Volume 25 (2013): Issue 5 (March 2013)

In this issue of Beiträge, we publish as a Commentary the oral presentation of Wolfram Röper entitled ‘Ten Years and More of ‘Biosciences’ in CORESTA - A Brief Historic Overview’, held at the CORESTA Meeting in Sapporo, Japan, September 23-27, 2012. We chose do so because this article deviates in its structure from a regular scientific publication in Beiträge. Nevertheless, we as Editors believe that this text is of interest to many scientists involved in tobacco research, in particular the younger colleagues, since, in our view, it impressively shows the increasing importance of the ‘biosciences’ for the development, evaluation and regulation of tobacco products. It is highly likely that the ‘biosciences’ will play an equal (or perhaps an even more important) role in the characterization of tobacco products in the future as did the physical and chemical sciences in the past.

We want to inform our readers that Professor John Gorrod has decided to step down as a member of the Advisory Board of Beiträge for reasons of age. Professor Gorrod, who is one of the best known nicotine researchers, was a member of the Advisory Board since September 2001. During that time, he had reviewed numerous manuscripts for Beiträge. We would like to thank Professor Gorrod for his valuable work for our Journal and wish him all the best for his well-earned retirement.

A method for the determination of menthol in a single puff of mainstream cigarette smoke is reported. A rotary smoking machine with a twin filter interface was used to smoke cigarettes with varying menthol and “tar” deliveries determined based on the Cambridge filter method. The twin filter interface mechanically switches to a new filter pad for collection of smoke from each cigarette puff. The individual filter pad extracts were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine menthol puff-by-puff deliveries. Menthol puff-by-puff profiles show an increase in menthol smoke delivery with increasing puff count, a trend consistent with total particulate matter and smoke delivery profiles of other mainstream smoke constituents. The sum of single puff menthol deliveries is comparable to the whole cigarette menthol smoke delivery as collected on a single filter pad. This method can also determine quantitative puff-by-puff deliveries of other mainstream cigarette smoke constituents.

Formation of dihydroxybenzenes in cigarette smoke is a subject of considerable interest because some dihydroxybenzenes are co-carcinogens, (e.g., catechol and certain alkylcatechols), and others such as hydroquinone can form metabolites that have toxic or carcinogenic properties. This present study describes the contribution of tobacco quinic acid (or (1S,3R,4S,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylic acid) and myo-inositol (or (1R,2R,3S,4S,5R,6S)-cyclohexane-1,2,3,4,5,6-hexol) to the formation of dihydroxybenzenes in cigarette smoke. The study is a continuation of a previous one showing the contribution of chlorogenic acid and rutin as precursors for these compounds (6). The yields of dihydroxybenzenes formed by pyrolysis of quinic acid and myo-inositol are relatively high and both quinic acid and myo-inositol can be present in some tobacco types at levels as high as 1% by weight. The level of these compounds makes them potentially important contributors to the formation of dihydroxybenzenes in cigarette smoke. Similar to the previous study on other dihydroxybenzene precursors from tobacco, this present study was done in three parts: 1) pyrolytic evaluation of the amount of dihydroxybenzenes in smoke generated from isolated quinic acid and myoinositol; 2) analysis of smoke from cigarettes made from a variety of tobaccos (14 single grades) and two blended cigarettes, followed by correlations of dihydroxybenzene yields from these cigarettes with the level of quinic acid and myo-inositol in the tobaccos; 3) addition of quinic acid or myo-inositol to several tobaccos followed by the smoking of the spiked cigarettes and measurement of the dihydroxybenzenes yield increase. The study performed on a variety of single-grade tobacco cigarettes and for two blended-tobacco cigarettes (one being the 2R4F Kentucky reference) shows that the contribution of quinic acid and of inositol to the formation of catechol and hydroquinone in smoke depends on the blend, as previously shown for chlorogenic acid and rutin. The study results suggest that quinic acid and myo-inositol may be major contributors to the formation of dihydroxybenzenes in cigarette smoke. Although the calculations do not provide precise numbers for the contribution of quinic acid and inositol to the formation of dihydroxybenzenes, these results suggest that the contribution could be as high as 50 to 60% of the level of dihydroxybenzenes.

Among the more than 5000 chemicals reported in cigarette smoke condensate (CSC), heterocyclic aromatic amines (HAAs) are considered to be a contributor to observed biological activity. HAAs are non-volatile and are reported at ppb levels in CSC. A new method for HAA analysis at the trace level is reported here. N, O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane was employed to derivatize amino groups by heating the reagent containing a sample of CSC at 80 °C for 30 min followed by analysis employing gas chromatography-mass spectroscopy (GC-MS) in the selected-ion-monitoring (SIM) mode. This derivatization method afforded symmetrical peak shapes on a ZB-50 stationary phase and achieved instrumental limits of quantification (LOQ) at 10:1 S/N from -1 ng/mL for AαC to120 ng/mL for Glu-P-1. The chemical identity of each derivative was confirmed by comparison of retention time and mass spectra of standards. The latter were characterized by the following ions: M·+ or [M-1]+, [M-15]+, and m/z 73 (i.e., trimethylsilyl). CSC and its base sub-fractions were studied using the GC-MS method. Ten HAAs were screened and five were quantified in cigarette smoke condensate, while 2-5 HAAs were quantified in each of three base sub-fractions. Values obtained with the new procedure agree well with values reported in the literature and with results obtained from a commercial laboratory via a different analytical method. The potential contribution of each HAA to the overall mutagenic activity observed for CSC and its base fractions is discussed. When considered together, HAAs account for only a small portion (-7.8%) of the observed mutagenicity of the CSC.

In June 2001, the CORESTA² Board formally decided to broaden the scope of CORESTA by engaging in biosciences (other than tobacco agronomy and phytopathology), starting with in vitro toxicity testing and biomarkers of tobacco (smoke) exposure. Until then, work addressing biological aspects of smoking had only been done since 1996 by a special committee within CORESTA, reporting directly to the Scientific Commission, the Smoking Behaviour Committee. Membership of that committee was - similar to ACAC³ - by invitation only. The broadened scope consequently led to the re-shaping of the then Study Groups ‘Smoke’ and ‘Technology’ into ‘Smoke Science’ (SS) and ‘Product Technology’ (PT).

Subsequently, three Sub-Groups (SG) and Task Forces (TF) were set up, reflecting this change: i) SG Smoking Behaviour (name change of former Committee in 2001), ii) TF ‘Nicotine Intake’ (2001, later on named ‘Nicotine Uptake’, disbanded in 2009) and iii) TF ‘In vitro Toxicity Testing of Tobacco Smoke’ (2002). Finally, a new SG ‘Biomarkers’ was launched in 2009 with a wider scope than its predecessor TF ‘Nicotine Uptake’. The work of these groups has had and still has significant impact on the scientific work within CORESTA, leading to numerous presentations at CORESTA meetings and publications in peer-reviewed journals.

This paper provides a brief analysis of some 270 presentations and posters addressing tobacco smoke toxicity, human smoking behaviour or biomarkers, delivered at CORESTA Congresses and SSPT Joint Meetings between 1993 and 2011. More than 50% of these papers covered different aspects of toxicology, mainly in vitro toxicity testing methodologies, smoke exposure systems and other equipments. Other papers described the influence of cigarette design parameters on smoke toxicity. Approaches to human risk assessment were presented, including the search for suitable in vitro models of the major smoking related human diseases.

CORESTA began discussing smoking behaviour topics at their Vienna meeting in 1995 and received five respective presentations there; indeed, the issue has various aspects, from smoking topography and human smoke yield to smoke uptake, deposition and retention, and… Why do people smoke at all?

As early as 1996, a presentation was given on the determination of urinary mutagenicity in volunteers exposed to ETS (environmental tobacco smoke), apparently indicating a need for CORESTA to engage in this field and to face new challenges. Indeed, our knowledge of biomarkers and how to measure them has increased considerably over the years, and there is a clear trend towards using this knowledge for conducting clinical studies into the assessment of ‘modified risk tobacco products’.