the entire architecture of the secondary and tertiary vasculature was completely destroyed. Likewise, the application of TPM from all brands of cigarette caused the disproportionate thinning of all primary and secondary blood vessels. A reduction in the total area and diameter of the primary, secondary, and tertiary blood vessels was observed after treatment with CSC and TPM. Histological evaluations revealed the loss of ectodermal and mesodermal integrity with both types of treatments. We also noted a profound inflammatory reaction restricted to the disk area with a novel filopodial deformity of the endoderm in the CSC treated groups. Scanty capillary plexus formation, deterioration of the extracellular matrix, and delayed migration of blood vessels were prominent findings among all treated groups. Results obtained from the CSC treated groups were more localized, while more generalized results were recorded in the TPM treated groups. Special caution should be taken for the presence of CSC and TPM while smoking during pregnancy or after surgery because it may severely affect the process of angiogenesis, which is vital to the maintenance of pregnancy and wound healing.     

In vitro bioactivity of combustion products from 12 tobacco constituents.

Twelve chemical components of tobacco leaf, representing 50% of its dry weight, were individually combusted and the bioactivities of their combustion products i.e. total particulate matter (TPM) were assayed using three in vitro tests. These components included carbohydrates, amino acids, proteins, polyphenols and carboxylic acids. The mutagenic potencies were assessed with the Salmonella mutagenicity assay (S. typhimurium TA98 and TA100). The induction of chromosomal damage, determined with the micronucleus test (IVMNT), and the neutral red uptake cytotoxicity test (NRU), were conducted on V79 hamster lung fibroblast cells. The Salmonella mutagenicity test and IVMNT were conducted with and without rat liver microsomal S9 fraction. Salmonella mutagenicity data confirmed the mutagenicity of TPM samples obtained from nitrogenous compounds (amino acids and proteins). The IVMNT showed that precursors of phenols in smoke (i.e. polyphenols) exhibited significantly higher levels of toxicity compared to other tobacco components. While S9 activation amplified the Salmonella mutagenicity response to combustion products, it significantly inhibited the toxicity measured with the IVMNT. NRU data demonstrated the increasing cytotoxicity induced following longer exposure time to TPM samples from nitrogenous and phenolic components. This study is the first to characterize the toxicity of the combustion products of major tobacco constituents. Our data suggest different mechanisms of toxicity and underline the relevance of using various bioassays.     

The effect of solvent and extraction methods on the bacterial mutagenicity of sidestream cigarette smoke

The mutagenic activity of sidestream cigarette smoke particles was estimated by testing sidestream cigarette smoke particles which had been collected under controlled burning conditions in the laboratory. Two different extraction methods (Soxhlet and ultrasonic agitation) and 3 different solvents (dichloromethane, methanol, and acetone) were compared for their efficiencies in the extraction of compounds from sidestream cigarette smoke particles which are mutagenic in the Ames test. The mutagenic activity of the sidestream smoke particles was estimated to be 15,000-20,000 revertants per cigarette in TA98 with metabolic activation and 12,000-17,000 revertants per cigarette in TA100 without metabolic activation. Only weak mutagenic activity was detected in TA98 without activation and in TA100 with activation. Under test conditions used, ultrasonic agitation produced the most consistent results and acetone extraction produced the highest levels of mutagenic activity.     

Paternal exposure to cigarette smoke condensate leads to reproductive sequelae and developmental abnormalities in the offspring of mice

Paternal smoking is associated with infertility, birth defects and childhood cancers. Our earlier studies using cigarette smoke condensate (CSC) demonstrated several deleterious changes in male germ cells. Here, we hypothesize that chronic paternal exposure to CSC causes molecular and phenotypic changes in the sire and the offspring, respectively. In this mouse study, CSC caused DNA damage and cytotoxicity in testes via accumulation of benzo(a)pyrene (B[a]P) and cotinine. Decreased expression of growth arrest and DNA damage inducible alpha (Gadd45a), aryl hydrocarbon receptor (Ahr), and cyclin-dependent kinase inhibitor 1A (P21) was seen in CSC exposed testes. Apoptotic germ cell death was detected by induction of Fas, FasL, and activated caspase-3. The CSC-exposed males displayed reduction in sperm motility and fertilizing ability and sired pups with reduced body weight and crown-rump length, and smaller litter size with higher numbers of resorption. This model of CSC exposure demonstrates testicular toxicity and developmental defects in the offspring.     

A comparative in vitro toxicity assessment of electronic vaping product e-liquids and aerosols with tobacco cigarette smoke

The use of electronic vaping products (EVPs) continues to increase worldwide among adult smokers in parallel with accumulating information on their potential toxicity and relative safety compared to tobacco smoke. At this time, in vitro assessments of many widely available EVPs are limited. In this study, an in vitro battery of established assays was used to examine the cytotoxic (Neutral red uptake), genotoxic (In vitro micronucleus) and mutagenic (Bacterial reverse mutation) responses of two commercial EVPs (blu GO™ disposable and blu PLUS+™ rechargeable) when compared to smoke from a reference cigarette (3R4F). In total, 12 commercial products were tested as e-liquids and as aerosols. In addition, two experimental base liquids containing 1.2% and 2.4% nicotine were also assessed to determine the effect of flavour and nicotine on all three assays.In the bacterial reverse mutation (Ames) and in vitro micronucleus (IVM) assays, exposures to e-liquids and EVP aerosols, with and without nicotine and in a range of flavourings, showed no mutagenic or genotoxic effects compared to tobacco smoke. The neutral red uptake (NRU) assay showed significantly reduced cytotoxicity (P < .05) for whole undiluted EVP aerosols compared to tobacco smoke, which by contrast was markedly cytotoxic even when diluted.The reduced in vitro toxicological responses of the EVPs add to the increasing body of scientific weight-of-evidence supporting the role of high-quality EVPs as a harm reduction tool for adult smokers.     

In vivo versus in vitro airway surface liquid nicotine levels following cigarette smoke exposure

Whole cigarette smoke (WCS) is composed of approximately 5% particulates and 95% vapors by weight and is difficult to reproduce quantitatively in the laboratory, where typically, routine in vitro application of smoke normally only utilizes the particulate phase. In this study, we used a system for exposing epithelial cells cultured at an air-liquid interface to WCS. We hypothesized that the use of WSC in vitro was more relevant to what is seen in vivo than methods of cigarette smoke application that only use a small fraction of WCS [i.e., aqueous extract or cigarette smoke condensate (CSC)]. To test this hypothesis, we compared nicotine and cotinine concentrations (measured by mass spectrometry) in the airway surface liquid (ASL) of human primary bronchial epithelial cultures (HBECs) exposed to serial dilutions of WCS to the concentrations found in induced sputum of human subjects who had recently smoked a cigarette; this was also compared to the concentrations found after an exposure to a concentration of CSC commonly used in vitro. When measured by mass spectrometry, nicotine levels were not significantly different in induced sputum versus the ASL of HBECs exposed in vitro to a 1:30 exposure of WCS. However, HBECs that had been exposed to CSC returned significantly lower concentrations of ASL nicotine. These results suggest that nicotine is a good dosimetry marker of WCS exposure and provides direct evidence that the use of WCS is more relevant than the use of CSC for in vitro systems.     

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by hte metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and β-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.     

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.     

Cigarette smoke condensate increases cathepsin-mediated invasiveness of oral carcinoma cells

Cigarette smoke, which contains several carcinogens known to initiate and promote tumorigenesis and metastasis, is the major cause of oral cancer. Lysosomal cathepsin proteases play important roles in tumor progression, invasion and metastasis. In the present work we investigated the effects of cigarette smoke condensate (CSC) on cathepsin (B, D and L) expression and protease-mediated invasiveness in human oral squamous cell carcinoma (OSCC) cells. Our results show that treatment of OSCC cells (686Tu and 101A) with CSC activated cathepsins B, D and L in a dose-dependent manner. Both expression and activity of these cathepsins were up-regulated in CSC-exposed versus non-exposed cells. Although cathepsin L had the lowest basal level, it had the highest induction in exposed cells compared to cathepsins B and D. Suppression of CSC-induced cathepsin B and L activities by specific chemical inhibitors decreased the invasion process, suggesting that these proteases are involved in the invasion process. Overall, our results indicate that CSC activates cathepsin B and L proteolytic activity and enhances invasiveness in OSCC cells, a response that may play a role in CSC-mediated tumor progression and metastasis dissemination.     

Cigarette smoke condensate induces cytochromes P450 and aldo-keto reductases in oral cancer cells

Our objective is to identify molecular factors which contribute to the increased risk of smokers for oral squamous cell carcinoma (OSCC). In the present study, we investigated the effects of cigarette smoke condensate (CSC) on gene expression profiles in different human oral cell phenotypes: normal epidermal keratinocytes (NHEK), oral dysplasia cell lines (Leuk1 and Leuk2), and a primary oral carcinoma cell line (101A). We determined differential gene expression patterns in CSC-exposed versus non-exposed cells using high-density microarray RNA expression profiling and validation by quantitative real-time RT-PCR. A set of 35 genes was specifically up- or down-regulated following CSC treatment (25microg/ml for 24h) by at least 2-fold in any one cell type. Notably, five genes of the cytochrome P450 (CYP1A1, CYP1B1) and aldo-keto reductase (AKR1C1, AKR1C3, AKR1B10) families were highly increased in expression, some of them 15- to 30-fold. The timing and extent of induction for these genes differed among the four cell phenotypes. A potential biological interaction network for the CSC response in oral cells was derived from these data, proposing novel putative response pathways. These CSC-responsive genes presumably participate in the prevention or repair of carcinogen-induced DNA damage in tobacco-related oral carcinogenesis, and may potentially be exploited for determining the severity of exposure and for correcting mutagenic damage in exposed tissues of the oral cavity.