Deborah Dillon

The mutagenic assessment of mainstream cigarette smoke using the Ames assay: A multi-strain approach

Salmonella typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were assessed for their suitability and use in conjunction with a Vitrocell(®) VC 10 Smoking Robot and 3R4F reference mainstream cigarette smoke. Little information exists on TA97, TA104, TA1535, TA1537 and TA102 using an aerosol 35mm spread-plate format. In this study, TA1535 and TA1537 were considered sub-optimal for use with a scaled-down format, due to low spontaneous revertant numbers (0-5 revertants/plate). In the context of a regulatory environment, TA97 is deemed an acceptable alternative for TA1537 and was therefore selected for whole smoke exposure in this study. However, there is no acceptable alternative for TA1535, therefore this strain was included for whole smoke exposure. TA1535, TA97, TA102 and TA104 were assessed for mutagenic responses following exposure to cigarette smoke at varying concentrations (using diluting airflow rates of 1.0, 4.0, 8.0 and 12.0L/min), and exposure times of 24 and 64min. A positive mutagenic response to cigarette smoke was observed in strain TA104 at both the 24 and 64min time points, in the presence of S-9, at the highest smoke concentration tested (1.0L/min diluting airflow). The three remaining strains were found to be unresponsive to cigarette smoke at all concentrations tested, in the presence and absence of metabolic activation. Cigarette smoke particulate deposition was quantified in situ of exposure using quartz crystal microbalance technology, enabling data to be presented against an associated gravimetric mass (μg/cm(2)). Finally, data obtained in this study were combined with previously published Ames data for TA98, TA100, YG1024, YG1042 and Escherichia coli (WP2 uvrA pKM101), generated using the same 35mm methodology. The combined data-set was used to propose an aerosol testing strategy, based on strain compatibility with the whole smoke aerosol, whilst maintaining the essence of the regulatory guidelines for the standard Ames assay.

Quantification of Cigarette Smoke Particle Deposition In Vitro Using a Triplicate Quartz Crystal Microbalance Exposure Chamber

There are a variety of smoke exposure systems available to the tobacco industry and respiratory toxicology research groups, each with their own way of diluting/delivering smoke to cell cultures. Thus a simple technique to measure dose in vitro needs to be utilised. Dosimetry—assessment of dose—is a key element in linking the biological effects of smoke generated by various exposure systems. Microbalance technology is presented as a dosimetry tool and a way of measuring whole smoke dose. Described here is a new tool to quantify diluted smoke particulate deposition in vitro. The triplicate quartz crystal microbalance (QCM) chamber measured real-time deposition of smoke at a range of dilutions 1 : 5–1 : 400 (smoke : air). Mass was read in triplicate by 3 identical QCMs installed into one in vitro exposure chamber, each in the location in which a cell culture would be exposed to smoke at the air-liquid interface. This resulted in quantification of deposited particulate matter in the range 0.21–28.00 μg/cm2. Results demonstrated that the QCM could discriminate mass between dilutions and was able to give information of regional deposition where cell cultures would usually be exposed within the chamber. Our aim is to use the QCM to support the preclinical (in vitro) evaluation of tobacco products.

A comparative assessment of cigarette smoke aerosols using an in vitro air–liquid interface cytotoxicity test

Abstract This study describes the evaluation of a modified air-liquid interface BALB/c 3T3 cytotoxicity method for the assessment of smoke aerosols in vitro. The functionality and applicability of this modified protocol was assessed by comparing the cytotoxicity profiles from eight different cigarettes. Three reference cigarettes, 1R5F, 3R4F and CORESTA Monitor 7 were used to put the data into perspective and five bespoke experimental products were manufactured, ensuring a balanced and controlled study. Manufactured cigarettes were matched for key variables such as nicotine delivery, puff number, pressure drop, ventilation, carbon monoxide, nicotine free dry particulate matter and blend, but significantly modified for vapor phase delivery, via the addition of two different types and quantities of adsorptive carbon. Specifically manufacturing products ensures comparisons can be made in a consistent manner and allows the research to ask targeted questions, without confounding product variables. The results demonstrate vapor-phase associated cytotoxic effects and clear differences between the products tested and their cytotoxic profiles. This study has further characterized the in vitro vapor phase biological response relationship and confirmed that the biological response is directly proportional to the amount of available vapor phase toxicants in cigarette smoke, when using a Vitrocell® VC 10 exposure system. This study further supports and strengthens the use of aerosol based exposure options for the appropriate analysis of cigarette smoke induced responses in vitro and may be especially beneficial when comparing aerosols generated from alternative tobacco aerosol products.

Application of a modified gaseous exposure system to the in vitro toxicological assessment of tobacco smoke toxicants.

Tobacco smoke is a complex mixture of over 6,000 individual chemical constituents. Approximately 150 of these have been identified as ‘tobacco smoke toxicants’ due to their known toxicological effects. A number of these toxicants are present in the gaseous phase of tobacco smoke. This presents a technical challenge when assessing the toxicological effects of these chemicals in vitro. We have adapted a commercially available tobacco smoke exposure system to enable the assessment of the contribution of individual smoke toxicants to the overall toxicological effects of whole mainstream cigarette smoke (WS). Here we present a description of the exposure system and the methodology used. We use the example of a gaseous tobacco smoke toxicant, ethylene oxide (EtO), a Group 1 IARC carcinogen and known mutagen, to illustrate how this methodology can be applied to the assessment of genotoxicity of gaseous chemicals in the context of WS. In the present study we found that EtO was positive in Salmonella typhimurium strain YG1042, a strain that is sensitive to tobacco smoke. However, EtO did not increase the mutagenicity of the WS mixture when it was added at greatly higher concentrations than those found typically in WS. The findings presented here demonstrate the suitability of this exposure system for the assessment of the mutagenic potential of gases in vitro. Whilst we have focused on tobacco smoke toxicants, this system has broad application potential in studying the biological effects of exposure to a wide range of gaseous compounds that are present within complex aerosol mixtures. Environ. Mol. Mutagen. 55:662–672, 2014.