Differences in Levels of Biomarkers of Potential Harm Among Users of a Heat-Not-Burn Tobacco Product, Cigarette Smokers, and Never-Smokers in Japan: A Post-Marketing Observational Study.

INTRODUCTION: Cigarette smoking is associated with the risk of certain diseases, but non-combustible products may lower these risks. The potential long-term health effects of the next-generation non-combustible products (heat-not-burn tobacco products (HNBP) or electronic vapor products) have not been thoroughly studied. The present study aimed to investigate the impact of biomarkers of potential harm (BoPH) of one of HNBP (a novel vapor product: NTV (novel tobacco vapor)), under the conditions of actual use. AIMS AND METHODS: This study was an observational, cross-sectional, three-group, multi-center study. Exclusive NTV users (NTV, n = 259), conventional cigarette smokers (CC, n = 100) and never-smokers (NS, n = 100) were enrolled. Biomarkers of tobacco smoke exposure (cotinine and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)) and BoPH including parameters of physical pulmonary functions relevant to smoking-related diseases were examined, and subjects answered a questionnaire on cough-related symptoms (J-LCQ) and health-related quality of life (SF-36v2®). RESULTS: Levels of cotinine, total NNAL and BoPH (high-density lipoprotein (HDL)-cholesterol, triglyceride, sICAM-1, WBC count, 11-DHTXB2, 2,3-d-TXB2, 8-epi-PGF2α, forced expiratory volume in 1 second (FEV1), % predicted value of FEV1 (%FEV1) and maximum midexpiratory flow (FEF25-75)) were significantly different in the NTV group as compared to levels in CC group (p < .05). Significantly higher levels of cotinine, total NNAL, and 2,3-d-TXB2, and lower levels of FEV1 and %FEV1, were observed among NTV users compared to the NS group. CONCLUSION: In a post-marketing study under actual use conditions, BoPH associated with smoking-related disease examined in exclusive NTV users were found to be favorably different from those of CC smokers, a finding attributable to a reduction in exposure to harmful substances of tobacco smoke. IMPLICATIONS: Cigarette smoking is associated with an increased risk of pulmonary diseases like COPD, cardiovascular diseases, and certain cancers. There is a growing body of evidence that HNBP reduces the exposure associated with smoking and that there is a favorable change in BoPH. However, long-term effects regarding the relative health risks to HNBP users compared to CC smokers have not been examined. This study provides post-marketing data under actual use conditions of the effects on biomarkers of potential harm in NTV, one of HNBP, exclusive users compared to CC smokers and never-smokers. The evidence suggests that exclusive NTV users have favorable levels of BoPH compared to CC smokers, and that is result from a sustained reduction in exposure to harmful substances of tobacco smoke.

Effects of reduced exposure to cigarette smoking on changes in biomarkers of potential harm in adult smokers: results of combined analysis of two clinical studies.

Purpose: Nonconventional vapor products (NVP), designed to reduce exposure to cigarette smoke toxicants (CSTs), could cause changes in biomarkers of potential harm (BoPH). Although, NVPs reduced CSTs exposure compared to conventional cigarettes (CC), the changes in the BoPH values varied among the studies. Hence, further information on BoPH using NVPs is needed. Material and methods: The data of two similarly designed studies using a kind of NVP, a noncombustion and nonheating inhaler type of smokeless tobacco product (NCIT) used under 31-day confinement, were pooled, and the differences in 15 BoPH between smokers and nonsmokers at baseline and between the 1 mg tar CC (CC1) group and NCIT group at Day 28/29 were analyzed. Results: At baseline, the levels of eight BoPH (red blood cells, white blood cells, 8-epi-prostaglandin F2α, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, 11-dehydrothromboxane B2, total cholesterol and glucose) were significantly different between smokers and nonsmokers. At Day 28/29, the levels of six BoPH were significantly different between NCIT and CC1 (8-epi-prostaglandin F2α, malondialdehyde, 11-dehydrothromboxane B2: CC1 > NCIT, total bilirubin, low-density lipoprotein cholesterol and total cholesterol: CC1 < NCIT). Conclusions: Reduced exposure to CSTs has favorable effects on BoPH associated with oxidative stress, antioxidant capacity and platelet activation/coagulation but not in lipid metabolism.

Pharmacokinetics of nicotine following the controlled use of a prototype novel tobacco vapor product.

The objective of this clinical study was to investigate the pharmacokinetics of nicotine following the use of a prototype novel tobacco vapor (PNTV) product in comparison to a conventional cigarette (CC1). The study was conducted in Japanese healthy adult male smokers, using an open-label, randomized, two-period crossover design, to assess the pharmacokinetics of nicotine after controlled use of a PNTV product or CC1. During the study period, blood samples were drawn from subjects for the measurement of plasma nicotine concentrations and nicotine intake was estimated from the mouth level exposure (MLE). The Cmax and AUClast following the use of PNTV product were 45.7% and 68.3%, respectively, of those obtained with CC1 and there were no significant differences in the tmax and t1/2 between PNTV product and CC1. The estimated MLE following the use of PNTV product was approximately two-thirds of that obtained following the smoking of CC1, but the relative bioavailability of PNTV product to CC1 was approximately 104%. The differences in Cmax and AUClast between PNTV product and CC1 therefore are explained by differences in nicotine intake. These results suggest that the PNTV product shows a similar pharmacokinetic profile to CC1, while delivering less nicotine following controlled use.

RecA protein recruits structural maintenance of chromosomes (SMC)-like RecN protein to DNA double-strand breaks.

Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recA(Q300R), is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recA(Q300R) fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecN(K35A) binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.

Exposure evaluation of adult male Japanese smokers switched to a heated cigarette in a controlled clinical setting.

The objective of this clinical study was to investigate changes in levels of biomarkers of exposure (BOEs) in healthy Japanese male smokers who switched to a prototype heated cigarette (HC). This was a controlled, semi-randomized, open-label, residential study conducted in Japan. A total of 70 healthy Japanese male smokers were enrolled. Following enrollment, subjects smoked their usual brand of cigarette for 2days and were subsequently randomized either to an HC group or a 10mg tar conventional cigarette (CC10) group for four consecutive weeks. Levels of BOEs for ten selected cigarette smoke constituents (nicotine, carbon monoxide (CO), benzene, 1,3-butadiene, acrolein, hydrogen cyanide, crotonaldehyde, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone [NNK], pyrene, 4-aminobiphenyl), and urine mutagenicity were measured at several time points during the study period. At the end of the study period, except for blood carboxyhemoglobin, levels of BOEs for the other nine constituents and urine mutagenicity were significantly lower in the HC group compared to the CC10 group. These results suggest that exposure to most cigarette smoke constituents, except CO, can be reduced by switching from CC10 to HC.

A Randomized Control Study in Healthy Adult Smokers to Assess Reduced Exposure to Selected Cigarette Smoke Constituents in Switching to the Novel Heated Tobacco Product DT3.0a.

This was a randomized, controlled, open-label, confinement study to assess change in exposure to selected cigarette smoke constituents in healthy adult cigarette smokers who switched to using a novel heated tobacco product (direct heating tobacco system, platform 3, generation 3, version a [DT3.0a]). Sixty nonmenthol cigarette smokers were randomized into 1 of the 4 study groups in which subjects switched to a nonmenthol type of tobacco stick used with DT3.0a, switched to a nonmenthol tobacco stick used with an in-market heated tobacco product device (THS), continued to smoke nonmenthol cigarettes, or stopped smoking. Furthermore, 30 menthol cigarette smokers were randomized into 1 of the 2 study groups in which subjects switched to a menthol tobacco stick used with DT3.0a (mDT3.0a) or continued to smoke menthol cigarettes. Fifteen biomarkers of exposure to selected harmful and potentially harmful constituents (HPHCs) were measured during the 5-day exposure period, followed by assessment of nicotine pharmacokinetics with the assigned product. Results indicated that switching to DT3.0a, THS, and mDT3.0a showed significant exposure reductions in most of the selected HPHCs as compared to continuing smoking cigarettes, with reductions being similar in magnitude to reductions observed with smoking cessation. For DT3.0a and mDT3.0a, nicotine pharmacokinetic parameters were not remarkably different from those obtained for cigarettes and the THS except that a longer time to maximum concentration was obtained following use of the mDT3.0a. In conclusion, switching from smoking cigarettes to DT3.0a or THS use reduced exposure to most of the selected HPHCs, and no remarkable differences were observed for the measurements obtained from different flavors of DT3.0a stick.

Assessment of the exposure to selected smoke constituents in adult smokers using in-market heated tobacco products: a randomized, controlled study.

The objectives of this clinical study were to demonstrate a reduction in exposure to selected harmful and potentially harmful constituents (HPHCs) in Japanese healthy adult smokers who switched to four in-market heated tobacco products. Eighty-nine smokers were randomly assigned for five days to one of six study groups: four groups who switched to one of the commercially available heated tobacco products; a group who continued to smoke their own brand of combustible cigarettes (CC); or a group who stopped smoking (SS). Fifteen biomarkers of exposure (BoE) to 14 HPHCs and pyrene were measured at baseline, Day 3 and Day 5 in 24 h urine and breath, under clinical confinement. Product consumption, nicotine uptake and subjective effects were also measured before and after product switching. On Day 5, significant reductions in most BoE relative to the CC group were observed after switching to heated tobacco products. No changes in BoE were observed between baseline and Day 5 in the CC group. Significantly, the magnitude of the reduction in exposure to most of the selected HPHCs observed in the heated tobacco product groups was close to that observed in the SS group.

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair.

BACKGROUND: DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. RESULTS: To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. CONCLUSION: These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

Role of the Schizosaccharomyces pombe F-Box DNA helicase in processing recombination intermediates.

In an effort to identify novel genes involved in recombination repair, we isolated fission yeast Schizosaccharomyces pombe mutants sensitive to methyl methanesulfonate (MMS) and a synthetic lethal with rad2. A gene that complements such mutations was isolated from the S. pombe genomic library, and subsequent analysis identified it as the fbh1 gene encoding the F-box DNA helicase, which is conserved in mammals but not conserved in Saccharomyces cerevisiae. An fbh1 deletion mutant is moderately sensitive to UV, MMS, and gamma rays. The rhp51 (RAD51 ortholog) mutation is epistatic to fbh1. fbh1 is essential for viability in stationary-phase cells and in the absence of either Srs2 or Rqh1 DNA helicase. In each case, lethality is suppressed by deletion of the recombination gene rhp57. These results suggested that fbh1 acts downstream of rhp51 and rhp57. Following UV irradiation or entry into the stationary phase, nuclear chromosomal domains of the fbh1Delta mutant shrank, and accumulation of some recombination intermediates was suggested by pulsed-field gel electrophoresis. Focus formation of Fbh1 protein was induced by treatment that damages DNA. Thus, the F-box DNA helicase appears to process toxic recombination intermediates, the formation of which is dependent on the function of Rhp51.

Investigation by microarray analysis of effects of cigarette design characteristics on gene expression in human lung mucoepidermoid cancer cells NCI-H292 exposed to cigarette smoke.

The effects of tobacco leaf types and the presence or absence of charcoal in the cigarette filters on gene expression were investigated using cigarette prototypes made of either flue-cured (FC) leaf or burley (BLY) leaf and Kentucky Reference 2R4F as a representative blend cigarette with cellulose acetate filters or charcoal filters. NCI-H292, human lung mucoepidermoid carcinoma cell line, was exposed to the total particulate matter (TPM) and gas/vapor phase (GVP) from each prototype for 8h and then the changes in gene expression from microarray data were analyzed. A number of genes associated with oxidative stress, inflammation, DNA damage and xenobiotic response were modified by the two fractions, TPM and GVP, from the three prototypes with cellulose acetate filters. Both TPM and GVP fractions strongly enhanced the gene expression of HMOX1, which is encoding the limiting enzyme in heme degradation and a key regulator of oxidative stress and inflammatory process. Comparing the effects of TPM and GVP fraction, TPM strongly activated Nrf2 pathway-mediated anti-oxidative stress reaction, whereas GVP caused notable DNA damage response. In comparison of FC and BLY, TPM from FC more strongly induced the expression of histone family proteins than that from BLY. GVP from FC markedly induced gene expression associated with HSP70-mediated inflammation relative to that from BLY. Charcoal included in the filter strongly reduced the effects of GVP from each cigarette on gene expression. However, charcoal did not modified the effects of TPM. As a whole, charcoal is a useful material for reducing the biological effects of GVP.

Degradation of Escherichia coli RecN aggregates by ClpXP protease and its implications for DNA damage tolerance.

Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in DeltaclpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance.