Oscar M. Camacho

Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke

Abstract Electronic cigarettes (E-cigarettes) are a potential means of addressing the harm to public health caused by tobacco smoking by offering smokers a less harmful means of receiving nicotine. As e-cigarettes are a relatively new phenomenon, there are limited scientific data on the longer-term health effects of their use. This study describes a robust in vitro method for assessing the cytotoxic response of e-cigarette aerosols that can be effectively compared with conventional cigarette smoke. This was measured using the regulatory accepted Neutral Red Uptake assay modified for air–liquid interface (ALI) exposures. An exposure system, comprising a smoking machine, traditionally used for in vitro tobacco smoke exposure assessments, was adapted for use with e-cigarettes to expose human lung epithelial cells at the ALI. Dosimetric analysis methods using real-time quartz crystal microbalances for mass, and post-exposure chemical analysis for nicotine, were employed to detect/distinguish aerosol dilutions from a reference Kentucky 3R4F cigarette and two commercially available e-cigarettes (Vype eStick and ePen). ePen aerosol induced 97%, 94% and 70% less cytotoxicity than 3R4F cigarette smoke based on matched EC50 values at different dilutions (1:5 vs. 1:153 vol:vol), mass (52.1 vs. 3.1 μg/cm2) and nicotine (0.89 vs. 0.27 μg/cm2), respectively. Test doses where cigarette smoke and e-cigarette aerosol cytotoxicity were observed are comparable with calculated daily doses in consumers. Such experiments could form the basis of a larger package of work including chemical analyses, in vitro toxicology tests and clinical studies, to help assess the safety of current and next generation nicotine and tobacco products.

Changes in levels of biomarkers of exposure observed in a controlled study of smokers switched from conventional to reduced toxicant prototype cigarettes

UNLABELLED Reduced toxicant prototype (RTP) cigarettes with substantially reduced levels of tobacco smoke toxicants have been developed. Evaluation of these prototype cigarettes included measurement of biomarkers of exposure (BoE) to toxicants in smokers switched from conventional cigarettes to the RTPs. A 6-week single-blinded randomised controlled study with occasional clinical confinement was conducted ( TRIAL REGISTRATION ISRCTN7215735). All smoking subjects smoked a conventional cigarette for 2-weeks. Control groups continued to smoke the conventional cigarette while test groups switched to one of three RTP designs. Clinical confinement and additional assessments were performed for all smoking groups after 2 and 4-weeks. A non-smoker group provided background levels of BoE. On average, smokers switched to RTPs with reduced machine yields of toxicants had reduced levels of corresponding BoEs. For vapour phase toxicants such as acrolein and 1,3-butadiene reductions of ⩾70% were observed both in smoke chemistry and BoEs. Reductions in particulate phase toxicants such as tobacco-specific nitrosamines, aromatic amines and polyaromatic hydrocarbons depended upon the technologies used, but were in some cases ⩾80% although some increases in other particulate phase toxicants were observed. However, reductions in BoEs demonstrate that it is possible to produce prototype cigarettes that reduce exposure to toxicants in short-term use.

The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter.

In vitro genotoxicity assays are often used to compare tobacco smoke particulate matter (PM) from different cigarettes. The quantitative aspect of the comparisons requires appropriate statistical methods and replication levels, to support the interpretation in terms of power and significance. This paper recommends a uniform statistical analysis for the Ames test, mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT); involving a hierarchical decision process with respect to slope, fixed effect and single dose comparisons. With these methods, replication levels of 5 (Ames test TA98), 4 (Ames test TA100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) resolved a 30% difference in PM genotoxicity.

Quantification of plasma microRNAs in a group of healthy smokers, ex-smokers and non-smokers and correlation to biomarkers of tobacco exposure

Abstract The stability of circulating miRNAs, their non-invasive sampling techniques and deregulation in diseases make them potential candidate biomarkers of biological effect. Here, we profiled the level of 84 plasma miRNAs in 30 smokers, 20 non-smokers and 20 ex-smokers. A robust statistical strategy was applied with replicate samples to account for reproducibility of the results. We identified differential expression of miR-124 and let-7a between the smoking and control groups. We further explored the dose–response relationship of miR-124 and let-7a with two biomarkers of tobacco exposure and found that this relationship was affected by adjustments based on age, pack-year and gender.

Assessing modified risk tobacco and nicotine products: Description of the scientific framework and assessment of a closed modular electronic cigarette

ABSTRACT Cigarette smoking causes many human diseases including cardiovascular disease, lung disease and cancer. Novel tobacco products with reduced yields of toxicants compared to cigarettes, such as tobacco‐heating products, snus and electronic cigarettes, hold great potential for reducing the harms associated with tobacco use. In the UK several public health agencies have advocated a potential role for novel products in tobacco harm reduction. Public Health England has stated that “The current best estimate is that e‐cigarettes are around 95% less harmful than smoking” and the Royal College of Physicians has urged public health to “Promote e‐cigarettes widely as substitute for smoking”. Health related claims on novel products such as ‘reduced exposure’ and ‘reduced risk’ should be substantiated using a weight of evidence approach based on a comprehensive scientific assessment. The US FDA, has provided draft guidance outlining a framework to assess novel products as Modified Risk Tobacco Products (MRTP). Based on this, we now propose a framework comprising pre‐clinical, clinical, and population studies to assess the risk profile of novel tobacco products. Additionally, the utility of this framework is assessed through the pre‐clinical and part of the clinical comparison of a commercial e‐cigarette (Vype ePen) with a scientific reference cigarette (3R4F) and the results of these studies suggest that ePen has the potential to be a reduced risk product. HighlightsDescription of a scientific framework to assess the risk profile of next generation products relative to cigarettes.Multi‐disciplinary studies comparing a commercial e‐cigarette (Vype ePen) with a scientific reference cigarette (3R4F).The test e‐cigarette aerosol is compositionally simpler, containing reduced levels of toxicants relative to cigarette smoke.Results from a range of multi‐disciplinary studies showed reduced responses for the test e‐cigarette relative to cigarettes.

Changes in levels of biomarkers of exposure and biological effect in a controlled study of smokers switched from conventional cigarettes to reduced-toxicant-prototype cigarettes

BACKGROUND Development of cigarettes that reduce exposure to harmful smoke constituents is a suggested tobacco harm reduction strategy, but robust methods for measurement of change are required. We investigated whether changes in biomarkers of exposure (BoE), effective dose (BoED) and biological effect (BoBE) could be detected after switching from conventional cigarettes to a reduced-toxicant-prototype cigarette (RTP). METHODS Regular smokers of 6-8mg ISO tar yield cigarettes were recruited in Hamburg, Germany, and supplied with a conventional 7mg ISO tar yield cigarette for 2weeks then switched to the same cigarette with a different tipping paper (control) or the RTP for 6months. Subjects smoked mostly at home and attended five residential clinic visits where urine and blood samples were collected for analysis. Primary endpoints were changes in specific biomarker levels compared with non-smoker background levels. Changes in daily cigarette consumption were also investigated. RESULTS BoE levels in controls generally increased over the study period, whereas most BoE and all BoED significantly declined in RTP smokers. Most BoBE data were similar across groups and/or too variable within individuals to detect changes. Increased daily cigarette consumption was affected by supply of free cigarettes, perceived shorter smoking time per cigarette than usual brands, and perceived reduced harm. CONCLUSIONS Despite increased cigarette consumption, reductions in BoE and BoED were detectable.

A system dynamics modelling approach to assess the impact of launching a new nicotine product on population health outcomes

&NA; In 2012 the US FDA suggested the use of mathematical models to assess the impact of releasing new nicotine or tobacco products on population health outcomes. A model based on system dynamics methodology was developed to project the potential effects of a new nicotine product at a population level. A model representing traditional smoking populations (never, current and former smokers) and calibrated using historical data was extended to a two‐product model by including electronic cigarettes use statuses. Smoking mechanisms, such as product initiation, switching, transition to dual use, and cessation, were represented as flows between smoking statuses (stocks) and the potential effect of smoking renormalisation through a feedback system. Mortality over a 50‐year period (2000–2050) was the health outcome of interest, and was compared between two scenarios, with and without e‐cigarettes being introduced. The results suggest that by 2050, smoking prevalence in adults was 12.4% in the core model and 9.7% (including dual users) in the counterfactual. Smoking‐related mortality was 8.4% and 8.1%, respectively. The results suggested an overall beneficial effect from launching e‐cigarettes and that system dynamics could be a useful approach to assess the potential population health effects of nicotine products when epidemiological data are not available. HighlightsScientists have developed model to assess the population health effects of launching new products.The model reinforces views that eCigarettes use is likely to benefit UK population health.Model forecast cigarette smoking prevalence falls from 12.4% to 9.7% in counterfactual scenario.Conservative inputs with no risk reduction for dual users.

E-cigarette Nicotine Delivery: Data and Learnings from Pharmacokinetic Studies.

OBJECTIVES E-cigarettes could potentially play a major role in tobacco harm reduction by delivering nicotine in a vapor containing significantly fewer toxicants than cigarette smoke and may aid smoking behavior changes such as reduction or cessation. METHODS We examined blood nicotine levels in smokers who were non-accustomed to e-cigarette use (Study 1) and accustomed e-cigarette users (Study 2). We compared nicotine levels when participants used a closed modular system e-cigarette to those when participants smoked a cigarette. RESULTS In Study 1, Cmax (geometric mean (CV)) during a 5-minute puffing period (10 puffs, 30 seconds apart) was 13.4 (51.4) ng/ ml for a regular cigarette. The e-cigarette Cmax was significantly lower (p .05) at 2.5 (67.8) ng/ml. In Study 2, during a 5-minute ad libitum puffing period, cigarette Cmax was 7.2 (130.8) ng/mL, and it was 7.8 (108.2) ng/mL for the e-cigarette. CONCLUSIONS Our data demonstrate heterogeneity of nicotine deliveries both between products and also with the same products used by different cohorts, eg, accustomed users versus smokers. Such differences must be taken into account when determining the likely behavioral impact, on smoking reduction and cessation, of nicotine delivery data and when planning e-cigarette nicotine pharmacokinetic studies.

Assessment of tobacco heating product THP1.0. Part 9: The placement of a range of next-generation products on an emissions continuum relative to cigarettes via pre-clinical assessment studies

ABSTRACT This series of nine papers described the operation and pre‐clinical assessment of a tobacco heating product THP1.0. This last paper contextualises the pre‐clinical assessment data on THP1.0 with data from other next generation products relative to cigarette smoke. The tobacco and nicotine risk continuum is a concept that ranks products according to their potential harm, with cigarettes at the highest risk extreme and Nicotine Replacement Therapy at the least risky extreme. Data generated in pre‐clinical studies on THP1.0 and a range of Next Generation Products (NGPs) may provide some initial indication of potential ranking of these products, although importantly, data from such studies are limited and cannot take into consideration several important aspects for risk such as long term product use patterns. In each of the studies, the responses to the emissions from THP1.0 were substantially reduced relative to cigarette smoke. Additionally, responses from THP1.0 were very similar to those from the other NGP emissions. A comparison of the results clearly showed the emissions from all the NGPs were considerably lower than those from cigarettes and all in around the same emissions level. These results show that THP1.0 could have the potential to be a reduced risk product compared to cigarettes, though further studies assessing the exposure, individual and population risk reduction profile would be required to substantiate this potential. HighlightsComparison of pre‐clinical data and a range of next generation products (NGPs).A tobacco heating product, THP1.0, showed substantially reduced responses in pre‐clinical tests in comparison to cigarettes.All tested NGPs have substantially reduced responses in pre‐clinical assessment studies in comparison to cigarettes.

Nicotine, cotinine, and β-nicotyrine inhibit NNK-induced DNA-strand break in the hepatic cell line HepaRG.

Recent in vitro work using purified enzymes demonstrated that nicotine and/or a nicotine metabolite could inhibit CYPs (CYP2A6, 2A13, 2E1) involved in the metabolism of the genotoxic tobacco nitrosamine NNK. This observation raises the possibility of nicotine interaction with the mechanism of NNK bioactivation. Therefore, we hypothesized that nicotine or a nicotine metabolite such as cotinine might contribute to the inhibition of NNK-induced DNA strand breaks by interfering with CYP enzymes. The effect of nicotine and cotinine on DNA strand breaks was evaluated using the COMET assay in CYP competent HepaRG cells incubated with bioactive CYP-dependent NNK and CYP-independent NNKOAc (4-(acetoxymethylnitrosoamino)-1-(3-pyridyl)-1-butanone). We report a dose-dependent reduction in DNA damage in hepatic-derived cell lines in the presence of nicotine and cotinine. Those results are discussed in the context of the in vitro model selected.