Volume 22 (2006): Edizione 3 (October 2006)

This article is a historical review and a vision for the future of tobacco plant research. This is the perspective of an experienced tobacco scientist who devoted his total professional career to tobacco research. From the very beginning, pioneering tobacco research was the foundation of plant science at the dawn of modern development, in such areas as light, nutrition, genetics, growth control, disorders and metabolism. Tobacco research led to current advancements in plant biotechnology. In addition, tobacco plant research contributed significantly to public health research in radioactive elements, mycotoxins, and air pollutants. However, public support for tobacco research has today greatly declined to almost total elimination because of a sense of political correctness. This author points out that tobacco is one of the most valuable research tools, and is a most abundant source of scientific information. Research with tobacco plants will contribute far beyond the frontiers of agricultural science: tobacco can be a source of food supply with nutrition value similar to that of milk; tobacco can be a source of health supplies including medical chemicals and various vaccines; tobacco can be a source of biofuel. All we need is to treat tobacco with respect; the use of tobacco is only in its initial stages.

Nicotine is the major alkaloid present in tobacco and the most frequently determined compound as a biomarker of tobacco exposure in both smokers and non-smokers exposed to environmental tobacco smoke. Current knowledge on the human metabolism and disposition kinetics of nicotine is reviewed, together with methods for the determination of nicotine and various metabolites in different human biological fluids and matrices. Only short-term biomarkers of nicotine exposure exist and long-term biomarkers of exposure such as the incorporation of nicotine and cotinine into human hair, toenails and deciduous teeth require further investigation. Determination of ‘nicotine boost’, the difference in blood nicotine concentrations that occur after smoking a single cigarette, provides an experimental indication of individual smoking behaviour, but is unsuitable for population studies. The determination of nicotine plus multiple phase I and phase II metabolites in 24-hour urine, often expressed as ‘nicotine equivalents’, provides the most accurate way to determine exposure to nicotine in smokers; however, few laboratories are equipped to perform the complex analysis required for this purpose. Nicotine equivalents can be used to estimate the uptake of nicotine from a cigarette in both individuals and in population studies. Despite recent advancements in analytical methodology and the possibility of determining multiple nicotine metabolites in various biological fluids, determination of cotinine, the major metabolite of nicotine, is likely to remain the most commonly used approach to assess exposure to tobacco smoke in both smokers and non-smokers. Representative data for cotinine in blood, saliva and urine of smokers and non-smokers are presented.

Methods based on the analyses of cigarette filters have been used to estimate ‘tar’ and nicotine yields to smokers. These methods rely on the measurement of filtration efficiencies (FEs). However FEs may be influenced by both cigarette design features e.g., type of filter and levels of filter ventilation, and human smoking behaviour factors such as puff flow-rates and cigarette butt lengths. Two filter analysis methods are considered in our study. One is based on the analysis of whole filters using average values of FEs obtained from a range of machine smoking regimes. The other, a ‘part filter’ method, analyses a 10 mm section from the mouth end of the filter where the FE remains relatively constant irrespective of puff flow rates and butt lengths. Human puffing behaviour records were obtained from 10 smokers, each smoking six commercial cigarettes ranging from 1 mg to 12 mg ‘tar’ yields [International Standard (ISO) values]. These records were used to drive a human smoke duplicator and the resulting ‘tar’ and nicotine yields obtained from duplication were compared with the estimates obtained from ‘whole’ and ‘part filter’ analysis. The results indicated that whilst both filter methods gave good correlations with nicotine and ‘tar’ yields obtained from smoke duplication, the ‘part filter’ method was less susceptible to the effect of nicotine condensation and changes in FEs and hence gave a more accurate assessment of yields than the ‘whole filter’ method.

Cigarette testing regulations based on more intensive puffing conditions than standard Federal Trade Commission/International Organisation for Standardization (FTC/ISO) conditions, together with intentional filter vent-blocking of cigarettes during testing, are currently required in some countries. Recently, an initial recommendation under the auspices of the Framework Convention on Tobacco Control, has called for international machine-testing of cigarettes with a 55 cc/30 s/2 s puffing regimen after 100% filter vent-blocking. While much is currently known regarding changes in smoke yields with different machine smoking parameters, a more limited understanding of potential changes in smoke composition exists. In the present work, the influence of smoking conditions on nicotine fate in a burning cigarette was studied by gas chromatography with atomic emission detection (GC-AED) using core-injected nicotine-d4. Tobacco rods were injected via a syringe to a fixed length with a constant volume of a methanol solution of known concentration of deuterated nicotine. Four different puffing conditions and two different vent-blocking conditions were studied. GC with mass spectrometric detection was used to identify the deuterium-labeled compounds that gave an enhanced deuterium AED-response. A comparison of the distribution of compounds containing deuterium in the mainstream smoke, sidestream smoke, and cigarette remains (butt and ash) of a full flavor cigarette brand under the four smoking conditions studied indicated that a greater percentage of labeled nicotine remained intact during the smoking process as the intensity of the puffing regimen increased. As smoking regimen intensity increased, the amounts of nicotine pyrolysis and oxidation products detected in sidestream smoke decreased, while marginal increases in these compounds were observed in mainstream smoke and in the cigarette butt. The sidestream/mainstream nicotine ratio decreased significantly as smoking intensity increased. Small amounts of intact nicotine were observed in the sidestream vapor phase; however, nicotine was not observed in mainstream vapor phase samples.

It has been suggested that ammonium compounds in tobacco generate nitrogen monoxide (NO) in cigarette smoke. This causes the smoke to retain the broncho-dilatory properties of the tobacco, which leads to an increased uptake of nicotine and thus to a potentially higher addiction to tobacco. The objective of this study was to ascertain putative correlations among the concentration of ammonium compounds in whole tobacco and the concentration of NO in mainstream smoke.

In 98 different cigarette brands marketed in the Netherlands, positive correlations were found between ‘tar’ and nicotine values (coefficient of variation, R² = 0.95), and between ‘tar’ and NO concentration (R² = 0.47). The quantity of ammonium compounds in tobacco (expressed as the amount of NH4+ present) varied, however, from 0.1 to 3.3 mg per gram of tobacco and was not associated with any of the parameters investigated here. In addition, five cigarette types were compared with respect to the levels of ammonium-compounds in the tobacco, the concentration of NO in the smoke and ‘tar’/nicotine ratio. The concentration of NO in the smoke from light menthol and light cigarettes (‘tar’ content < 9 mg/cig) was significantly lower than that from their regular equivalents (‘tar’ content > 9 mg/cig). As expected, the ‘tar’/nicotine ratio of regular cigarettes was significantly higher than the ratio in light cigarettes.

This study shows that the whole tobacco in the various cigarette brands differed in the amount of ammonium compounds it contained, but these amounts bore no relation to the level of NO and the level of nicotine and ‘tar’ in the smoke. Other factors that affect the burning process, such as nitrate content and product design may have made the association between ammonium compounds in tobacco and the level of NO in mainstream smoke less clear.

The use of a tobacco agar medium (TAM) was investigated to visually differentiate Cryptococcus species from Rhodotorula and Candida species that can be isolated from tobacco. This study was first conducted with pure isolates of each of the major yeast species that have been isolated from tobacco. All Cryptococcus strains that were tested produced colonies with different degrees of pigmentation ranging from light to dark brown or black. All Candida and Pichia colonies were white to off-white. Candidaparapsilosis colonies were easily differentiated since they had rough contours and surfaces. All Rhodotorula colonies were pink or orange. In order to validate the use of this medium, tobacco was spiked with a mixed culture of Cryptococcus, Candida, Rhodotorula and Pichia. TAM allowed visual detection and enumeration of the four yeast genera based on colony colour and/or morphology.

Among the polycyclic aromatic hydrocarbons (PAHs), a major class of identified cigarette mainstream smoke (MSS) components, are several shown to be tumorigenic in laboratory animals and suspect as possible tumorigens to humans. To date, nearly 540 PAHs have been completely or partially identified in tobacco smoke [Rodgman and Perfetti (1)]. A detailed chronology is presented of studies on four much discussed PAHs identified in tobacco smoke, namely, benz[a]anthracene (B[a]A), its 7,12-dimethyl derivative (DMB[a]A), dibenz[a, h]anthracene (DB[a, h]A), and benzo[a]pyrene (B[a]P). Of the four, DMB[a]A, DB[a, h]A, and B[a]P are considered to be potently tumorigenic on mouse skin painting and subcutaneous injection. Opinions on the tumorigenicity of B[a]A to mouse skin vary. DMB[a]A is frequently used in tumorigenicity studies as an initiator. Examination of the number of tobacco smoke-related citations listed for these four PAHs reveals the enormous effort devoted since the early 1950s to B[a]P vs. the other three. An annotated chronology from 1886 to date describes the tobacco smoke-related research pertinent to these four PAHs, their discovery, isolation and/or identification, quantitation, and contribution to the observed biological activity of MSS or cigarette smoke condensate (CSC). Much of the major literature on these four PAHs in tobacco smoke is presented in order to permit the reader to decide whether the current evidence is sufficient to classify them as a health risk to smokers. There has certainly been a tremendous effort by researchers to learn about these PAHs over the past several decades. Each of these PAHs when tested individually has been shown to possess the following biological properties: 1) Mutagenicity in certain bacterial situations, 2) tumorigenicity in certain animal species, to varying degrees under various administration modes, and 3) a threshold limit below which no tumorigenesis occurs. For more than five decades, it has been known that some of the PAHs, when co-administered in pairs of a potent tumorigen plus a non-tumorigen or weak tumorigen, show inhibitory effects on the tumorigenicity of the most potent, e.g., B[a]A plus DB[a, h]A; B[a]A plus B[a]P; anthracene plus DB[a, h]A. Over the period studied, some regulatory agencies considered these tobacco smoke PAHs to be serious health concerns, others did not.

With respect to cigarette MSS, certainly the ‘danger is in the dose’ for any MSS component tested singularly to be tumorigenic. But is the level of any of these MSS PAHs high enough to be of concern to smokers? The information herein presented indicates that over the last five decades the following has occurred: 1) The per cigarette yields of these four PAHs have decreased substantially, 2) compared to CSC or Federal Trade Commission (FTC) ‘tar’, their per cigarette yields have also decreased to a point that they may be below any significance biologically, and 3) the specific tumorigenicity in mouse skin-painting studies of the CSC has decreased. These are the three criteria originally proposed to define the ‘less hazardous’ cigarette. Actually, criterion 1) was first directed only at B[a]P. Previous studies highlighted the concern that some regulatory bodies had in attempting to understand why lung cancer and other forms of cancer seemed more prevalent in smokers. But cigarette smoking alone could not reconcile the evidence. Social, ethnic, environmental, and economic factors are also very important in understanding the entire biological effect. In fact, the level of B[a]P in CSC could only explain about 2% of its specific tumorigenicity observed in skin-painted mice and the combination of the levels of all the known tumorigenic PAHs in CSC could only explain about 3% of its tumorigenicity. Despite an 18-month study in the late 1950s, the search for a ‘supercarcinogen’ in MSS and CSC to explain the observed biological effects was unsuccessful. In addition, the exceptional study on MSS PAHs by United States Department of Agriculture (USDA) personnel in the 1970s indicated no ‘supercarcinogen’ was present. Only recently has the concept of complex mixtures in relation to the understanding of the complexity of carcinogenesis taken hold. Perhaps the reason why MSS is less tumorigenic than expected in humans is because of the presence of other MSS components that inhibit or prevent tumorigenesis. For example, it is well known that MSS contains numerous anticarcinogens present in quantities significantly greater than those of the PAHs of concern. When one reviews the history of these four PAHs in MSS or CSC it is clear that many unanswered questions remain.