Margareta Curvall

Screening of tabacco smoke constituents for mutagenicity using the Ames' test

To clarify the mutagenic activity of individual smoke components, 239 compounds, representative of the gaseous and semivolatile phases of tobacco smoke, were assayed for mutagenicity towards 4 histidine-requiring mutants of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537). All compounds were tested qualitatively both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced rats. Without S-9, only 2,3-dimethylindole and 2,3,5-trimethylindole showed mutagenic activity that was not enhanced by the metabolic activation system. 2,6-Diaminotoluene and coronene, which like the above compounds are not documented carcinogens were found to be mutagenic for strain TA 98 with S-9. Mutagenic activity was also observed for the previously known mutagens benz[a]pyrene, chrysene, benz[a]-anthracene, perylene and beta-naphthylamine, on exposure to strains TA 98 and/or TA 100 with S-9.

Simultaneous determination of nicotine and cotinine in plasma using capillary column gas chromatography with nitrogen-sensitive detection

A rapid and sensitive method is described for the stimultaneous determination of nicotine and its principal metabolite, cotinine, in plasma. A one-step extraction procedure is employed and the quantitative analyses are performed by capillary column gas chromatography using a thermionic specific detector. Other special measures to avoid contamination from external sources such as atmosphere, solvents and laboratory equipment, which constitutes the major limiting factor of nicotine assay, were also undertaken. The structural analogues of nicotine and cotinine, N-methylanabasine and N-ethylnorcotinine, are used as internal standards. Moreover, a micromethod, which requires only 0.1 ml of plasma and found to be suitable for analysis of cotinine in finger-tip samples of blood, is described. LInearity over the concentration ranges 5-100 ng of nicotine per ml of plasma and 5-500 ng of cotinine per ml of plasma is demonstrated. The precision of the method has been investigated by determining the reproducibility at different levels of nicotine and cotinine within the working ranges, for both 1-ml and 0.1-ml samples of plasma.

In vitro studies of biological effects of cigarette smoke condensate: II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects. The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1-F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones. The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.

Simulation and evaluation of nicotine intake during passive smoking: Cotinine measurements in body fluids of nonsmokers given intravenous infusions of nicotine

The technique of monitoring cotinine concentrations in body fluids as a means of measuring nicotine intake during passive smoking has been evaluated in two studies, both of which used intravenous infusion to simulate nicotine intake. In the first study, nicotine and cotinine were given separately, for 1 hour in four different intravenous doses (3.2, 15.4, 30.9, and 61.7 nmol/min) to each nonsmoker. In the second study, nicotine and cotinine were infused for 4 hours; each subject received five different doses of nicotine (1.5, 3.1, 6.2, 10.8, and 15.4 nmol/min) and one of cotinine (10.8 nmol/min). The concentration of cotinine was constant in both plasma and saliva from 1 to 4 hours after the nicotine infusion; the plateau levels of cotinine were found to be linearly and directly related to the nicotine intake. The ratio of salivary to plasma cotinine was 1:1.27. A linear relationship was also found between nicotine and cotinine infusion rates and the AUC values for cotinine. The fraction metabolized to cotinine was found to be about 0.5. The results from these studies show that: (1) there is a linear relationship between the plateau concentration of cotinine and the amount of nicotine infused over a period of 1 up to 4 hours; (2) salivary cotinine provides the same information on nicotine intake as does plasma cotinine; and (3) single measurements of either plasma or salivary cotinine concentrations at 1 to 4 hours after the exposure could be used to predict the nicotine intake during 1 to 4 hours of environmental tobacco smoke exposure.

In vitro studies of the biological effects of cigarette smoke condensate. III. Induction of SCE by some phenolic and related constituents derived from cigarette smoke: A study of structure-activity relationships

Since our earlier studies of 23 individual weakly acidic constituents of cigarette smoke indicated that benzenes having vicinal oxygenation or a conjugated double bond induce sister-chromatid exchanges (SCE), we have now selected and examined a complementary set of 27 smoke constituents for their SCE-inducing properties. Of the 50 compounds tested in all, 23 were found to induce SCE, and these include all benzaldehydes but one and the majority of the compounds having a conjugated carbon-carbon double bond as well as several of the guaiacols. These groups of active compounds comprise important flavourants such as vanillin, ethylvanillin, isoeugenol and guaiacol. The structure-activity relationships encountered here may be useful in predicting the SCE-inducing activity of related compounds.

Swedish snus and the GothiaTek ® standard

Some smokeless tobacco products, such as Swedish snus, are today considered to be associated with substantially fewer health hazards than cigarettes. This risk differential has contributed to the scientific debate about the possibilities of harm reduction within the tobacco area. Although current manufacturing methods for snus build on those that were introduced more than a century ago, the low levels of unwanted substances in modern Swedish snus are largely due to improvements in production techniques and selection of raw materials in combination with several programs for quality assurance and quality control. These measures have been successively introduced during the past 30-40 years. In the late 1990s they formed the basis for a voluntary quality standard for Swedish snus named GothiaTek®. In recent years the standard has been accepted by the members of the trade organization European Smokeless Tobacco Council (ESTOC) so it has now evolved into an industrial standard for all smokeless tobacco products in Europe.The initial impetus for the mentioned changes of the production was quality problems related to microbial activity and formation of ammonia and nitrite in the finished products. Other contributing factors were that snus came under the jurisdiction of the Swedish Food Act in 1971, and concerns that emerged in the 1960s and 1970s about health effects of tobacco, and the significance of agrochemical residues and other potential toxicants in food stuffs.This paper summarizes the historical development of the manufacture of Swedish snus, describes the chemical composition of modern snus, and gives the background and rationale for the GothiaTek® standard, including the selection of constituents for which the standard sets limits. The paper also discusses the potential future of this voluntary standard in relation to current discussions about tobacco harm reduction and regulatory science in tobacco control.

Effect of tobacco smoke compounds on the plasma membrane of cultured human lung fibroblasts.

The ability of compounds derived from tobacco and tobacco smoke to increase the permeability of the membranes of human lung fibroblasts has been studied by measuring the release of an intracellular marker after short term exposure. Of the 464 compounds tested, about 25% gave rise to severe membrane damage. The most active compounds, when divided according to functionality, were found within the groups of amines, strong acids and alkylated phenols, whereas nitriles and polycyclic aromatic hydrocarbons were found completely inactive. A pronounced effect of the chain length on the activity was observed for the aliphatic alcohols, aldehydes and acids, and all monocyclic aromatic compounds but benzonitriles and benzoic acids showed an increase in activity with increasing alkylsubstitution. It is concluded that tobacco smoke contains a number of membrane damaging substances. These membrane active compounds could not only cause direct toxic reactions but also potentiate the toxic effect by promoting the cell membrane penetration of other toxic substances in tobacco smoke.

Effects of tobacco smoke compounds on the noradrenaline induced oxidative metabolism in isolated brown fat cells

The effect on cell metabolism of 320 individual smoke components have been investigated by measuring their inhibition of noradrenaline induced respiration in isolated hamster brown fat cells. The compounds are representative of the gaseous and semivolatile phases of tobacco smoke. The strongest inhibitors were found within the groups of aliphatic alcohols, aldehydes and acids, of alkylated phenols and indoles and of alpha,beta-unsaturated aliphatic aldehydes and ketones. Some of the aliphatic aldehydes and acids significantly increased the basal respiration of the cells, probably by acting as substrates and/or uncoupling of mitochondrial respiratory control.

Evaluation of the biological activity of cigarette‐smoke condensate fractions using six in vitro short‐term tests

The biological activity of the volatile part of the particulate phase of cigarette-smoke condensate, the semivolatile fraction, has been examined, since the constituents of this material are accessible to selective filtration. Such a process offers a possibility to reduce the biological activity of total cigarette smoke without appreciably affecting the taste. Cigarette-smoke condensate, obtained from domestic American blend type cigarettes, was therefore separated into a nonvolatile and a semivolatile fraction, and the latter was fractionated by liquid-liquid extractions into four subfractions; acids, phenols, bases, and neutrals. The biological activity of these fractions was investigated using six in vitro short-term tests, of which two, the Ames test and the induction of sister chromatid exchanges, provided information on their genotoxicity, and the other four provided information on their cytotoxicity by measuring inhibition of cell growth, inhibition of oxidative metabolism, membrane damage, and ciliotoxicity. Sister chromatid exchanges were found to be induced by the total condensate, the nonvolatile and the semivolatile fractions, and the subfractions derived from the semivolatile fraction, except the bases. The Ames test showed the total condensate and the nonvolatile fraction to contain direct-acting base-pair mutagens as well as indirect-acting frameshift mutagens. While the semivolatile fraction was found nonmutagenic, two of its subfractions, acids and phenols, were shown to contain base-pair mutagens, which did not require metabolic activation. The total condensate and the nonvolatile and semivolatile fractions showed similar activity in the four cytotoxicity tests. Of the semivolatile subfractions, the acids and the phenols exhibited the highest activity and the bases the lowest; the toxicity observed for the neutrals varied with the test system used.

Conjugation Pathways in Nicotine Metabolism

Nicotine, cotinine and trans-3′-hydroxycotinine are excreted as glucuronic acid conjugates in urine of tobacco users. The average ratios between free and conjugated alkaloids in 24-hour urine samples from tobacco users are 1.0, 0.5 and 2.3 for nicotine, cotinine and trans-3′-hydroxycotinine, respectively. Nicotine, cotinine and trans-3′-hydroxycotinine and their glucuronides together with the N-oxides of nicotine and cotinine, account for more than 90% of the ingested dose of nicotine.