Walter T. Morgan

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 ofthis 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 μg/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.

Environmental tobacco smoke (ETS): A market cigarette study

Abstract The environmental tobacco smoke (ETS) yield of selected analytes was determined for the 50 top-selling U.S. cigarette brand styles (1991) and the University of Kentucky Research cigarette, K1R4F. ETS was generated by smokers in an environmental test chamber. Analytes determined included real-time measurements of nicotine, 3-ethenylpyridine, respirable suspended particles (RSP), carbon monoxide, and total hydrocarbons by flame ionization detector response (FID). Real-time RSP values were determined independently by a piezoelectric balance and real-time aerosol monitor (RAM). Additional analytes determined on a time-integrated basis included: nicotine, 3-ethenylpyridine, myosmine, RSP, ultraviolet particulate matter (VUPM), fluorescent particulate matter (FPM), solanesol, scopoletin, formaldehyde, acetaldehyde, acetone, catechol, ammonia, 34 volatile organic compounds (VOCs), and total VOCs (estimated by GC/mass spectrometric response). In general, lowering mainstream tar resulted in lower ETS emissions. The current study showed that ETS-RSP and nicotine were not predictive of each other. In fact, this ETS market brand style comparison showed a poor relationship between ETS nicotine and ETS-RSP. ETS analyte yields are summarized by mainstream tar categories, and overall sales-weighted average yields are calculated. Sales-weighted average ETS-RSP yields for full flavor (FF), full flavor low tar (FFLT), and ultra low tar (ULT) were 14.86, 12.30, and 10.51 mg/cig, respectively. The average RSP yield for all cigarettes evaluated was 13.67 mg/cig. These results, based on 65.3% of the U.S. cigarette market, should enable better estimations of the contribution of ETS to indoor air.

Determination of volatile organic compounds and ETS apportionment in 49 homes

Abstract Forty-nine nonsmoking married women participated in a home personal exposure study for 28 volatile organic compounds (VOCs) and total volatile organic compounds (TVOCs). The women were selected and classified according to 18 socioeconomic categories based on age (18–34 y, 35–49 y, 50–64 y), family income (

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

40K), and husbands smoking status. Of the 29 analytes, 21 demonstrated no statistically significant difference in concentration between nonsmoking and smoking homes. One VOC, trichloroethylene, was elevated in the nonsmoking homes and seven VOCs, benzene, styrene, pyridine, 2-picoline, 3-picoline, 3-ethylpyridine, and 3-ethenylpyridine were elevated in the smoking homes. A correlation matrix and a factor analysis indicate that benzene and styrene were not significantly correlated or associated with 3-ethyenylpyridine, a proposed vapor phase environmental tobacco smoke (ETS) marker. All of the nitrogenous bases were significantly correlated with 3-ethenylpyridine. Benzene, styrene, and TVOC were not significantly correlated with the number of cigarettes smoked; however, 3-ethenylpyridine was significantly correlated with the number of cigarettes smoked. A Pearson correlation analysis indicated that gas heat and smoking husband were significantly correlated with elevated benzene concentrations, but a multiple regression model for benzene accounted for less than 30% of the total variance. ETS variables accounted for only 8% of the total variance. In the smoking homes, an apportionment technique was evaluated for selected VOCs in order to determine the median percentage of each analyte attributable to ETS. The results, with percentages attributable to ETS were TVOC (5.5%), benzene (13.2%), styrene (12.6%), pyridine (40.7%), 2-picoline (67.1%), 3-picoline (90.1%), 4-picoline (37.2%), and 3-ethylpyridine (62.0%). Indoor air sources other than ETS were also identified for limonene, tetrachlorethylene, 1,4-dichlorobenzene, and alkylbenzenes.

Determination of volatile organic compounds and respirable suspended particulate matter in New Jersey and Pennsylvania homes and workplaces

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.

National incidence of smoking and misclassification among the U.S. married female population

Abstract One hundred-four self-reported nonsmoking married women participated in a home and workplace personal environmental tobacco smoke (ETS) exposure study for 33 volatile organic compounds (VOCs), total volatile organic compounds (TVOCs), respirable suspended particulate matter (RSP), and ETS-RSP. The women were selected and classified according to socioeconomic categories based on age (25–39 y and 40+ y), total annual household income (

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

40K), and reported ETS exposure status at home and at work (SH = smoking home, NSH = nonsmoking home, SW = smoking work, and NSW = nonsmoking work). Saliva samples were collected at the start and at the end of the study for cotinine determinations. Five participants (4.8% of the total), married to smokers and working in smoking workplaces, were excluded because they had average salivary cotinine concentrations greater than 10 ng/mL indicating that they were likely smokers. The background correction factor for cotinine (SH/NSH) or Z, indicated that total exposure was 4.8 times greater for those living with a smoker versus those not living with a smoker. Apportionment of TVOCs indicated that 3.4% of the TVOCs in the smoking homes and 0.8% of the TVOCs in the smoking workplaces were attributable to ETS. Apportionment of benzene and styrene indicated that 11.4% and 13.4%, respectively, were attributable to ETS in smoking homes; 11.5% and 6.2%, respectively, were attributable to ETS in smoking workplaces. RSP apportionment based on solanesol particulate matter (Sol-PM) indicated that 28.7% of the RSP in smoking homes and 22.7% of the RSP in smoking workplaces were attributable to ETS. RSP apportionment based on scopoletin particulate matter (Sco-PM) indicated that 12.9% of the RSP in smoking homes and 9.6% of the RSP in smoking workplaces were attributable to ETS. Median daily and weekly exposures to VOCs, TVOCs, and RSP were calculated from the concentrations determined and tended to follow the trend: SH > NSH > SW > NSW. The home/work exposure differential (SH/SW) indicated that ETS exposure was higher for living with a smoker than for working with a smoker by a factor of 3.7 for RSP and ETS-RSP and 2.4 for VOCs and TVOCs.

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

Because of a lack of representative data on smoking status misclassification among U.S. married females, a two-part study was conducted. Part I was conducted to obtain nationally representative estimates of the percentage of U.S. women who report themselves to be current, former, and never smokers, to determine the concordance of smoking habits among spouse pairs, and to establish field quotas and probability weightings for Part II. Part II was conducted to determine smoker misclassification rates using salivary cotinine as an indication of active smoking. Part I, conducted in January 25-29, 1992, utilized random-digit dialing telephone interviewing throughout the 48 contiguous United States. Part II, conducted from February 19, 1992 to March 7, 1992, was a mall-intercept study in nine geographically disperse U.S. cities and it involved interviewing and saliva collection. Among married U.S. women, 25% reported they were current smokers, 22% reported they were former smokers, and 53% reported they were never smokers. Using a cotinine concentration of either > 35 ng/ml or > 106 ng/ml to indicate regular smoking, 3.61% and 2.55% of regular smokers, respectively, reported themselves to be never smokers. The concordance ratio, an important parameter in correcting for non-differential misclassification bias, was found to be 5.52. In addition, an indication of substantial differential misclassification was found between exposed and unexposed populations. This type of misclassification bias has previously not been accounted for in the adjustment of epidemiology-based risk assessments of tobacco smoke exposure and lung cancer. Taken together, these data suggest that misclassification bias alone is likely to explain any lung cancer risk elevation observed in the U.S. epidemiology of environmental tobacco smoke exposure among nonsmoking women.

A collaborative study for the determination of Tobacco specific nitrosamines in Tobacco

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.

Quantitative Electron Paramagnetic Resonance: The Importance of Matching the Q-factor of Standards and Samples

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.