Switching from usual brand cigarettes to a tobacco-heating cigarette or snus: Part 2. Biomarkers of exposure.

A randomized, multi-center study of adult cigarette smokers switched to tobacco-heating cigarettes, snus or ultra-low machine yield tobacco-burning cigarettes (50/group) was conducted, and subjects' experience with the products was followed for 24 weeks. Differences in biomarkers of tobacco exposure between smokers and never smokers at baseline and among groups relative to each other and over time were assessed. Results indicated reduced exposure to many potentially harmful constituents found in cigarette smoke following product switching. Findings support differences in exposure from the use of various tobacco products and are relevant to the understanding of a risk continuum among tobacco products (ClinicalTrials.gov Identifier: NCT02061917).

Switching from usual brand cigarettes to a tobacco-heating cigarette or snus: Part 3. Biomarkers of biological effect.

A randomized, multi-center study of adult cigarette smokers switched to tobacco-heating cigarettes, snus or ultra-low machine yield tobacco-burning cigarettes (50/group) for 24 weeks was conducted. Evaluation of biomarkers of biological effect (e.g. inflammation, lipids, hypercoaguable state) indicated that the majority of consistent and statistically significant improvements over time within each group were observed in markers of inflammation. Consistent and statistically significant differences in pairwise comparisons between product groups were not observed. These findings are relevant to the understanding of biomarkers of biological effect related to cigarette smoking as well as the risk continuum across various tobacco products (ClinicalTrials.gov Identifier: NCT02061917).

Toxicological evaluation of smokeless tobacco: 2-year chronic toxicity and carcinogenicity feeding study in Wistar Han rats.

UNLABELLED: A comprehensive 2-year oral chronic toxicity/carcinogenicity study was conducted with smokeless tobacco using modern toxicological test methods and well-accepted standards. The study included a 1-year interim subgroup to assess toxicity at that intermediate time point. Test groups consisted of a tobacco blend (B) used in snus, and an aqueous tobacco extract of that tobacco blend (E) administered at 0.2, 2, or 5 mg nicotine/kg body weight/day via dosed feed to male and female Wistar Han rats. The dosages were selected to simulate potential exposure in humans ingesting smokeless tobacco or an aqueous extract of smokeless tobacco (the latter intended to simulate a snus extract, to enable bridging these data to snus epidemiology data). The following endpoints were evaluated: clinical observations, body weights, feed consumption (FC), ophthalmic exams, toxicokinetics, clinical pathology, gross pathology, and histopathology. During the 2-year study, clear treatment-related, dose-responsive effects included: (1) increases in plasma nicotine and cotinine (indicating that animals were appropriately exposed to levels relevant to human exposure) and (2) decreases in body weights with some alterations in FC. At the 2-year time point, two tumor types (in the highest B doses) displayed statistically significantly increased incidence trends vs. CONTROLS: (1) uterine carcinoma in females and (2) epididymal mesothelioma in males. Three tumor types displayed statistically significantly decreased incidence trends: (1) mammary gland adenomas in females, (2) skin basal cell carcinomas in females, and (3) thyroid follicular cell adenomas in males. These increases (and decreases) in tumor trends were interpreted as not being treatment-related because: (1) there were no preneoplastic or related non-neoplastic histopathological findings in the treated rats at the 1-year or 2-year time points to suggest that any of these neoplastic findings were treatment-related and (2) the tumor morphologies and incidences were generally within the expected range of historical controls for Wistar Han rats. Findings from this study indicate that chronic exposure of male and female Wistar Han rats to either a tobacco blend used in snus, or a tobacco extract of that blend does not lead to increased toxicity or carcinogenicity, based on the specified outcomes measured.

The time course of expression of genes involved in specific pathways in normal human bronchial epithelial cells following exposure to cigarette smoke.

This study was conducted to determine the time course of gene expression associated with specific signaling pathways in normal human bronchial epithelial (NHBE) cells after exposure to 2 concentrations of 2R4F tobacco mainstream smoke (MSS). Expression of 84 genes representing 18 signal transduction pathways was quantitated in MSS- and air-exposed cultures using real-time polymerase chain reaction (PCR) arrays at 1, 4, and 24 hours following exposure. A confidence score, calculated based on statistical analysis of the degree and reproducibility of expression changes, was used to identify potential biologically significant changes in gene expression. Stimulation of NIAP, an apoptosis inhibitor, suppression of NFKB1 and MYC, representing pro-apoptotic activity, and down-regulation of TCF7 and up-regulation of KLK2, representing anti-/pro-inflammatory responses, were altered 1 hour after exposure to the high concentration of MSS. At the 4-hour time point, the pattern had changed such that 10 different genes were now up-regulated and an additional gene was now down-regulated. Significant changes included genes involved in inflammatory response (LTA, SELPLG, and IL8), repair and wound-healing activity (MMP10), and growth activity (GREB1, EGR1), suggesting repair in this period. By 24 hours, the only up-regulated genes in common with the 4-hour profile were SELPLG and IL8, suggesting continued inflammatory signaling. These results suggest that identification of specific gene expression-based biomarkers of MSS toxicity is promising for investigating specific mechanisms of cellular damage. As expected, the expressed signals were dependent on the concentration of MSS and the postexposure times.

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.

Effect of creatinine and specific gravity normalization techniques on xenobiotic biomarkers in smokers' spot and 24-h urines.

Renal excretion mechanisms are xenobiotic-specific; therefore, accurate exposure assessment requires an understanding of relationships of xenobiotic biomarker concentration and excretion rate to urine flow, specific gravity and creatinine concentration. Twenty-four-hour urine collection for xenobiotic exposure assessment is considered the "gold standard" procedure. Random spot-urine collection is convenient and minimizes subject compliance concerns but requires that normalization techniques be employed to account for diuresis and diurnal variation in xenobiotic biomarker excretion. This paper examines and makes recommendations concerning normalization techniques and conditions under which spot-urine results most accurately reflect 24-h urine results. Specific gravity, creatinine, and xenobiotic biomarkers were determined in smokers' spot and 24-h urines. Normalization techniques were applied, variance-component analyses were performed to estimate variability, spot urines were pooled mathematically to simulate 24-h urines and analyses of variance were performed to evaluate spot urines' ability to reflect 24-h urine concentrations. For each xenobiotic biomarker concentration, log-linear relationships were observed with urine flow, specific gravity, and creatinine. For most xenobiotic biomarker excretion rates, log-linear relationships were observed with urine flow; creatinine, however, was unaffected by urine flow. The conventional creatinine ratio-normalization technique demonstrated greater variability (within-day, between-day and between-subject) than other normalization techniques. Comparisons of simulated 24-h urines to spot urines suggest that spot-urine collection be performed only between 2 p.m. and 2 a.m. and that the modified specific-gravity-adjusted-creatinine ratio-normalization technique and the creatinine-regression normalization technique yield the best agreement between spot- and simulated 24-h urine results.

Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples.

Matrix-degrading and pro-inflammatory changes in human vascular endothelial cells exposed to cigarette smoke condensate.

Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation of this finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 microg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation.

Relationship between FTC 'tar' and urine mutagenicity in smokers of tobacco-burning or Eclipse cigarettes.

The US Federal Trade Commission (FTC) classifies domestic cigarettes into one of three 'tar' categories based on 'tar' and nicotine levels. The objective of the present study was to determine urine mutagenicity in groups of smokers of ultra-low 'tar' (ULT), full-flavor low 'tar' (FFLT) and full-flavor 'tar' (FF) filtered cigarettes after switching to primarily tobacco-heating Eclipse cigarettes. Sixty-seven smokers maintained a specified diet and consumed ad libitum their usual brands of cigarettes, switched to Eclipse, and switched back to their usual brands. Twenty-four hour urine samples were collected weekly, concentrated on XAD-2 resin, and tested in the Ames mutagenicity assay using bacterial strains TA98 and YG1024 with S9 metabolic activation. Daily consumption of cigarettes was not significantly different (at P<0.05) between FTC 'tar' categories and average daily cigarette consumption did not change significantly in any smoker group after switching to Eclipse cigarettes. Average urine mutagenicity was 47% less (P<0.05) for ULT than for FFLT usual brand smokers as measured by the more sensitive strain YG1024, although no significant differences (P<0.05) were observed in urine mutagenicity between usual brand FTC 'tar' categories as measured by strain TA98. The reduction in urinary mutagens in the more sensitive strain, YG1024, observed in ULT smokers as compared with higher 'tar' categories suggest reduced exposure to mutagens. Usual brand salivary cotinine in the ULT group was significantly lower (P<0.05) than the FF group and the FFLT group. Salivary cotinine did not differ significantly (at P<0.05) among the smoker groups when smoking Eclipse compared to usual brand. After switching to Eclipse, the following reductions in urinary mutagenicity were observed: ULT, 70.1+/-6.4% (TA98), 70.9+/-6.2% (YG1024); FFLT, 77.1+/-2.4% (TA98), 73.6+/-2.0% (YG1024); and FF, 76.1+/-3.5% (TA98), 71.4+/-4.0% (YG1024). Across all 'tar' categories, cigarette smokers experienced significant reductions (P<0.05) in urine mutagenicity, but not salivary cotinine, upon switching to Eclipse. The reduction in urine mutagenicity when smoking Eclipse provides supporting evidence that Eclipse may present less risk of cancer compared to cigarettes currently in the market.