A Systematic Review and Meta-analysis of the Association between E-cigarette Use among Cigarette Smokers and Quit Attempts Made to Abstain from Cigarette Smoking.

Objective: Following AMSTAR 2 and PRISMA guidelines, in this synthesis of evidence we sought to identify and characterize any associations between e-cigarette use among cigarette smokers and cigarette smoking quit attempts. Methods: We queried 3 databases from January 1, 2007 to January 5, 2021. Search results were screened using the PICOS review method. Included studies examined e-cigarette use and cigarette smoking quit attempts across e-cigarette use statuses. Risk of bias was assessed according to the Agency for Healthcare Research and Quality Evidence-Based Practice Center approach. Finally, 4 random-effects models compared e-cigarette users and non- e-cigarette-users in terms of past year and prospective (6 to 12 months) cigarette smoking quit attempts. Results: We qualitatively synthesized 17 adjusted studies for this review. Two meta-analyses showed past year quit attempts were significantly associated with current e-cigarette users and 2 prospective data analyses found no significant association. Conclusions: The results of the meta- analyses emphasize temporality in the association between e-cigarette use and cigarette smoking quit attempts. Numerous methodological limitations, including inadequate definitions of e-cigarette use and non-adjustment for confounding variables, limit the confidence in conclusions that can be drawn on the causal association between e-cigarette use and cigarettes smoking quit attempts.

Assessing the Evidence on the Differential Impact of Menthol versus Non-menthol Cigarette Use on Smoking Dependence in the US Population: A Systematic Review and Meta-analysis.

Background: Menthol's effect on cigarette smoking behaviors is an intensely scrutinized US public health issue. This systematic review and meta-analysis examined the question: Does menthol cigarette use have a differential impact on smoking dependence compared with non-menthol cigarette use? Methods: We consulted 6 databases from inception to October 15, 2021. We included articles comparing menthol versus non-menthol cigarette smokers against predefined smoking dependence outcomes. Risk of bias was assessed using the AHRQ Evidence-Based Practice Center approach. We applied a random-effects model to pool adjusted odds ratios. Results: We synthesized 37 demographically adjusted studies. Meta-analytic results suggested non-menthol smokers were equally/more likely to report daily versus non-daily smoking; menthol use was associated with needing a cigarette within one hour; cigarettes per day was not associated with menthol use; menthol use was associated with a low (vs high) Heaviness of Smoking Index score; and results were either non-significant or associated menthol use with lower TTFC. Conclusions: Despite consistently good or fair quality adjusted studies across several measures, results were discordant depending on measures used and means of measurement. Overall, the evidence is insufficient to draw clear conclusions on a differential association between menthol (vs non-menthol) cigarette use and smoking dependence.

Assessing the Evidence on the Differential Impact of Menthol versus Non-menthol Cigarette Use on Initiation and Progression to Regular Smoking: A Systematic Review and Meta-analysis.

Background: Despite numerous assessments of the public health impact of menthol cigarettes, a rigorous synthesis related to menthol cigarettes and behavioral outcomes is lacking. This systematic review and meta-analysis examined the question: Does menthol cigarette use have a differential impact on initiation and progression to regular smoking compared to non-menthol cigarette use? Methods: We consulted 6 databases from their inception to October 15, 2021. We included articles comparing menthol versus non-menthol smokers among 4 predefined smoking initiation and progression outcomes. We assessed risk of bias was using the Agency for Healthcare Research and Quality Evidence-Based Practice Center approach. We applied a random-effects model to pool adjusted odds ratios. Results: We qualitatively synthesized 16 adjusted studies across the outcomes. Results from one meta-analysis suggested no difference between menthol and non-menthol smokers in likelihood to report daily versus non-daily smoking. Conclusion: This systematic review and meta-analysis did not identify a consistent, statistically significant, or differential association between menthol use and progression to regular smoking. Varying definitions of outcome measures and lack of longitudinal evidence limited the confident conclusions that could be drawn from this evidence base.

Reporting and methodological quality of systematic literature reviews evaluating the associations between e-cigarette use and cigarette smoking behaviors: a systematic quality review.

INTRODUCTION: Several published systematic reviews have examined the potential associations between e-cigarette use and cigarette smoking, but their methodological and/or reporting quality have not yet been assessed. This systematic quality review followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and AMSTAR (A MeaSurement Tool to Assess systematic Reviews) 2 to evaluate the quality of systematic reviews investigating potential associations between e-cigarette use and cigarette smoking. MATERIALS AND METHODS: PubMed/MEDLINE, Embase, and PsycINFO were searched from 01 January 2007 to 24 June 2020. Methodological quality was assessed using AMSTAR 2, and reporting quality was assessed using PRISMA guidelines. RESULTS: Of 331 potentially relevant systematic reviews, 20 met predefined inclusion criteria. Most reviews (n = 15; 75%) reported on e-cigarette use and cigarette smoking cessation, while three reported on e-cigarette use and cigarette smoking initiation (15%); and two reported on cigarette smoking initiation and cessation (10%). According to AMSTAR 2 guidelines, 18 of the 20 reviews (90%) were "critically low" in overall confidence of the results, while two were ranked "low." Additionally, reporting quality varied across the reviews, with only 60% reporting at least half of the PRISMA items. DISCUSSION: Methodological limitations were identified across reviews examining potential associations between e-cigarette use and cigarette smoking behaviors, indicating that findings from these reviews should be interpreted with caution. CONCLUSIONS: Future systematic reviews in this field should strive to adhere to AMSTAR 2 and PRISMA guidelines, to provide high quality syntheses of the available data with transparent and complete reporting.

Assessing the evidence on the differential impact of menthol versus non-menthol cigarette use on smoking cessation in the U.S. population: a systematic review and meta-analysis.

BACKGROUND: The potential impact of menthol versus non-menthol cigarette use on smoking behaviors is an intensely scrutinized topic in the public health arena. To date, several general literature reviews have been conducted, but findings and conclusions have been discordant. This systematic review followed PRISMA guidelines to examine the Key Question, "Does menthol cigarette use have a differential impact on smoking cessation compared with non-menthol cigarette use?" METHODS: Six databases-Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, Database of Abstracts of Reviews of Effects, MEDLINE, Embase and PsycInfo-were queried from inception to June 12, 2020. Articles comparing menthol versus non-menthol cigarette smokers in terms of at least one predefined smoking cessation outcome were included. Risk of bias was assessed using the Agency for Healthcare Research and Quality Evidence-Based Practice Center approach. A random-effects model utilizing the DerSimonian and Laird method to pool adjusted odds ratio was applied. Variations among pooled studies were assessed using Cochran's Q statistic, and heterogeneity was quantified using the inconsistency index (I2). RESULTS: Forty-three demographically adjusted studies (22 rated "good", 20 rated "fair", and one study rated "poor" individual study quality) comparing menthol and non-menthol smokers were qualitatively synthesized across the following measures (study count; strength of evidence): duration of abstinence (2; low); quit attempts (15; insufficient); rate of abstinence/quitting (29; moderate); change in smoking quantity/frequency (5; insufficient); and, return to smoking/relapse (2; insufficient). Overall, the qualitative synthesis failed to show a consistent trend for an association between menthol cigarette use and smoking cessation across outcomes. Meta-analyses found no difference between menthol and non-menthol cigarette use and either quit attempts or abstinence. CONCLUSIONS: Given the lack of consistency or statistical significance in the findings-combined with a "low" overall strength of evidence grade, based on deficiencies of indirectness and inconsistency-no consistent or significant associations between menthol cigarette use and smoking cessation were identified. Recommendations for future studies include increased focus on providing longitudinal, adjusted data collected from standardized outcome measures of cessation to better inform long-term smoking cessation and menthol cigarette use. Such improvements should also be further considered in more methodologically rigorous systematic reviews characterized by objectivity, comprehensiveness, and transparency with the ultimate objective of better informing public health and policy decision making.

Assessing comprehension and perceptions of modified-risk information for snus among adult current cigarette smokers, former tobacco users, and never tobacco users.

INTRODUCTION: Snus, a low nitrosamine smokeless tobacco product, presents less risks to health than cigarettes. Effectively communicating such risk information could facilitate smokers switching completely to snus, thereby benefiting public health. METHODS: This study assessed comprehension and perceptions of modified-risk information regarding snus. Adult cigarette smokers, former tobacco users, and never tobacco users (N = 3,922) from a US internet panel viewed an advertisement stating that smokers who switched completely to snus could greatly reduce risk of lung cancer, respiratory disease, heart disease, and oral cancer. Respondents answered questions regarding the modified-risk information and rated perceived risks of snus relative to cigarettes and other smokeless tobacco products. RESULTS: Across the four diseases mentioned in the advertisement, most respondents (49.7%-68.6%, across tobacco user groups) understood that snus presents less risk than cigarettes but is not completely safe. Some indicated snus presents the same risk as cigarettes; this was highest for oral cancer (33.7%-42.02%) and lowest for lung cancer (15.4%-23.1%) and respiratory disease (15.6%-23.4%). Majorities understood snus is addictive (77.7%-87.9%), quitting all tobacco is the best option for smokers (83.6%-93.1%), and non-users of tobacco should not use snus (80.4%-87.8%). Only 2.1%-5.8% indicated smokers would receive a health benefit if they continued to smoke while using snus. CONCLUSIONS: The modified-risk information, conveying that snus presents less risk than cigarettes but is not completely safe, was understood by majorities of respondents. Differential risk beliefs across diseases suggest responses were shaped not only by the modified-risk information, but also by intuitions and pre-existing beliefs about tobacco products.

Measures of dependence in menthol and nonmenthol smokers - A comprehensive narrative review.

Introduction. More than a decade ago, concerns were raised that menthol in cigarettes might enhance addiction to smoking. This article provides a comprehensive review of published studies examining cigarette dependence among menthol and nonmenthol smokers. The purpose of the review is to evaluate the scientific evidence to determine if menthol increases cigarette dependence. Materials and Methods. The published literature was searched in 2019 for studies that provide evidence on cigarette dependence among menthol compared to nonmenthol smokers. Included in this review are published studies that compare menthol and nonmenthol smokers based on widely accepted and validated measures of dependence, or other established predictors of dependence (age of smoking initiation [first cigarette]/age of progression [regular/daily smoking]) and indicators of dependence (smoking frequency, cigarettes smoked per day, time to first cigarette after waking, night waking to smoke, smoking duration). Results and Conclusion. Based on a review of the available studies, including those with adjusted results and large representative samples, reliable and consistent empirical evidence supports a conclusion that menthol smokers are not more dependent than nonmenthol smokers and thus menthol in cigarettes does not increase dependence.

Assessing the Likelihood and Magnitude of a Population Health Benefit Following the Market Introduction of a Modified-Risk Tobacco Product: Enhancements to the Dynamic Population Modeler, DPM(+1).

Researchers and those responsible for evaluating and implementing policies intended to reduce population harm must assess the potential for both intended and unintended consequences associated with those policies. Such assessments should be based on the combined dimensions of magnitude, and thus likelihood, of shifts in exposure patterns needed to produce a population benefit or harm, and magnitude of the expected population benefit or harm. In response to this assessment need, we provide a conceptual description of the dynamic population modeler, DPM(+1), as well as illustrative analyses that estimate the effects on all-cause mortality, life expectancy, and quality of life-adjusted life expectancy if exposure patterns in the population shift from a higher risk product (e.g., cigarettes) to a lower, or modified, risk tobacco product (MRTP) in specified ways. Estimates from these analyses indicate that, within a single birth cohort, switching completely from cigarette smoking to MRTP use is more likely to lead to a population-level survival benefit than initiating tobacco use with an MRTP instead of cigarettes. This is because tobacco initiation rarely occurs beyond young adulthood, whereas continuing smokers exist in all subsequent age categories, leading to a greater cumulative effect. In addition, complete switching to MRTP use among a small proportion of smokers in each age category offsets the survival deficit caused by unintended shifts in exposure patterns, such as MRTP initiation among never tobacco users followed by transitioning to cigarette smoking and/or cigarette smokers switching to MRTP use instead of quitting.

Measures of initiation and progression to increased smoking among current menthol compared to non-menthol cigarette smokers based on data from four U.S. government surveys.

There are no large-scale, carefully designed cohort studies that provide evidence on whether menthol cigarette use is associated with a differential risk of initiating and/or progressing to increased smoking. However, questions of whether current menthol cigarette smokers initiated smoking at a younger age or are more likely to have transitioned from non-daily to daily cigarette use compared to non-menthol smokers can be addressed using cross-sectional data from U.S. government surveys. Analyses of nationally representative samples of adult and youth smokers indicate that current menthol cigarette use is not associated with an earlier age of having initiated smoking or greater likelihood of being a daily versus non-daily smoker. Some surveys likewise provide information on cigarette type preference (menthol versus non-menthol) among youth at different stages or trajectories of smoking, based on number of days smoked during the past month and/or cigarettes smoked per day. Prevalence of menthol cigarette use does not appear to differ among new, less experienced youth smokers compared to established youth smokers. While there are limitations with regard to inferences that can be drawn from cross-sectional analyses, these data do not suggest any adverse effects for menthol cigarettes on measures of initiation and progression to increased smoking.

Evaluating the association between menthol cigarette use and the likelihood of being a former versus current smoker.

Menthol in cigarettes has been examined for its potential to affect smoking dependence, measured primarily as number of cigarettes smoked per day and time to first cigarette after waking; the ability to quit smoking constitutes an additional measure of dependence. Successful quitting among menthol compared to non-menthol cigarette smokers is difficult to determine from the literature, due in part to the various definitions of quitting used by researchers. Nevertheless, intervention and follow-up studies of smoking cessation treatments generally indicate no differences in quitting success among menthol compared to non-menthol smokers, while cross-sectional studies suggest some differences within race/ethnicity groups. The association between menthol cigarette use and likelihood of being a former versus current smoker was examined based on data from the National Health Interview Survey and Tobacco Use Supplement to the Current Population Survey. Analyses stratified by race/ethnicity and limited to smokers who had quit at least one year prior to survey participation provided inconsistent results with regard to menthol cigarette use and quitting, both within surveys (i.e., comparing race/ethnicity groups) and between surveys (i.e., same race/ethnicity group across surveys). Evidence suggesting the existence or direction of an association between menthol in cigarettes and quitting depended on the data source.

Patterns of menthol cigarette use among current smokers, overall and within demographic strata, based on data from four U.S. government surveys.

The National Health and Nutrition Examination Survey, National Survey on Drug Use and Health, National Health Interview Survey and Tobacco Use Supplement to the Current Population Survey provide estimates of the proportions of U.S. smokers who currently use menthol cigarettes, overall and within demographic strata. Among adult past-month, regular and daily smokers, menthol cigarette use ranges from 26% to 30%, with statistically higher proportions of female versus male smokers (8-11 percentage points higher) currently using menthol cigarettes. Compared to adult smokers overall, statistically higher proportions of non-Hispanic Black smokers (72-79%) and statistically lower proportions of non-Hispanic White smokers (19-22%) currently use menthol cigarettes, with no differences among smokers of other race/ethnicity groups (18-20% to 28-30%, depending on the survey). Higher proportions of younger adult past-month, regular and daily smokers (aged 18-25years) currently use menthol cigarettes compared to older adult smokers (aged 26-29years and/or ⩾30years); however, differences are small in magnitude, with the vast majority of adult smokers (70-75%) who currently use menthol cigarettes being aged ⩾30years. Comparisons between youth and adult smokers are provided, although data for youth smokers are less available and provide less consistent patterns of menthol cigarette use.

Primary measures of dependence among menthol compared to non-menthol cigarette smokers in the United States.

Previously published studies provide somewhat inconsistent evidence on whether menthol in cigarettes is associated with increased dependence. The National Health and Nutrition Examination Survey, National Survey on Drug Use and Health, National Health Interview Survey, and Tobacco Use Supplement to the Current Population Survey collect data on current cigarette type preference and primary measures of dependence, and thus allow examination of whether menthol smokers are more dependent than non-menthol smokers. Analyses based on combined data from multiple administrations of each of these four nationally representative surveys, using three definitions for current smokers (i.e., smoked ⩾1day, ⩾10days and daily during the past month), consistently demonstrate that menthol smokers do not report smoking more cigarettes per day than non-menthol smokers. Moreover, two of the three surveys that provide data on time to first cigarette after waking indicate no difference in urgency to smoke among menthol compared to non-menthol smokers, while the third suggests menthol smokers may experience a greater urgency to smoke; estimates from all three surveys indicate that menthol versus non-menthol smokers do not report a higher Heaviness of Smoking Index. Collectively, these findings indicate no difference in dependence among U.S. smokers who use menthol compared to non-menthol cigarettes.

Safety assessment of diammonium phosphate and urea used in the manufacture of cigarettes.

A tiered testing strategy has been employed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the mainstream smoke or biological activity that results from burning cigarette tobacco. The foundation of this evaluation strategy is comparative testing, typically including chemical and biological assessments. In the manufacture of cigarettes, diammonium phosphate (DAP) and urea have been historically used as ingredients added to tobacco, to reconstituted tobacco sheet, and to other processed tobaccos. As part of ongoing stewardship efforts, a toxicological assessment of cigarettes with and without DAP and urea was conducted. Chemical and biological analyses were conducted for test cigarettes added 0.5% DAP and 0.2% urea in the final blend and also for those added 1.0% DAP and 0.41% urea in the final blend compared to reference cigarettes without added DAP or urea. Principal components of this evaluation included a determination of selected mainstream smoke constituent yields, an Ames assay in Salmonella typhimurium strains TA98 and TA100, a sister chromatid exchange assay in Chinese hamster ovary cells, a 13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats, and a 30-week dermal tumor-promotion evaluation of mainstream cigarette smoke condensate in SENCAR mice. Comparative evaluations demonstrated that the addition of DAP and urea to cigarettes at up to 1% and 0.41%, respectively, does not alter the biological activity compared to reference cigarettes without DAP or urea.

DBA/2 mouse skin is unresponsive to dermal tumor promotion by cigarette smoke condensate.

Previous studies demonstrated that repetitive application of cigarette smoke condensate (CSC) to 7,12-dimethylbenz[a]anthracene (DMBA)-initiated SENCAR mouse skin for 29 weeks at doses of 10, 20 and 40 mg "tar"/application results in time- and dose-dependent dermal tumor formation. To evaluate CSC-induced tumor promotion in other mouse skin models, male DBA/2 mice were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (300 microg) or DMBA (75 or 150 microg) followed by promotion with 1R4F CSC at concentrations ranging from 9 to 45 mg "tar"/application. Both MNNG and DMBA have previously been shown to adequately initiate tumor development. Study end-points included clinical signs, body weights, and mass tracking. Neither the DMBA-initiated/acetone-promoted control groups, nor DMBA-initiated/CSC-promoted groups produced grossly observable skin tumors. For MNNG-initiated groups, a total of four tumors were observed. Based on these findings, it would appear the DBA/2 mouse was unresponsive to CSC dermal tumor promotion. It is not possible, based on the study design employed, to determine the underlying basis for the apparent resistance exhibited by this mouse strain to CSC-induced tumor promotion.

Altered methylation in gene-specific and GC-rich regions of DNA is progressive and nonrandom during promotion of skin tumorigenesis.

Altered DNA methylation, an epigenetic mechanism, likely contributes to tumorigenesis, with an inverse relationship existing between methylation in a promoter region and transcription. Using the SENCAR two-stage mouse skin tumorigenesis model, altered methylation was characterized in precancerous tissue and in tumor tissue. Mouse skin was initiated with 7,12-dimethylbenz[a]anthracene and promoted three times a week with 3, 9, 18, or 27 mg cigarette smoke condensate (CSC) for 4, 8, or 29 weeks; tumors were collected at 29 weeks. In addition, reversibility of changes in methylation was assessed following cessation of the promoting stimulus. DNA was isolated, and GC-rich methylation was assessed quantitatively via methylation-sensitive restriction digestion, arbitrarily primed PCR, and electrophoretic separation of PCR products. Analysis focused on regions of altered methylation (RAMs), which persisted from 4 to 8 weeks and from 8 weeks to tumor tissue. Persistent RAMs (i.e., seen in precancerous tissue and carried forward to tumors) are likely to play a key role in tumorigenesis. Twenty-two CpG sites in the upstream region of the Ha-ras promoter were unmethylated in control skin, 27 mg CSC, and tumor tissue. At two CpG sites closer to the transcriptional start site the incidence of hypomethylation increased with the dose of CSC. Hypomethylation was detected in all tumor samples. Expression of Ha-ras increased with 18 and 27 mg CSC promotion and more so in tumor tissue. These data support our hypothesis that tumor promotion involves instability of the epigenome, providing an environment where changes in the methylation status of specific regions of the genome accumulate progressively and contribute to the clonal expansion of initiated cells that leads to tumor formation.

Short-term biomarkers of cigarette smoke condensate tumor promoting potential in mouse skin.

Previous studies demonstrated that cigarette smoke condensates (CSCs) possessing significantly different tumorigenic potentials according to a standardized 30-week mouse skin tumor-promotion protocol could likewise be discriminated utilizing short-term indices of sustained hyperplasia and/or inflammation (G. M. Curtin et al., 2004, Toxicol. Sci. 81, 14-25). The current study employed a truncated initiation-promotion protocol to further evaluate CSC-induced hyperplasia, examining issues related to time course of induction, existence of a threshold and suitable dynamic range for detectable responses, and reversibility. Condensate application (9-36 mg "tar"/200-microl application, thrice-weekly for 3-15 weeks) induced treatment-related increases for epidermal thickness, proliferative index as assessed by 5-bromo-2'-deoxyuridine (BrdU) labeling, and ornithine decarboxylase (ODC) expression. Interestingly, observed increases for interfollicular BrdU labeling and ODC expression were partially reversed but still elevated upon cessation of promotion, while increases within the perifollicular epidermis remained elevated at a level similar to that observed during CSC application. In particular, assessments based on perifollicular ODC expression would appear to provide a greater opportunity for test article discrimination based on a rapid time to induction, a low threshold and expanded dynamic range of responses, and the potential to account for irreversible changes. These findings are particularly intriguing based on reports suggesting that ODC expression may be necessary for tumor promotion and that mouse skin tumors originate primarily within the perifollicular epidermis.

Increased DNA methylation in the HoxA5 promoter region correlates with decreased expression of the gene during tumor promotion.

Promoter-region DNA methylation inhibits transcription. A two-stage SENCAR (sensitive to mouse carcinogenesis) mouse skin carcinogenicity model was used to examine gene-specific changes in methylation during skin tumor promotion. Analysis was performed on 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin promoted with 9, 18, 27, or 36 mg cigarette smoke condensate (CSC) for 9 wk, or 27 mg CSC for 9 wk and sacrificed 6 wk afterwards (recovery group). Additionally, tumors arising following promotion with 27 mg CSC for 29 wk were assessed. Gene array analysis identified differentially expressed genes. Expression of HoxA5, a tumor suppressor gene, was decreased following 9 wk of treatment with 27 mg CSC, and returned to control levels during recovery. HoxA5 promoter methylation was measured with the enzymatic regional methylation assay (ERMA). DNA was bisulfite-modified, PCR-amplified with primers containing dam sites, incubated with [14C-methyl] S-adenosyl-L-methionine (SAM) and dam methyltransferase for DNA quantification, then incubated with [3H-methyl] SAM and SssI methylase to quantify methylation status. Higher 3H/14C ratios indicate increased methylation. The 3H/14C ratios of animals promoted with 27 or 36 mg CSC (48.2 +/- 6.9 and 24.2 +/- 6.1, respectively) were higher than the control or recovery group ratios (12.3 +/- 0.1 and 12.6 +/- 0.3, respectively); sequence analysis supported these findings. Increased methylation of p16 or O6 methylguanine methyltranferase (MGMT) was detected in 4/8 (50%) of the tumor samples from mice promoted with 27 mg CSC. These data suggest that increased DNA methylation contributes to the downregulation of HoxA5, and combined with hypermethylation of p16 or MGMT, this might facilitate the clonal expansion of increasingly aberrant cells during promotion.

Short-term in vitro and in vivo analyses for assessing the tumor-promoting potentials of cigarette smoke condensates.

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Lung tumorigenicity in A/J and rasH2 transgenic mice following mainstream tobacco smoke inhalation.

Hypothesizing that their respective genetic backgrounds would confer an increased sensitivity to lung tumorigenesis, the plausibility of selected rodent models for the inhalation testing of mainstream tobacco smoke (MTS) was evaluated. Strain A/J and rasH2 transgenic (Tg) mice were exposed to MTS from Kentucky 1R4F research cigarettes using either a whole-body or nose-only exposure regimen. The whole-body regimen consisted of a 20-week exposure period [0.200 mg wet total particulate matter/liter (WTPM/l), 6 h/day, 5 days/week]; nose-only dosing proceeded for 28 weeks [0.040, 0.125, or 0.400 mg WTPM/l, 3 h/day, 5 days/week]. Both regimens included a 16-week recovery period. Gross and microscopic examinations of the lungs were used to evaluate tumor formation, with experimental results supporting the following conclusions: 1. Evaluation of MTS-induced tumorigenicity based on gross evaluation versus microscopic confirmation provides strikingly disparate results, indicating that serial sectioning is necessary for a definitive assessment of lung tumors. 2. While the dosing regimens employed do not allow for a definitive comparison, whole-body exposure appeared to be more effective for inducing statistical changes in tumor multiplicity and incidence compared to nose-only exposure. 3. Exposure-related stress, evidenced as reductions in both body weight gain and background tumor formation, represents a potential confounder during inhalation testing of MTS tumorigenicity, with additional investigation warranted to validate the specificity of exposure-related responses. 4. Comparative findings between A/J and rasH2 Tg mice suggest that the former may be overly sensitive to exposure-related stress, potentially influencing tumorigenic responses.

Progressive alterations in global and GC-rich DNA methylation during tumorigenesis.

DNA methylation plays a key role in the regulation of gene expression, and failure to maintain normal patterns of methylation often contributes to carcinogenesis. We have characterized progressive methylation changes during the promotion stage of carcinogenesis using a SENCAR mouse skin initiation/promotion tumorigenesis model. Mice were initiated with a dermal application of 75 microg dimethylbenz[a]anthracene (DMBA) and promoted with 9, 18, 27, and 36 mg cigarette smoke condensate (CSC) thrice weekly for time periods up to 29 weeks, when a large increase in tumor number was produced by the highest three doses. Global and GC-specific methylation were assessed using SssI methylase and arbitrarily primed PCR, respectively. Changes in GC-specific methylation were dose- and time-dependent. CSC doses required to detect these changes were 27 mg at 6 weeks and 18 mg at 9 weeks. This effect appears to be reversible; changes in GC-specific methylation were less marked after 9 weeks promotion with 27 mg CSC followed by 6 weeks of recovery in comparison to 9 and 15 weeks promotion with 27 mg CSC and no recovery period. Both tumor and non-tumor tissue promoted with 27 mg CSC for 29 weeks exhibited changes in GC-specific methylation that were more pronounced in tumors. Tumor tissue was globally hypomethylated, whereas non-tumor tissue did not exhibit changes in global methylation. In conclusion, as expected for a mechanism underlying tumor promotion, CSC alters methylation in a threshold-exhibiting, reversible, progressive fashion during promotion. Progressive alterations in global and GC-rich methylation appear to be mechanistically important during tumor promotion.