Chromosome aberrations and urinary thioethers in smokers

Mutation Research, 240 (1990) 289-293

289

Elsevier MUTGEN 01534

Chromosome aberrations and urinary thioethers in smokers B. Sinu6s, M. Izquierdo and J. Perez Viguera Department of Pharmacology, Medical School, University of Zaragoza, E-5OOO9-Zaragoza(Spain)

(Received2 May 1989) (Revisionreceived21 November1989) (Accepted23 November1989)

Keywords: Chromosomeaberrations; Urinary thioethers; Smokers; Cytogeneticeffects;Internal dosimetry

Summary Chromosome aberrations, classified as chromosome- and chromatid-type aberrations, were evaluated in 94 individuals. The concentration of thioethers in the urine was also determined. The sample consisted of 41 non-smokers (control group) and 53 smokers, 25 smoked 10-20 cigarettes/day (subgroup IIA) and 28 smoked more than 20 cigarettes/day (subgroup IIB). Our aim was to perform internal dosimetry on smokers using a cytogenetic test, and a test for urinary excretion of thioethers, in order to determine the relation between these 2 methods of dosimetry. Our results show a higher frequency of chromosome aberrations (p < 0.0001) and a higher excretion of urinary thioethers ( p < 0.0001) in smokers as compared to non-smokers. However, linear regression between these parameters was not statistically significant. In view of the variation between different individuals with regard to the amount of urinary thioethers, it seems more accurate to perform the biomonitoring of smoking by analyzing chromosome aberrations.

Tobacco smoking is the most important cause of lung cancer (U.S. Office on Smoking and Health, 1982). It also appears to be responsible for a high percentage of cancers of the mouth (Hammond and Horn, 1958), pharynx, esophagus, bladder, pancreas and kidneys (Wigle et al., 1980). Both the International Agency for Research on Cancer (IARC, 1982) and the American Conference of Governmental Industrial Hygienists (ACGIH, 1980) identified at least 38 known or suspected carcinogens in tobacco smoke in the particulate phase, and 16 in the volatile phase.

Correspondence: Prof. Blanca Sinu6s Porta, Departamento d6 Farmacologia, Facultad de Medieina, Calle Domingo Miral s/n, E-50009Zaragoza(Spain).

In addition, one must consider many other substances which, during biotransformation, lead to reactive, intermediate metabolites (Guenthner and Oesch, 1981). The mechanism of carcinogenesis appears to be related to the production of epoxides by biotransformation and their possible covalent binding to DNA. This binding produces chromosome damage, which could give rise to neoplasia (Chasseaud, 1979; Van Doom et al., 1981). Among the methods for biomonitoring the exposure to potential mutagens and carcinogens those for evaluating biological effects are based on short-term tests, using cytogenetic parameters as indicators (Van Sittert and De Jong, 1985). The formation of conjugates of those electrophilic products with glutathione which can then bind covalently to DNA, RNA and proteins rep-

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290 resents a detoxification pathway (Van Doorn et al., 1981). Our aim is to perform internal dosimetry in smokers, analyzing chromosome aberrations and measuring the concentration of thioethers in the urine and to find out whether there is any correlation between these 2 end points. Materials and methods

Individuals Blood and urine samples were taken from 94 individuals. Two groups were established: group I, 41 non-smokers, 24 men and 17 women, aged 18-55 (average __+SD, 33.63 _+ 11.06) and group II, 53 smokers. Group II was subdivided into 2 groups: subgroup IIA, 25 individuals who smoked 10-20 cigarettes/day, 12 men and 13 women, aged 18-61 (average _+ SD, 30.2 + 12.77), and subgroup IIB, 28 individuals who smoked more than 20 cigarettes/day, 18 men and 10 women, aged 18-60 (average + SD, 36.21 _+ 13.42). All subjects were healthy and were examined and interviewed, by the same persons, concerning diseases, drug intake, exposure to ionizing radiation, smoking habits, viral infection or exposure to other known or suspected mutagenic agents in order to eliminate any factor which might interfere with the results. Likewise, age distribution was homogeneous in all groups. Cytogenetic methods Culture. Heparinized blood (2 ml) was obtained by puncture of an arm vein. 0.5 ml was added to 5 ml culture medium containing TC-199 (Gibco) with 15% fetal bovine serum and antibiotics: penicillin 100/~g/ml and 100 units/ml streptomycin. The cultures were stimulated by adding phytohemagghitinin (PHA-M) (Difco, 3%) at a final concentration of 250 units/ml. 24 h after culture initiation, 5-bromo-2'-deoxyuridine (BrdUrd) was added at a final concentration of 5 /~g/ml medium. All cultures were incubated at 3 7 ° C for 72 h. Preparations were made using differential staining using the method described by Perry and Wolff (1974).

Chromosome aberrations. Only M 1 metaphase cells were considered to be scored on coded slides. To analyze morphological alterations of the chromosomes, 100 mitoses were studied per individual. Lesions were classified as follows: (1) chromatid-type aberrations, including chromatid gaps and chromatid breaks, and (2) chromosome-type aberrations, including isochromatid-chromatid breaks a n d / o r acentric fragments, dicentric chromosomes + associated fragments, and centric rings + associated fragments. Neither numerical aberrations nor balanced translocations and inversions were counted since only metaphases with 46 chromosomes were considered, and no banding analysis was performed. Urinary thioethers Urine samples of about 100 ml were taken on the same day as blood samples. The samples were kept for a maximum of 3 months at - 2 0 ° C. Urinary thioethers were determined using the method described by Van Doorn et al. (1980), based on the Ellman reaction (Ellman, 1959). Urine samples were centrifuged for 5 rain at 3000 X g before processing. Aliquots of 5.0 ml of clear urine were transferred to glass-stoppered tubes, and the p H was adjusted to 1.5-2.0 with 4 N HC1. After adding 8.0 ml of ethyl acetate, the layers were shaken vigorously for 15 rain using a shaker. The layers were separated by centrifugation at 3000 x g for 5 rain. The ethyl acetate layer was removed, and the procedure was repeated with another 8 ml of ethyl acetate. The collected ethyl acetate layers were evaporated to dryness with a rotary evaporator. The residue was dissolved in 2.0 ml distilled water. Alkaline hydrolysis was performed with 1.0-ml samples in brown-glass screw-capped tubes by adding 0.5 ml of 4 N NaOH, saturating with nitrogen, and keeping the closed tubes in a boiling waterbath for 50 rain. The tubes were cooled in ice for 10 min and 0.5 ml of 4 N HC1 was added while mixing. 5 rain later, the SH concentration was determined according to Ellman (1959) with slight modifications. A 0.25-ml aliquot of the aqueous solution was added to a freshly made mixture of 2.0 ml of 0.5 M phosphate buffer (pH = 7.l) and 0.3 ml of a

291 TABLE 1 CHROMOSOME ABERRATIONS Group

I (non-smokers) II (smokers) IIA (mild smokers) IIB (heavy smokers)

Chromatid-type

Chromosome-type

Gaps

Breaks

Isochromatid breaks or acentric fragments

Dicentrics + associated fragments

Centric rings + associated fragments

0.75 +_0.83 0.96 + 1.05 0.56 + 0.82 1.32 + 1.12

0.19 +__0.45 0.34 _+0.73 0.2 +_0.5 0.46 + 0.88

0.12 _+0.4 0.71 +_1.02 0.72 _+0.93 0.71 ___1.i 1

0.19 ± 0.46 0.51 ± 0,82 0.28 +_.0,45 0.71 _+1

0.17 _+0.38 0.53 +_0.74 0.28 _+0.45 0.75 ± 0.88

Frequency/100 cells. Values are given as mean + standard deviation.

5,5'-dithio-bis-(2-nitrobenzoic acid) solution (0.4 mg D T N B per ml of 1% sodium citrate solution). Absorbances were read at 412 nm on a Pye Unicam SP 30 UV spectrophotometer. Corrections were made for the contribution of the extract and the D T N B solution. SH concentrations were calculated from the corrected absorbance and the molar absorbance of the reference compound, N-acetylcysteine. Thioether concentrations in urine samples were expressed in mmole S H / m o l e creatinine. N o corrections were made for the presence of thiols, disulfides or thioesters in the extracts. Creatinine assay The creatinine content of the urine samples was measured using a colorimetric method based on the reaction of creatinine with alkaline picrate (Test-combination, Boehringer Mannheim). Urinary thioether concentrations were determined in samples with a creatinine content of 5.0 mmole/1 or higher. Statistical methods Statistical analyses were performed by comparison of averages between groups and subgroups, and by linear regression. The statistical differences were calculated using the A N O V A test or the M a n n - W h i t n e y U test, depending on whether or not the data fit a normal distribution. Correlations were evaluated using Spearman's test. Results The mean value of total chromosome aberrations was greater in the smokers (group II) than in

the non-smokers (group I) (Table 1) ( F = 16.18, p < 0.0001). The mean values of this parameter increased with increasing tobacco consumption (Table 1), and the difference was significant when comparing the control group (I) and subgroup IIB (smokers > 20 cigarettes/day) ( F = 16.71, p < 0.0001), and the 2 subgroups of smokers (IIA and IIB) ( F = 7.89, p < 0.05). However, the difference between the non-smokers and subgroup IIA (smokers of 10-20 cigarettes/day) was not significant ( F = 0.81, p > 0.05). The chromosome-type aberrations behaved in a similar way: they were more frequent in the group of smokers, and the average values increased with increasing tobacco consumption (Table 1). There was a significant difference between the control group (I) and the smokers (lI) (z = - 3 . 9 5 , p < 0.05), and between the control group and subgroup IIB (z = - 4 . 1 1 , p <0.05). The difference between the 2 subgroups of smokers ( I I A - I I B ) was also significant (z = - 1.58, p < 0.05), as was that between the control group and subgroup IIA (10-20 cigarettes/day) (z = - 2.41, p < 0.05).

TABLE 2 M E A N VALUES A N D S T A N D A R D URINARY THIOETHERS

DEVIATION

Group

Urinary thioethers (mmole S H / m o l e creatinine)

I (non-smokers) II (smokers) IIA (mild smokers) IIB (heavy smokers)

2.62_+ 1.87 11.19+_ 3.48 10.80_+ 3.47 11.49+3.53

OF

292 The frequencies of chromatid-type aberrations increased with smoking and there were statistically significant differences between smokers and non-smokers (z = - 1 . 0 6 , p < 0.05), between non-smokers and subgroup fiB (z = - 2 . 3 7 , p < 0,05) and between subgroups IIA and IIB (z = - 2 . 6 8 , p < 0.05). However, there was no difference between the control group and subgroup IIA (z = - 0 . 7 1 , p > 0.05) (Table 1). The concentration of urinary thioethers was higher in smokers than in non-smokers (Table 2) ( F = 123.25, p < 0.0001). The difference between the control group and each of the 2 subgroups of smokers was significant: subgroup IIA ( F = 38.30, p < 0.0001) and subgroup IIB ( F = 50.25, p < 0.0001). There was no difference between the 2 subgroups of smokers ( I I A - I I B ) ( F = 0.26, p > 0.05) (Table 2). There was no significant linear regression between urinary thioether elimination and chromosome aberrations, either in non-smokers (r = 0.136, p > 0.05) or in smokers (r = 0.053, p > 0.05). Discussion

Our results show higher frequencies of chromosome aberrations in smokers than in nonsmokers, and agree with data from other authors. Venerna (1959) observed an increase in chromosome aberrations and acentric fragments in smokers; Obe and Herha (1978) found a greater frequency of chromosome aberrations in lymphocyte chromosomes from smokers compared with non-smokers and N a k a y a m a et al. (1985) reported DNA-strand breaks in smokers. N a k a y a m a et al. (1985) suggested that this is due to the effect of electrophilic substances in tobacco, such as catechol, methyl derivatives and hydroquinone. However, there are contradictory reports. Hedner et al. (1983) found no increase in chromosome aberrations in lymphocytes form smokers although they found an increase in a subgroup of smokers that was occupationally exposed to other genotoxic substances. This agrees with Bigatti et al. (1985), who suggested that tobacco could exert a weak additional effect in individuals who were already exposed to other substances. In our case, the increase in chromosome aberrations observed

in smokers can probably be attributed to smoking, since any individuals who might be affected by other factors were excluded from the sample, thus indicating that tobacco could be an important inducer of chromosome aberrations. Concentrations of urinary thioethers have been determined by other authors (Vainio et al., 1978; Van Doorn et al., 1982; Kilpikari and Savolainen, 1982; Lafuente and Mallol, 1986). We found higher excretion of urinary thioethers in smokers and the test was discriminatory in terms of exposure, since the concentrations of urinary thioethers increased with tobacco consumption. Our results agree with those reported by Lafuente and Mallol (1986), Vainio et al. (1978), Van Doorn et al. (1982) and Kilpikari and Savolainen (1982). We found no correlation between the concentrations of urinary thioethers and chromosome damage, due probably to pharmacokinetic differences between individuals with regard to enzyme activities. On the other hand, the application of urinary thioether concentration to the biological monitoring of internal exposure to reactive intermediates on an individual basis has been limited by the fact that it lacks chemical specificity and suffers from relatively high background values (Onkenhout et al., 1986; Heinonen et al., 1983). In summary, our data show that it is not possible to predict the frequencies of chromosome damage from urinary thioether excretion, the cytogenetic test being more discriminating for biological monitoring of tobacco smoking exposure.

References

ACGIH (1980) TLV's, threshold limit values of chemical substances in work-room air adopted by ACGIH for 1980, American Conference of Governmental industrial Hygienists, Cincinnati, OH. Bigani, P., L. Lamberti, G. Ardito, F, Armellino and C. Malanetto (1985) Chromosome aberrations and sister chromatid exchanges in occupationally exposed workers, Med. Lav. 76, 334-339. Chasseaud, L.F. (1979) The role of glutathione and glutathione-S-transferases in the metabolism of chemical carcinogens and other electrophilic agents, Adv. Cancer Res., 29, 175-275. Ellman, G.L. (1959) Tissue sulphydryl groups, Arch. Biochem. Biophys., 82, 70-77.

293 Guenthner, T.M., and F. Oesch (1981) The effects of modulation of microsomal epoxide hydrolase activity on microsome-catalyzed activation of benzo[a]pyrene and its covalent binding to DNA, Cancer Lett., 11, 175-183. Hammond, E.C., and D. Horn (1958) Smoking and death rates - report on forthy-four months of follow-up of 187,783 men. I. Total mortality, J. Am. Med. Ass., 166, 1159-1172. Hedner, K., B. H~3gstedt, A.M. Kolning, E. Mark-Vendel, B. Str~3mbeck and F. Mitelman (1983) Sister chromatid exchanges and structural chromosome aberrations in relation to smoking in 91 individuals, Hereditas, 98, 77-81. Heinomen, T., V. KytiSniemi, M. Sorsa and H. Vainio (1983) Urinary excretion of thioethers among low-tar and medium-tar cigarette smokers, Int. Arch. Occup. Environ. Health, 52, 11-16. IARC (1982) International Agency for Research on Cancer, Chemicals, Industrial Processes and Industries Associated with Cancer in Humans, IARC Monogr., Suppl. 4, IARC, Lyon. Kilpikari, I., and H. Savolainen (1982) Increased urinary excretion of thioether in new rubber workers. Br. J. Ind. Med., 39, 401-403. Lafuente, A., and J. Mallol (1986) Thioethers in urine during occupational exposure to tetrachloroethylene, Br. J. Ind. Med., 43, 68-69. Nakayama, T., M. Kaneko, M. Kodama and C. Nagata (1985) Cigarette smoke induces DNA single-strand breaks in human cells, Nature (London), 314, 462-464. Obe, G., and J. Herha (1978) Chromosomal aberrations in heavy smokers, Hum. Genet., 41, 259-263. Onkenhout, W., E.J. Van Bergen, J.H. Van Der Wart, G.P. Vos, W. Buijs and N.P. Vermeulen (1986) Identification and quantitative determination of four different mercapturic acids formed from 1,3-dibromopropane and its

1,1,3,3-tetradeutero analogue by the rat, Xenobiotica, 16, 21-33. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. U.S. Office on Smoking and Health (1982) The health consequences of smoking: cancer, A report of the Surgeon General, Publ. DHHS (PHS) 82-50179, U.S. Government Printing Office, Washington, DC. Vainio, H., H. Savolainen and I. Kilpikari (1978) Urinary thioether of employees of a chemical plant, Br. J. Ind. Med., 35, 232-234. Van Doom, R., P.J. Borm, C.M. Leijdekkers, P.T. Henderson, J. Reuvers and T.J. Van Bergen (1980) Detection and identification of S-methylcysteine in urine of workers exposed to methyl-chloride, Int. Arch. Occup. Environ. Health, 46, 99-109. Van Doom, R., C.M. Leijdekkers, R.P. Bos-Brouns and P.T. Henderson (1981) Detection of human exposure to electrophilic compounds by assay of thioether detoxication products in urine, Ann. Occup. Hyg., 24, 77-92. Van Doom, R., C.M. Leijdekkers, S.M. Nossent and P.T. Henderson (1982) Excretion of TTCA in human urine after administration of disulfiram, Toxicol. Lett., 12, 77-92. Van Sittert, N.J., and G. De Jong (1985) Biomonitoring of exposure to potential mutagens and carcinogens in industrial populations, Food Chem. Toxicol., 23, 23-31. Venema, G. (1959) The influence of substances extracted from cigarette-smoke on mitotic processes in Allium cepa, Chromosome, 10, 679-685. Wigle, D.T., Y. Mao and M. Grace (1980) Relative importance of smoking as risk factor for selected cancers, Can. J. Publ. Health, 71,269-275.