(Wynder et al 1967) This is why cigarettes with low-tar yields have been devel- ..... Wynder JK, Ernst L, Hoffmann D ( 1967) Tobacco and tobacco smoke In: ...
International Archives of
Int Arch Occup Environ Health (1983) 52:11-16
Oand CtIV1g
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© Springer-Verlag 1983
Urinary Excretion of Thioethers Among Low-tar and Medium-tar Cigarette Smokers* Tuula Heinonen, Vuokko Kyt 6 niemi, Marja Sorsa, and Harri Vainio Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29, Finland
Summary The urinary excretion of thioethers was studied among smokers of medium-tar cigarettes (16 3 mg/cig ) and low-tar cigarettes (5 4 mg/cig ) The test persons (26 smokers and 11 nonsmokers) were healthy young men in the military service The smokers had an increased excretion of thioethers into the urine (P=O002 with Mann-Whitney U-test) when compared with the nonsmokers No differences in the amount of urinary thioethers were detected between the low-tar and medium-tar cigarette smokers The smokers (varying from 2 0 to 8 5 mmol/mol creatinine) had more individual variation than the nonsmokers (varying from 1 5 to 4 5 mmol/mol creatinine) Furthermore, the individual variation in the group of low-tar cigarette smokers did not markedly differ from the individual variation of medium-tar cigarette smokers. Key words: Biological monitoring Cigarette smoking
Glutathione conjugates
Thioethers -
Introduction Cigarettes contain hundreds of compounds, many of which are biologically highly reactive (Schmeltz et al 1974) The exact mechanism by which cigarette smoking leads to malignancies is still unknown, but among the possible causal factors the tarry fraction of cigarette smoke is considered to be important (Wynder et al 1967) This is why cigarettes with low-tar yields have been developed However, other factors (such as the pyrolysis products of proteins, nitrosation of polycyclic hydrocarbons or formation of free radicals) may be involved in the carcinogenicity of cigarette smoke (Borg 1972; Green 1977 ; Matsumoto et al. 1977 ; Mizusaki et al 1977 ;Nagao et al 1977) Therefore, it is not yet clear whether only reductions in the amount of tar in cigarettes are sufficient to decrease substantially the health hazards of smoking. * This study has been supported by a research grant (24/60) from the National Research Council for Sciences, Academy of Finland Offprint requests to: Dr Marja Sorsa (address see above)
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Glutathione, a cellular nucleophile, reacts easily with electrophilic compounds (Chasseaud 1979) Such electrophilic compounds may also be abundant in cigarette smoke Glutathione conjugates are excreted into the urine or the bile as mercapturic acids or other thioethers (Chasseaud 1976) Recently, a review has been published about studies where the increased concentration of urinary thioethers was used to indicate exposure to electrophilic compounds, a class of chemicals including suspected mutagens and carcinogens (van Doorn et al. 1981a) When compared with nonsmokers, smokers have also had increases in the excretion of thioethers (van Doorn et al 1979). The aim of this study was to clarify the excretion of thioethers in the urine of smokers and nonsmokers under a controlled situation, using volunteer smokers and nonsmokers Another purpose of the study was to compare the extent of excreted thioethers between low-tar cigarette smokers and medium-tar cigarette smokers. Materials and Methods Exposure Conditions The cigarettes used were two commercial self-burning American-type filtered brands The tar contents were 5 4 mg/cigarette for the low-tar cigarettes and 16 3mg/cigarette for the medium-tar cigarettes. The age of the male subjects was 19 0 0 7 (mean SD) years, and they were on a similar diet throughout the whole study period, with the exception of occasional free weekends from military service. After interviews, the subjects were divided into 3 groups: nonsmokers (11 persons, Group C), persons who usually smoked medium-tar cigarettes (13 persons, Group A); and persons who usually smoked low-tar cigarettes before the experiment (13 persons, Group B) All the urine samples were collected in the evening at 22:00 h The number of cigarettes smoked was recorded daily Altogether four samples were taken from the subjects So that a base-line level could be determined, the first two samples were taken at the beginning of the experiment, on two consecutive days when the smokers had smoked the medium-tar cigarettes for 4 or 5days. Group A then continued to smoke medium-tar cigarettes for the next 3weeks whereas Group B smoked low-tar cigarettes during this period The third urine sample was taken after this period Group A then switched to smoking low-tar cigarettes, and Group B smoked mediumtar cigarettes for 3 weeks The fourth urine sample was taken after the second 3-week period. The nonsmokers' urine samples were taken simultaneously with the smokers' urine samples. The urine samples were stored in deep freeze (-20 °C) until their analysis about 6 months later The number of urine samples for the thioether analyses was not always four per persons, because enough urine was not always available after other analyses (cf Falck 1982). Determination of Thioethers The content of thioethers was measured from centrifuged urine, principally following the method developed by van Doorn et al (1981b) The measurements were made in the following way: 5 ml of urine was acidified with 4 MHC 1 to pH 1 5-2 ; the urine was then extracted with 10 ml of ethyl acetate using a mechanical shaker for 15 min The organic layer was separated, and the extraction procedure was repeated The combined organic layers were evaporated at 370 C under reduced pressure using a Heindolph evaporator An adapter of six samples was used The residue was dissolved in 2 0 ml of distilled water, using sonication for 30 s The free sulphydryl groups were measured from the water extract with Ellman's reaction immediately after sonication: 0 25 ml of water extract was added to a test tube, which contained 2 0 ml of 0.5 M phosphate buffer (p H 7 1) and 0 3ml of DTNB solution (0 4 mg 5,5'-dithiobis-( 2-nitro-
13
Smoking and Urinary Thioethers a) smokers
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D
A.
43
b) nonsmokers
-
a M 2
15 z
0
Fig la, b Distribution of the individual concentrations of urinary thioethers among smokers (a) and nonsmokers (b)
C 0 C
ay P
FI
5
U.
2
I
4
6
8
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10
Thioethers/Creatinine (mmol/mol)
Table 1 The concentration of urinary thioethers of three test groups after periods of smoking low-tar or medium-tar cigarettes Thioethers/creatinine (mmol/mol) Group A
Group B
Group C
4 7± 06 10
4 6 + 06 10
2 7 ± O3 7
3 W medium-tar n
4 1± O 8 5
3 9 O5 10
2 4 ±O 4 8
3 W low-tar n
5 1±O 8 9
3 0+ O 4 5
3 1±O 5 8
Base-level n
Base-level = the samples were taken 5 and 6 days after beginning the experiment 3 W medium-tar = 3 weeks medium-tar cigarettes for Groups A and B 3 W low-tar = 3 weeks low-tar cigarettes for Groups A and B The samples of nonsmokers (Group C) were taken simultaneously The value are the mean ± SEM, N= number of persons
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T Heinonen et al. a) medium-tar cigarette smokers
.> 2 .C 'a
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.2 .
25
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20
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b) low-tar cigarette smokers
15
_
. 15 O C
10 5
S
|
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.
4
I
I
6
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1
10
Fig 2 a, b Distribution of the individual concentrations of urinary thioethers among medium-tar cigarette smokers (a) and lowtar cigarette smokers (b)
Thioethers/Creatinine (mmol/mol)
benzoic acid)/ml 1%citrate solution) The absorbance of the Ellman reaction tubes was read at 412 nm using double beam spectrophotometer against a blank (2 0 ml phosphate buffer, pH 7.1, + 0 3 ml DTNB + 0 25 ml distilled water). After this, 1 0 ml of water extract was placed in glass screw-capped tubes, 0 5-ml 4 MNa OH was added, and the air space of the tubes was replaced by pure nitrogen The hydrolysis was performed in a bath of boiling water for 50 min The tubes were then cooled in an ice bath for 10 min, 0 5 ml of 4M HCI buffered with 0 65 M phosphate buffer (pH 7 1) was added, the tubes were mixed, and a sample of 0 25 ml was taken immediately to measure the concentration of free sulphydryl groups as already described. When the content of urinary thioethers was calculated, corrections were made for the concentration of the free sulphydryl compounds in the urine Furthermore, the correction for the colour of the urine extracts was made as follows: 2 0 ml phosphate buffer, p H 7 1, + 0 3 ml distilled water + 0 25 ml urine extract (hydrolyzed or non-hydrolyzed) Absorbance of the tubes was measured at 412 nm against a blank (2 0 ml phosphate buffer, p H 7 1, + 0 55 ml distilled water) The content of urinary thioethers was expressed as mmol/mol urinary creatinine The concentration of creatinine was measured according to the method of Clark and Thompson (1949). The statistical significance between smokers and nonsmokers or medium-tar cigarette smokers and low-tar cigarette smokers was tested with Mann-Whitney U-test. Results Compared with the nonsmokers (n = 11), the smokers (n = 26) had a significant increase (P=O 002) in the excretion of thioethers The mean values (+ SEM)
Smoking and Urinary Thioethers
15
were 4 3 ± 0 4 mmol/mol urinary creatinine for the smokers and 2 8 ± 0 2 for the nonsmokers The variation between individuals (one value represents the mean of all urine analyses per person) among the smokers was higher (from 2 0 to 8.5 mmol/mol creatinine) than the individual variation among the nonsmokers (from 1 5 to 4 5 mmol/mol creatinine, cf Fig la, b). No differences was to be seen in the content of urinary thioethers between smokers of low-tar and medium-tar cigarettes, either in the base levels or after the periods of smoking low-tar or medium-tar cigarettes (Table 1) Furthermore, the individual variation of the medium-tar cigarette smokers did not markedly differ from that of the low-tar cigarette smokers (Fig 2a, b) The number of cigarettes smoked daily per person varied from 10 to 25 cigarettes No clear correlation could be detected in the number of cigarettes smoked and the content of urinary thioethers (data not shown). Discussion The measurement of the content of urinary thioethers has been used as a method for monitoring exposure to potentially genotoxic agents in rubber industry workers (Kilpikari and Savolainen 1982; Vainio et al 1978), workers at chemical waste incinerators (van Doorn et al 1981 b), and workers exposed to methyl chloride (van Doorn et al 1980) In agreement with the earlier results of van Doorn et al (1979), we detected a significantly increased excretion of thioethers (a 54 % increase) in smokers compared with nonsmokers Furthermore, the results indicate that cigarette smoking must be taken into account as a confounding or modifying factor if thioethers are used in monitoring occupational exposures. The interindividual variation of thioether content was higher among smokers than among nonsmokers The greater variation may reflect the individual differences in the metabolism of xenobiotics, or it may reflect individuals' different smoking habits. No differences were observed in the excretion of thioethers between low-tar and medium-tar cigarette smokers This suggests that the tar content of cigarettes should not be regarded as the decisive factor when cigarette smokers' exposure to electrophilic and potentially alkylating agents in the cigarettes is estimated. Thus, according to this criteria, the "healthiness" of low-tar cigarettes remains questionable. Acknowledgements The authors greatfully acknowledge the help of our colleague, Dr Kai Falck, in getting the urine samples.
References Borg DC (1972) Electron spin resonance applied to free radicals of physiological, pharmacological, or biochemical interest In: Swartz HM, Bolton JK, Borg DC (eds) Biological applications of electron spin resonance Wiley-Interscience, New York, pp 340-341 Chasseaud LF (1976) Conjugation with glutathione and mercapturic acid excretion In: Arias IM, Jakoby WB (eds) Glutathione: metabolism and function, vol 6 Raven Press, New York, pp 77-114
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Chasseaud LF (1979) The role ofglutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents In: Klein Y, Weinhouse S (eds) Advances in cancer research, vol 29 Academic Press, New York, pp 175-255 Clark LC Jr, Thompson HL (1949) Determination of creatine and creatinine in urine Anal Chem 21:1218-1221 van Doorn R, Bos RP, Leijdekkers ChM, Wagenaas-Zegers JLG, Henderson PTh (1979) Thioether concentration and mutagenicity of urine from cigarette smokers Int Arch Occup Environ Health 43 :159-166 van Doorn R, Borm PJA, Leijdekkers ChM, Henderson PTh, Reuvers J, van Bergen TJ (1980) Detection and identification of S-methylcysteine in urine of workers exposed to methyl chloride Int Arch Occup Environ Health 46: 99-109 van Doorn R, Leijdekkers ChM, Bos RP, Brouns RME, Henderson PTh (1981a) Detection of human exposure to electrophilic compounds by assay of thioether detoxification products in urine Ann Occup Hyg 24: 77-92 van Doorn R, Leijdekkers ChM, Bos RP, Brouns RME, Henderson PTh (1981b) Enhanced excretion of thioethers in urine of operators of chemical waste incinerators Br J Ind Med 38:187-190 Falck K (1982) Urinary mutagenicity caused by smoking In: Sorsa M, Vainio H (eds) Mutagens in our environment Liss, New York, pp 387-400 Green CR (1977) Neutral (oxygenated) compounds in cigarette smoke and their possible precursors Recent Adv Tobacco Sci 3 :94 Kilpikari I, Savolainen H (1982) Increased urinary excretion of thioether in new rubber workers Br J Ind Med 39: 401-403 Matsumoto T, Yoshida D, Mizusaki S, Okamoto H (1977) Mutagenic activity of amino acid pyrolysates in Salmonella typhimurium TA 98 Mutation Res 48: 279-286 Mizusaki S, Okamoto H, Akiyama A, Fukura Y (1977) Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate Mutation Res 48: 319-325 Nagao M, Honda M, Seino Y, Yahagi T, Kawachi T, Sugimura T (1977) Mutagenicities of protein pyrolysates Cancer Lett 2: 335-340 Schmeltz I, Hoffman D, Wynder EL (1974) Toxic and tumorigenic agents in tobacco smoke: analytical methods and modes of origin Trace, Subst Environ Health 8: 281-295 Vainio H, Savolainen H, Kilpikari I (1978) Urinary thioether of employees of a chemical plant Br J Ind Med 35: 232-234 Wynder JK, Ernst L, Hoffmann D (1967) Tobacco and tobacco smoke In: Studies in experimental carcinogenesis Academic Press, New York Received August 25, 1982 / Accepted March 3, 1983
