Volume 24 (2010): Issue 1 (April 2010)

This issue marks a turning point in the history of BeiträgezurTabakforschung International. From its first issue in 1961, our Journal has been published and funded by the Verband der Cigarettenindustrie (VdC). After the liquidation of the VdC in 2007 we had to find a new publisher as well as sponsors, who are willing to provide the funds required for the continuation of the publishing process. During the last two years, we asked various corporations to financially support Beiträge and thus ensure its survival. Our call has been honoured by a number of the addressees approached so that we were able to continue our work for the Journal. We would like to express our cordial thanks for the opportunity to dedicate this issue to all our friends and sponsors who made this possible.

This study examined the potential to minimize the cytotoxic or genotoxic effects that dimethylsulfoxide (DMSO), when used as solvent, has on the in vitro interleukin-8 (IL-8) release, mammalian cell cytotoxicity and micronuclei formation, and bacterial mutagenesis induced by cigarette smoke wet total particulate matter (WTPM). The use of DMSO as a solvent vehicle for test articles of limited water solubility is widely applied in in vitro assays due to its moderate toxicity to test organisms and its excellent solvent properties for both polar and non-polar compounds, such as WTPM. A significant DMSO dose-dependent depletion in the IL-8 release was observed, with or without the addition of WTPM, at concentrations spanning those typically employed in in vitro assays. DMSO at 3.6% reduced cell viability 40-50%. Overall, DMSO at final concentrations of 0.5% and 4.0% resulted in about 50% and 90% depletion of final IL-8 levels, respectively. DMSO-induced cytotoxicity was evident only at concentrations of 1.5% or more, a concentration higher than that typically employed in such testing. The WTPM-induced cytotoxicity was equivalent at low ranges of DMSO concentrations. DMSO concentrations of 3.6% or higher resulted in an increase of cytotoxicity by 20-25%. DMSO alone did not give rise to bacterial mutagenicity at doses from 0% to 3.9%; however, WTPM exposure with increasing levels of DMSO resulted in increased toxicity of the WTPM at doses of DMSO greater than 6.9%, as indicated by lower revertant counts. This effect suggests that for Ames assay analysis of WTPM collected in DMSO, the level of DMSO should be minimized to prevent lower revertant counts due to DMSO-induced toxicity. DMSO alone gave a dose-dependent increase in the background micronuclei percentage, with a statistically significant increase at 4%. In the presence of WTPM, DMSO at 3% concentration resulted in a significantly higher micro-nucleus frequency, suggesting a possible clastogenic role of DMSO or potential cell permeability differences. In conclusion, this study should provide guidance for the range of DMSO concentrations when used as a solvent for WTPM-mediated in vitro toxicological assays and therefore help in properly designing and conducting in vitro studies that utilize DMSO as the extraction solvent of choice.

Phytol or (2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecen- 1-ol (MW = 296.53 a.u.), is naturally present in tobacco at levels around 100-150 µg/g dry leaf, where it is bound in the form of an ester to a porphyrine type group (known as chlorin) with several substituents and chelated to Mg²⁺ ion, to form chlorophyll. This study evaluated the formation of PAHs when several levels of phytol up to ten times higher than typical phytol level were added on 3R4F cigarettes. The cigarettes were smoked under two different smoking conditions. One regimen (indicated as 35/60) used 35 mL puff volume, 2 s puff duration, each puff taken every 60 s, and in the other used 60 mL puff volume, 2 s puff duration, each puff taken every 30 s (indicated as 60/30). The increase in the PAHs level upon phytol addition was not significant. A statistical evaluation of the dependence of total PAHs and the added level of phytol showed that the hypothesis of a zero slope for the dependence line phytol/total PAHs cannot be rejected (with a p = 0.101 for 35/60 smoking and p = 0.626 for 60/30 smoking). Still, for 35/60 smoking conditions, an increase of about 14% in total levels of PAHs was noticed when the added phytol level was 1.5 mg phytol per cigarette. However, this PAHs increased level was not necessarily determined by the phytol addition, and can be caused by random procedural/analytical errors (as indicated by the statistical analysis) or by the modification of cigarettes during phytol addition. Also, the observed level is much lower than the expected 100% increase in the PAHs level, based on literature prediction. Intensive smoking did not show an increase in PAHs level at all. Flash pyrolysis of free phytol and of chlorophyll a provided results that indicated that phytol bound in chlorophyll is not likely to generate different PAHs level compared to free phytol. Thus, phytol has been shown not to be a significant contributor/ precursor to the PAHs formation in cigarette smoke.

We studied the changes in the activity of the enzymes polyphenol oxidase (PPO) and peroxidase (POX), the concentration of total soluble phenolic compounds and soluble protein content in different tobacco cultivars (Virginia and Burley) during curing. The curing procedure was a special combination of open-air-curing and fluecuring methods and a long fermentation period. In the curing period the changes in data suggested a combination of the biochemical processes and the direct effect of oxygen. A slight increase then a decrease in the concentrations of both total soluble phenolic compounds and the soluble protein content were detected. In this period we found no correlation between the concentration of total soluble phenolic compounds, the decreasing PPO and the increasing POX activity. In the fermentation period a deactivation of the enzymes (PPO and POX) and a decrease in the concentration of both total soluble phenolic compounds and soluble protein content were found, because the fermentation is not a biochemically-regulated process. Moreover, these results suggest that the end of curing period is the most favourable time for protein isolation from different tobacco cultivars.

The efficacy of pheromone traps is affected by trap placement and pest biology, as well as by the trap and lure design. We evaluated the effect of trap height and placement in relation to surfaces on tobacco moth catches using release-capture experiments. Six traps were mounted vertically in a 9 × 15 × 4.2-5.8 m shed on a wall at heights of 0, 1, 2, 3, 4 and 5 m. More catches were obtained near the ceiling and at 1 m above the floor in the dark. Catches at 1 m were negligible when light shone through the upper windows. In a 42.3 × 36.5 × 4 m tobacco warehouse, the respective efficacies of aerially suspended traps and surface-mounted traps were examined. The number of catches obtained using traps mounted on pillars was significantly higher than that obtained in traps suspended from poles. These results suggest practical considerations for monitoring in warehouses. We recommend eliminating any night lighting and placing traps on surfaces, such as walls and stored commodities, at higher positions that are within reach (1 m) to facilitate convenient inspection.

Cigarettes with similar design features but with either cellulose acetate or dual carbon filters were made at 1-mg and 13-mg “tar” levels, as determined under the ISO smoking procedure. Products were smoked under the ISO, Massachusetts and Canadian smoking regimes to provide per-cigarette and per-puff yields of twelve vapour phase (VP) smoke components. The yields generated at the lit end of the cigarette and the significant yield reductions caused by filter ventilation, selective (carbon) adsorption, tobacco rod ventilation and diffusion were estimated in a modelling approach. For a “1-mg tar” carbon-filtered product it was estimated that the VP generated at the lit end was reduced by 99.4% to a machine yield of 17 µg/cig under ISO smoking conditions. Under the Canadian regime with 100% vent blocking, the estimated total VP was lowered 20% by tobacco rod effects and 15% by carbon filter adsorption giving a machine yield of 3487 µg/cig. The carbon filter adsorbed less efficiently partly due to the artificially high smoke temperatures through the filter that would probably not be tolerated by human smokers. Under the Massachusetts regime with 50% vent blocking, conditions better associated with human smoking, the total VP was lowered 51% by filter ventilation, 22% by tobacco rod effects and 17% by carbon filter adsorption giving a machine yield of 659 µg/cig. Ventilation is used to achieve “tar”/nicotine/carbon monoxide yield ceilings at 10/1/10 mg based on the current ISO smoking method. If future regulations were to mandate further reductions in VP then this will only be selectively achieved by increasing filter or tobacco rod ventilation/porosity or by using selective adsorption. It is inevitable that manufacturers will need to add further ventilation into their product to comply with such regulations and this should be reflected in any smoking regime. Furthermore, regimes with 100% vent blocking, that do not produce data reflecting the significant reductions in VP yields, provided to the smoker by ventilation, are misleading and their results will not correlate with relevant biomarker data. When proposing a different smoking regime, it is essential to understand the generation and transfer of smoke within cigarettes and factors involved in the subsequent data interpretation as described in this work. For regulatory evaluation purposes, cigarette characterisation using a regime that removes ventilation, one of the main design tools, is more misleading than the current ISO regime or one with partial vent blocking.