Curt R. Enzell

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.

The pharmacokinetics of cotinine in plasma and saliva from non-smoking healthy volunteers

SummaryCotinine is a major metabolite of nicotine in man. Its disposition kinetics has been followed in plasma and saliva from nine nonsmokers, 23 to 56 years of age. Cotinine 5, 10 and 20 mg was given intravenously and orally to each subject, and plasma, saliva and urine samples were collected for 96 h.The kinetics of cotinine was best described by a multi-compartment model with three distinct phases both in plasma and saliva. Regardless of the mode of administration, there was no indication of dose-dependent kinetics. Mean total plasma clearance was 63.8 ml·h−1·kg−1 and mean renal clearance was 4.7 ml·h−1·kg−1, i.e. only 10% of the dose was excreted unchanged in the urine. The volume of distribution, as calculated from the plasma curves, was slightly greater than the body weight, 1.1 l·kg−1. The concentration of cotinine was 20 to 40% higher in unstimulated mixed saliva than in plasma during the absorption, distribution and elimination phases. As the clearance and distribution values in saliva were directly proportional to the corresponding values in plasma, similar terminal half-life values were obtained in the two body fluids, 15.5 and 16.8 h for plasma and saliva, respectively.Thus the kinetics of cotinine is linear after intravenous and after oral dosing, and salivary concentrations give the same information about cotinine disposition in the body as do plasma concentrations.

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.

The chemistry of the natural order cupressales—XXI : Cuparene and cuparenic acid, two sesquiterpenic compounds with a new carbon skeleton

Abstract An aromatic sesquiterpene, cuparene (IV), has been isolated from Chamaecyparis thyoides , Biota orientalis and Widdringtonia . Cuparene gives terephthalic acid on oxidation with dilute nitric acid and (+)-camphonanic acid (III) on prolonged treatment with ozone and is therefore 1-(4-methylphenyl)-1:2:2-trimethyl cyclo pentane. Oxidation of cuparene with chromium trioxide gives the corresponding substituted benzoic acid, cuparenic acid (V). This acid has been found to be identical with one of the sesquiterpene acids (“acid III”) previously isolated from Widdringtonia species and now also from Chamaecyparis thyoides .

Biodegradation of carotenoids - an important route to aroma compounds

In the group of compounds referred to as degraded carotenoids the majority of the members are volatile compounds comprising 13 to 9 carbon atoms. A fair number of these are important aroma constituents occurring in many higher plants, some of which are widely used and of considerable economic importance. The discussion will be focused on the formation of selected representatives encountered in tobacco, since this is the richest single source of these compounds and also since it comprises a large number of degraded diterpenoids of known structure and stereochemistry providing additional information on possible degradative pathways. Although degraded carotenoids constitute a large group of compounds, only the function of a few mainly those which play a vital role in life processes is known. Many, however, possess highly attractive aroma properties and it is primarily selected members of this group and their formation which will be considered here. The degraded carotenoids retaining the largest number of original carbon atoms are the nor-. carotenoids, which have 39, 38 or 37 carbon atoms. However, these are of little interest here, since neither do they appear to be precursors of any of the aroma compounds under consideration, nor do the reactions leading to their formation have any bearing on the generation of the aroma compounds. In contrast, both the formation and the structures of the apo-carotenoids are of importance, since, being present in higher plants and frequently possessing the same end groups as the four carotenoids predominant here, they are potential precursors of the carotenoid-like aroma compounds. Remarkably little, however, is known about their immediate precursors and the reactions by which they are formed. In fact, it is still unclear whether they result from direct cleavage of the bonds indicated in Fig. 1, or by a stepwise mechanism of the Glover-Redfearn type. Moreover, it is still an open question in virtually all cases whether the cleavage is enzyme-assisted or accomplished by singlet oxygen or autooxi dation. More detailed information on enzymatic in-chain cleavages is only available for the conversion of /3carotene to retinal, which is postulated to occur as shown in Fig. 2 (Ref. 1). However, has also been shown that a tea enzyme preparation in the presence of tea flavanols converted C-labelled -. carotene into /%-ionone, and several other, unidentified volatile substances including some derived exclusively from the central part of the polyene chain (Ref. 2). Furthermore, soy bean lipoxygenase is known to effect the conversion of violaxanthin into 3-hydroxy-,3-ionone epoxide (Ref. 3). It seems therefore that there are site-specific as well as non-sitespecific enzymes capable of effecting cleavage of the polyene chain. 693 024

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.

Effects of tobacco and tobacco smoke constituents on cell multiplication in vitro

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxicity test system for tobacco and tobacco smoke constituents. Some 250 compounds, representative of these materials, were examined by exposing cells to different concentrations of these constituents and measuring the inhibition of culture growth, which was related to corresponding effects encountered for positive standards. When employing the present cell toxicity test system possible effects of factors such as penetration, distribution and microsomal metabolism of the compounds studied, are not taken into account. The most active constituents were found to be unsaturated aldehydes and ketones, phenols and indoles. The good correlation obsered between functional groups and toxicity permits, within the range of functionalities studied, prediction of the toxicity for a compound of known structure.

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.

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.