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Genotoxicity studies on professional hair colorists exposed to oxidation hair dyes
Mutation Research 394 Ž1997. 153–161
Genotoxicity studies on professional hair colorists exposed to oxidation hair dyes S. S¸ ardas¸ ) , N. Aygun, ¨ A.E. Karakaya Gazi UniÕersity, Faculty of Pharmacy, Department of Toxicology, 06330 Ankara, Turkey Received 2 January 1997; revised 6 March 1997; accepted 18 July 1997
Abstract The cytogenic repercussions of occupational exposure to oxidation hair dyes were assessed by using three assays in professional hair colorists. The assays were sister chromatid exchanges ŽSCE. in circulating lymphocytes to evaluate the interchange of DNA replication products at apparently homologous chromosomal loci, single cell gel electrophoretic ŽSCGE. assay to detect the presence of DNA strand breaksralkali-labile damage, and the Ames assay using Salmonella typhimurium strain TA98 to detect the urine mutagenicity. The ability of these assays to detect genetic damage caused by oxidation hair dyes in man compared with closely matched controls produced the following findings. Ži. The SCE assay could not detect the mutagenic effect in lymphocytes of exposed subjects from whom complete data were obtained. However, subjects Žcontrols and exposed. with a history of smoking had slightly increased SCEs than the non-smokers in both groups. Žii. The extent of DNA migration ŽSCGE assay. did not distinguish between the samples in either the exposed or control subjects. Like the SCE results, the exposed and control smoker subjects showed a greater proportion of damaged lymphocytes with apparent migration of DNA. Žiii. No clear differences in the mutagenic activity of the urine samples were observed between the exposed and control subjects. But, pooling exposed and controls together, a positive and significant variation in the urinary mutagenic effect was observed with the number of cigarettes smoked per day. q 1997 Elsevier Science B.V. Keywords: Sister chromatid exchange; Single cell gel electrophoresis; Comet assay; Ames assay; Oxidation hair dye; Occupational exposure
1. Introduction The most common dyeing systems for coloring the hair are the permanent Žoxidation. hair dyes, lasting for several months. They are usually compounded of 7 to 12 aromatic substances. For additional substances and a sophisticated discussion of hair dye chemistry, the reader is referred to Marzulli and Green w1x. There is evidence that many hair dye constituents Žincluding certain phenylenediamines, )
Corresponding author.
nitrophenylenediamines, diaminotoluenes and diaminoanisoles. are mutagenic, and some are carcinogenic to rodents w2–6x. Marketed hair dyes were first reported to be mutagenic in connection with a biochemistry class experiment conducted by Dr. Bruce Ames at the University of California, Berkeley w7x. The mutagenicity of some hair dyes has also been demonstrated in mammalian cells in culture w8–10x. These compounds readily penetrate the human skin, and some have been detected in the urine of people who have dyed their hair under normal conditions of use w2,11–13x. The skin penetration capacity of 2,4-
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 3 5 - 6
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diaminotoluene Ž2,4-TDA. became a matter of public interest following newspaper publicity in 1970, and this hair dye ingredient was removed from hair dye formulations voluntarily by some manufacturers. Considerable controversy concerning the safety of hair dyes led the United States to mandate a cancer warning label on hair dyes containing 2,4-TDA and diazo dyes w14x. General provisions for cosmetic products, including hair dyes, are compelled in the Council Directive of the European Economic Community ŽEEC. in 1976 on the Approximation of the Laws of the Member States Relating to Cosmetic Products w15x, which is amended constantly. Therefore diazo dyes were gradually banned, withdrawn or severely restricted in different countries in different years. The cosmetic regulations in Norway, Finland and Turkey are closely modelled on the EEC Directive and in 1992 under the EEC Cosmetics Directive, diazo dyes were also banned in Turkey w16x. Since the numerous individual chemicals used in hair colorants have varied over time, data on humans or animals in mutagenicity studies differ depending on the year of publication. Some authors ŽBurnett et al. w17x, Kirkland et al. w18x, Turanitz et al. w19x, Hofer et al. w20x, Babish et al. w21x. failed to show mutagens detectable in a sensitive microbial system or in sister chromatid exchanges frequencies between pre-dyeing samples due to authority over cosmetics. In the latest report in series of Office of Population Censuses and Surveys w22x covering the years 1979–80 and 1982–83, male hairdressers and barbers in England and Wales were reported to have had increased mortality from cancers at all sites at ages 15–64 Ž45 deaths, 21.6 expected. and from lung cancer Ž21 deaths, 7.9 expected.. Single female hairdressers also showed a significant excess of cancers at all sites at ages 15–64. Hairdressers may also be exposed to volatile solvents, propellants and aerosols from hair sprays and other care products. Most of the hair dyes sold on the Turkish market are oxidation-type, and we have previously reported the ingredients analyzed by HPLC and were able to detect 2,4-TDA w23x; the range of detectable concentration in the urine of professional hairdressers, who usually did not wear gloves when applying hair dyes, was 16–67 mgrl w13x.
As a follow-up to our above studies, the objective of this study was to apply the following three monitoring methodologies simultaneously to a group of professional hair colorists applying marketed oxidation dyes after new regulatory aspects in Turkey, and to unexposed controls: Ži. sister chromatid exchange ŽSCE. analysis on human peripheral blood lymphocytes which allows the estimation of DNA damage produced by mutagens and carcinogens w24x; Žii. the single cell gel electrophoresis ŽSCGE. technique, which is a promising tool for estimation of DNA damage at the individual cell level in in vivo or in vitro studies w25–28x; Žiii. Mutagenicity assessment of urine by the Ames test w29x, which represents an indication of the absorbtion, metabolism and excretion of the potential mutagens by the body. The role of several possible confounders was taken into account.
2. Materials and methods 2.1. Subjects The study consisted of 15 professional male hair colorists selected from the busiest hairdressing saloons for dye application in Ankara Turkey. Their ages ranged between 17 and 43 years and were in the profession for 2–25 years. They were applying different mixtures of permanent Žoxidation. dyes every day, in order to achieve the required color balance of their female customers. None of them were wearing gloves during 2–10 applications a day ŽFig. 1 shows the cracked and dyed hands of an applier., and 6 of
Fig. 1. Cracked and dyed hands of a hair dye applier.
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them were smokers. The controls were closely matched by age, smoking habits and were male office employees. At the end of the week monitored, total urine for 500 ml was collected on Friday and at the time of urine delivery, 6 ml of peripheral venous blood was drawn, into heparinized tubes. Blood samples were processed immediately after collection; urine was filtered, and stored at y208C until assayed. 2.2. SCE analysis 0.8 ml heparinized whole blood were added within the same day of sampling to 10 ml TC 199 Medium ŽSeromed. supplemented with 20% fetal calf serum ŽSeromed ., 1% PHA ŽSigma ., 25 m M 5bromodeoxyuridine ŽSigma. and 100 U penicillin Ž10 mgrml.. Cultures were incubated in the dark for 72 h at 378C. The cells were exposed to 5 = 10y7 colchicine for the final 2 h. Lymphocytes were collected, fixed and air-dried, and stained with the fluorescence plus the Giemsa technique of Perry and Wolff w30x. SCEs were analyzed in 20 cells containing 46 chromosomes in each preparation, and the mean SCE frequency was calculated as SCEs per cell of each subject. SCEs were scored blind by two independent investigators. 2.3. SCGE assay In order to isolate peripheral blood lymphocytes from heparinized whole blood samples, 5 ml of blood was carefully layered over 8 ml lymphocyte separation medium in a Sterilin Universal ŽBibby Sterilin. and centrifuged at 2000 = g for 15 min. After the plasma layer was removed and saved, the buffy layer was carefully removed and the cells were washed with TC 199 medium and then collected by 10 min centrifugation at 1000 = g. Lymphocytes were resuspended at approximately 10 7rml in TC 199 medium with 20% vrv plasma and 10% vrv dimethylsulfoxide ŽDMSO.. Lymphocytes were transferred to microfuge tubes and stored at y808C. 2.3.1. Slide preparation Slides were prepared in duplicate. 120 ml normal 0.5% agarose in PBS was layered on to a precleaned microscope slides, immediately covered with a cov-
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erslip and allowed to solidify. 1500–10 5 cells were mixed with 75 ml of 0.5% low-melting-point agarose in PBS at 378C, the coverslip removed and the mixture added to the slide. The coverslip was replaced and the slide placed on ice for 5 min. After solidification of the agarose the coverslip was gently removed, a top layer of 75 ml low-melting-point agarose added, the coverslip replaced and the slide returned to ice. Once the top layer had solidified, the coverslip was removed and the slide gently immersed in cold lyzing solution Ž2.5 M NaCl, 100 mM EDTA, 1% N-lauroylsarcosine, 10 mM Tris– HCl, pH 10 to which 1% Triton X-100 and 10% DMSO had been added fresh.. The slides were left at 48C for at least 1 h. 2.3.2. Electrophoresis and staining The slides were removed from the lyzing solution and placed close together in a horizontal gel electrophoresis tank ŽPharmacia, GNA 100. near the anode. It was filled with fresh electrophoresis buffer Ž300 mM NaOH, 1 mM EDTA, pH 13. to a level of 0.25 cm above the slides which were then left to soak for 40 min in the alkali. Electrophoresis was carried out for 20 min at 19 V 300 mA using a consort E 425 electrophoresis power supply. All the above steps were carried out under dimmed yellow light to avoid any additional DNA damage occurring. After electrophoresis, the slides were gently removed from the tank and neutralizing buffer Ž0.4 M Tris– HCl, pH 7.5. added drop-wise to the slides three times, allowing the slides to sit for 5 min each time. Excess liquid was blotted from each slide and the DNA was stained with 50 ml ethidium bromide Ž40 mgrml.. A clean coverslip was then placed over the slide. Slides were stored in a closed container at 48C and analyzed within 24 h, as gel dehydration over longer storage times led to deterioration in slide quality. 2.3.3. Image analysis Slides were examined at 200 = magnification under a fluorescent microscope ŽZeiss. equipped with an excitation filter of 546 nm and a barrier filter of 590 nm. Fifty-five cells per sample, selected at random, were analyzed under constant sensitivity. For each cell, the length of migration of any damaged DNA Žtotal length of head and tail minus nuclear
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diameter. was determined w31,32,25x. Quantitation was carried out by two independent investigators using the photographs of comets. 2.4. Urine mutagenicity analysis Frozen urine samples of 15 professionalists and 10 closely matching controls were thawed at room temperature and filtered through a Whatman ŽNo. 1. filter. A small volume was reserved for creatinine measurement w33x. Polymeric adsorbent bead XAD-2 was pretreated by three washes with reagent grade acetone, four washes with methanol, and ten washes with distilled water. Urine was passed successively through the XAD-2 at a rate of 4 mlrmin. Beads were washed with five volumes of distilled water to remove residual histidine and bound material was eluted from the individual columns with 15 volumes of acetone solvent. Solvent was removed by low heat under a steam of nitrogen. The XAD-bound residues were dissolved in DMSO, and concentrates were stored at y208C until assayed for mutagens w34–37x. 2.4.1. Bacterial tester strain and S9 Salmonella typhimurium strain TA98 was obtained from Dr. B.N. Ames ŽBiochemistry Department, University of California, Berkeley. for which Ames reported positive results with hair dyes and smoking w7x. The strain was stored and tested before use for the maintenance of characteristics and for the sensitivity to standard mutagens as described by Maron and Ames w36x. Microsomal enzyme fraction ŽS9. was prepared from 5-week-old rats treated with Na-phenobarbital plus b-naphthoflavone combination w38x. 2.4.2. Test procedure Bacteria were inoculated in to 50 ml of nutrient broth ŽOxoid. and grown overnight at 378C. The assays contained 100 ml of S9 and urine concentrate in DMSO. 130 units of b-glucuronidase were added to each 100 ml of urine. The mixture was incubated at 378C for 20 min, then 2.0 ml of top agar containing biotin and histidine were added and the mixture was poured onto the standard Ames plates for the selection of histidine ŽHisq. revertants w36x. The mutagenic activity of the urine samples tested was expressed as R srR c , which is the ratio between the
number of revertant colonies counted in the treated plates, and the number of spontaneous revertants in the presence of solvent and S9 mix only. The mutagenic activity was also corrected for the creatinine content of the urine sample tested, according to the formula R s y R crCr, where R s is the number of revertants in the sample, R c that in solvent control, and Cr the amount of creatinine Žg. in the urine sample w39x. 2.5. Statistical eÕaluation SCE results were evaluated by using one- or two-way analyses of variance ŽANOVA Program. and multiple regression analyses using a straight-line model ŽNew regression program. to examine the degree of linear relationship between variables. Urine mutagenicity statistics were based on distribution-free Mann–Whitney U test and matched pairs were analyzed with equivalent distribution-free Wilcoxon’s test for paired samples w40x.
3. Results and discussion Table 1 shows individual information and the mean SCE and the SCGE frequency for professional hair colorists and controls. As a whole group the mean SCE frequency was 4.05 " 1.2 among the exposed and 4.78 " 0.24 among the unexposed, the difference being not statistically significant. An observed tendency for positive variation of SCGE with exposure to oxidation hair dyes and controls also did not reach statistical significance. The whole mean length of DNA migration was 1.17 mm in exposed and 1.49 mm in controls Ž R 2 s 0.33, p s 0.14 and R 2 s 0.37, p s 0.10, respectively.. However, the results obtained by SCGE and the mean SCE frequency observed in professional colorists and controls seem to show similar trends with measures of DNA damage increasing with the frequency and history of smoking ŽFig. 2 and Fig. 3 show the SCE and the grade of damage ŽSCGE., respectively, in a professional hair colorist.. It can be clearly seen in Table 1 that mean SCE frequencies in smokers in exposed group Ž4.69 " 1.03. and controls Ž5.81 " 0.46. were higher than SCE frequencies of nonsmokers in both exposed Ž3.42 " 0.32. and control
S. S¸ardas¸ et al.r Mutation Research 394 (1997) 153–161
157
Table 1 SCGE and SCE frequency in lymphocytes of professional hair colorists and controls
Exposed group Smokers 1 2 3 4 5 Mean " SD Non-smokers 6 7 8 9 10 11 12 13 Mean " SD Control group Smokers 1 2 3 4 5 Mean " SD Non-smokers 6 7 8 9 10 11 12 13 Mean " SD
Age
Cigarettes smokedrday
Employment Žyears.
Length of DNA migration Žmm.
Mean DNA migration Ž55 cells.
Mean SCErcell Ž20 cells.
26 17 18 43 19
10 ) 10 ) 10 ) 20 10 )
7 4 4.5 25 6
1.5 2.3 1.9 2.8 1.7 2.04 " 0.51
0.027 0.042 0.035 0.051 0.030
4.15 4.60 4.0 1 6.51 4.22 4.69 " 1.03
17 21 21 19 17 18 19 30
– – – – – – – –
3.5 6 7 5 4 2 2 14
– – 1.3 – – – 1.2 – 0.31 " 0.57
– – 0.023 – – – 0.021 –
3.00 3.55 3.10 3.50 3.25 4.01 3.30 3.66 3.42 " 0.32
29 19 20 45 21
10 ) 20 10 ) 10 ) 10 )
– – – – –
2.2 2.9 2.0 3.1 2.4 2.52 " 0.46
0.040 0.052 0.036 0.056 0.043
5.66 6.49 5.20 5.84 5.89 5.81 " 0.46
18 20 23 22 20 18 20 27
– – – – – – – –
– – – – – – – –
1.4 – 1.2 – – – – 1.2 0.47 " 0.65
0.025 – 0.021 – – – – 0.021
4.30 3.26 4.22 4.14 4.38 3.74 3.50 4.00 3.94 " 0.40
groups Ž3.94 " 0.40.. Also, the length of DNA migration in smokers in both exposed Ž2.04 mm. and controls Ž2.52 mm. were higher than the SCGE evaluations of non-smokers exposed Ž0.31 mm. and controls Ž0.47 mm.. The results expressed in Table 1 are for 13 professionals, since two of the blood samples were clotted and the atttempt to analyze those were unsuccessful. However, there was no
problem with their urine samples; therefore the following mutagenic activities are expressed for all 15 professionals. All testing was performed in triplicate using Salmonella strain TA98rS9 fraction from rats; induced Na-phenobarbital, b-glucuronidase and creatinine amounts of all subjects are shown in Table 2. The mean number of revertant colonies" SD for the TA98 strain in hair dye professionals with the addi-
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Fig. 3. Grade of damage ŽSCGE. in smoker professional hair dyer. Fig. 2. SCE frequency of a smoker professional hair dyer.
tion of S9 mix were 3.80 " 8.23, and for controls 1.67 " 0.98. Without S9 mix the means were 1.50 " 2.26 for hair dye professionals and 1.24 " 0.81 for controls. The mutagenic activities in the exposed groups do not differ significantly from their own controls Ž p ) 0.05.. However, the revertant colony counts in the urine of smokers in exposed hairdressers Ž8.42 " 12.10, 2.96 " 3.65. and controls Ž2.41 " 0.94, 1.53 " 1.25. were higher than non-smoker exposed Ž0.72 " 0.67, 0.54 " 0.68. and non-smoker controls Ž1.17 " 0.69, 1.05 " 0.36. with or without the presence of S9 mix, respectively. Our results show that no mutagenic activity on S. typhimurium strain TA98 was detectable in the urine of exposed subjects, which may be attributed to the occupational exposure to the oxidation hair dyes. Our results also indicate a noticeable and variable excretion of mutagens active on strain TA98 in the urine of smokers, independently of occupational exposure. Fig. 4 shows a S. typhimurium TA98 plate of a smoker exposed professional hair dyer. Therefore, professional hair colorists do not receive significant systemic doses of mutation with the
present formulations that could be detected by SCE, SCGE or Ames test. However, the only significant differences observed in this study were those between smokers and non-smokers; this was found in exposed professionals as well as controls, using three different tests. The evaluation of the performance of SCE and SCGE assay in the detection of the in vivo damage caused by smoking was similar to a number of previous studies w41–43x and significant increases in the mean frequency of SCE have also been reported in various occupational exposed and smoker subjects compared with their smoker controls w44– 46x. Ames assay also appears to be good choice for monitoring smokers, as also confirmed by Kado et al. w47x, Ames et. al. w7x and many others. There are no published data on hair dyes in humans as yet using the recently developed SCGE technique. Existing data on humans by Kirkland et al. w18x. SCE was studied in the peripheral lymphocytes of a small group of volunteers comprising 13 women and one man immediately before and 6 h and 7 days after one normal application of hair dyes, all of which were mutagenic to S. typhimurium TA 1538rTA 98, and no consistent increase in the number of SCE per cell was recorded. Hofer et al. w20x
Notes to Table 2: Whole mean of exposed subjects Žmean " SD. s 3.80 " 8.23 ŽS9 q .; 1.50 " 2.26 ŽS9 y .. Whole mean of controls subjects Žmean " SD. s 1.67 " 0.98 ŽS9 q .; 1.24 " 0.81 ŽS9 y .. b-G, b-glucuronidase; NM, non-mutagenic. a R srR c s Mutagenic activity expressed as the ratio between the number of mutants produced by the sample and the number of spontaneous mutants in the solvent control. b R s y R crCr s Corrected mutagenic activity expressed as the ratio between net induced revertants and grams of creatinine in the sample Žsee Section 2..
Non-smokers 5 6 7 8 9 10 Mean " SD
Control group Smokers 1 2 3 4 Mean " SD
Non-smokers 7 8 9 10 11 12 13 14 15 Mean " SD
Exposed group Smokers 1 2 3 4 5 6 Mean " SD
– – – – – –
10–20 10 ) 10 ) 10 )
– – – – – – – – –
10–20 10 ) 10 ) 10 ) 20 10 )
Cigarettes smokedr day
510 460 530 520 505 510
500 570 510 390
540 504 490 540 470 530 500 546 510
524 480 490 510 520 490
Urine volume Žml.
6.63 5.98 6.89 6.76 6.57 6.63
6.50 7.41 6.63 5.07
7.02 6.55 6.37 7.02 6.11 6.89 6.50 7.08 6.63
6.81 6.24 6.37 6.63 6.76 6.37
b -G Ž m l.
0.2243 0.1746 0.0358 0.1179 0.2310 0.1114
0.1207 0.0472 0.1255 0.1988
0.1022 0.1442 0.1642 0.1076 0.1952 0.0876 0.0934 0.1498 0.0593
0.1225 0.0369 0.1873 0.0818 0.0848 0.1276
Creatinine amount Žgr100 ml. Cr
9 57 29 36 26 12
50 72 43 52
4 3 5 45 4 24 27 5 33
374 24 27 21 558 16
S9 Žq .
16 45 35 29 18 21
10 72 24 34
13 1 2 11 6 4 11 32 53
176 2 47 29 94 4
S9 Žy .
Rs
29 28 29 19 19 23
29 19 23 23
26 25 20 26 26 20 19 25 25
22 20 26 19 19 20
S9 Žq .
Salmonella typhimurium TA98
Table 2 Mutagenic activity of urinary extracts from hair dye exposed subjects on S. typhimurium TA98 " S9 mix
29 29 29 22 22 23
29 22 23 23
26 23 15 26 26 15 19 23 23
18 15 26 19 22 15
S9 Žy .
Rc
0.31 1.97 1.00 1.89 1.37 0.52 1.17 " 0.69
1.72 3.79 1.87 2.26 2.41 " 0.94
0.15 0.12 0.25 1.73 0.15 1.20 1.42 0.20 1.32 0.72 " 0.67
17.00 1.20 1.04 1.10 29.40 0.80 8.42 " 12.10
S9 Žq .
0.55 1.55 1.20 1.31 0.81 0.91 1.05 " 0.36
0.34 3.27 1.04 1.48 1.53 " 1.25
0.50 0.04 0.13 0.42 0.23 0.27 0.58 0.39 2.30 0.54 " 0.68
9.78 0.13 1.80 1.53 4.27 0.27 2.96 " 3.65
S9 Žy .
R srR c a
Mutagenic activity
NM 160.00 28.00 144.00 30.00 NM
174.00 1122.00 159.00 146.00
NM NM NM 177.00 NM 46.00 86.00 NM 135.00
2873.00 108.00 5.34 24.45 6356.00 NM
S9 Žq .
NM 92.00 168.00 59.00 NM NM
NM 1059.00 8.00 55.00
NM NM NM NM NM NM NM 60.00 506.00
1290.00 NM 112.00 122.00 849.00 NM
S9 Žy .
R s y R crCr b
Corrected mutagenic activity
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by the consumer, and we believe that the results reported here by three different tests ŽSCE, SCGE and Ames assay. have special significance since they provide information on smoker subjects, but the slow penetration and release of currently permitted dyes from the skin into the systemic circulation seems unlikely to have significant detectable genotoxic effects in occupational exposure. References Fig. 4. S. typhimurium TA98 plate of a smoker professional hair dyer.
studied chromosomal aberrations in lymphocytes from 6 women and 4 men who volunteered to have their hair dyed 13 times at intervals of 3 to 6 weeks with commercial preparations, and no difference was observed between the control and treated groups. In conjunction with this study, Turanitz et al. determined the SCE in blood samples taken before the first exposure, after a sham dyeing and after the first three and the last three actual dyeing procedures. No evidence was found of any effect of repeated hair dyeing on the frequency of SCE w19x. In a study in the USA, mutagenicity was determined in urine specimens collected prior and during 24 h period immediately after application of several hair coloring products containing high levels of dyes and dye intermediates w17x. Concentrated ŽXAD-2 resin. urine samples did not increase the number of reverse mutations in S. typhimurium TA 1538 in the presence of exogenous metabolic system from rat liver ŽS9.. Another study was conducted in New York State, USA on cosmetologists who were occupationally exposed to a wide range of chemicals including hair dyes, and a control group of dental personnel w21x. Urine samples were tested for mutagenicity in S. typhimurium TA 100 in the presence and absence of S9. In the presence of S9, there was no difference between the groups, but without S9 the frequency of mutagenic urine samples was 15% higher among cosmetologists than dental personnel. In conclusion, the available studies relate to exposures that occurred at different years, during which period there were changes in both the types and quantities of products used by the hairdressers and
w1x F.N. Marzulli, S. Green, Hair dye toxicity, J. Environ. Path. Toxicol. 1 Ž1978. 509–530. w2x IARC, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man, Vol. 16, Some Aromatic Amines, and Related Nitro Compounds, Hair Dyes, Colouring Agents and Miscellaneous Industrial Chemicals, IARC, Lyon, 1978. w3x National Cancer Institute, Bioassay of 2,4-Diaminoanisole Sulfate for Possible Carcinogenicity, Carcinogenesis Technical Report No. 84, DHEW Publ. NCI-TR-84, Washington. DC, 1978. w4x National Cancer Institute, Bioassay of 4-Amino-2-nitrophenol for Possible Carcinogenicity, Carcinogenesis Technical Report No. 94, DHEW Publ. NCI-TR-94, Washington, DC, 1979. w5x National Cancer Institute, Bioassay of 2-Nitro-p-phenylenediamine for Possible Carcinogenicity, Carcinogenesis Technical Report No. 169, DHEW Publ. NCI-TR-169, Washington, DC, 1979. w6x C. Burnett, Evaluation of toxicity and carcinogenicity of hair dyes, J. Toxicol. Environ. Health 6 Ž1980. 247–257. w7x B. Ames, H.O. Kammen, E. Yamasaki, Hair dyes are mutagenic: identification of a variety of mutagenic ingredients, Proc. Natl. Acad. Sci. USA 6 Ž1975. 2423–2427. w8x K.A. Palmer, A. Denunzio, S. Green, The mutagenic assay of some hair dye components using thymidine kinase locus of L5178Y mouse lymphoma cells, J. Environ. Pathol. Toxicol. 1 Ž1977. 87–91. w9x W.F. Benedict, Morphological transformation and chromosome aberrations produced by two hair dye components, Nature 260 Ž1976. 657–662. w10x D.J. Kirkland, S. Venitt, Cytotoxicity of hair colorant constituents; chromosome damage induced by two nitrophenylenediamines in cultured Chinese hamster cells, Mutation Res. 40 Ž1976. 47–56. w11x E. Kiese, E. Rauscher, The absorption of p-toluenediamine through human skin in hair dyeing, Toxicol. Appl. Pharmacol. 13 Ž1968. 325–331. w12x H.I. Maibach, M.A. Leaffer, W.A. Skinner, Percutaneous penetration following use of hair dyes, Arch. Dermatol. 111 Ž1975. 1444–1445. w13x S. S¸ ardas¸, A.E. Karakaya, B. S¸ ener, Determination of 2,4-diaminotoluene in human urine, J. Fac. Pharm. Gazi 3 Ž1986. 89–96.
S. S¸ardas¸ et al.r Mutation Research 394 (1997) 153–161 w14x J.F. Corbett, Hair dye toxicity, in: Orfanos, Montagna, ŽEds.., Hair Research, Springer, Berlin, 1980, pp. Stuttgen ¨ 529–535. w15x Commission of the European Communities, Council Directive 76r768rEEC of July 1976 on the approximation of the laws of the Member State relating to cosmetic products, Off. J. Eur. Commun. L262 Ž1976. 169–200. w16x Commision of the European Communities, Fifteenth Communities Directive of 21 October Ž92r86rEEC. on the approximation of the laws of the Member States relating to cosmetic products, Off. J. Eur. Commun. L325 Ž1992. 18–22. w17x C.M. Burnett, C.M. Fuchs, J.F. Corbett, Mutagenicity studies on urine concentration from female users of dark hair color products, Drug Chem. Tox. 2 Ž3. Ž1979. 283–293. w18x D.J. Kirkland, J.R. Honeycombe, S.D. Lawler, S. Venitt, C. Crofton-Sleigh, Sister-chromatid exchanges before and after hair dyeing, Mutation Res. 90 Ž1981. 279–286. w19x K. Turanitz, R. Kovac, H. Tuschl, E. Palvicek, Investigations on the effect of repeated hair dyeing on sister chromatid exchanges, Food Chem. Toxicol. 21 Ž1983. 791–793. w20x H. Hofer, N. Bornatowics, E. Reindl, Analysis of human chromosomes after repeated hair dyeing, Food Chem. Toxicol. 21 Ž1983. 785–789. w21x J.G. Babish, J.M. Scarlett, S.E. Voekler, W.H. Gutermann, D.J. Lisk, Urinary mutagens in cosmetologists and dental personnel, J. Toxicol. Environ. Health 34 Ž1991. 197–206. w22x Office of Population Censuses and Surveys, Occupational Mortality Decennial Supplement, 1978–80, 1982–83, Her Majesty’s Stationery Office, London, UK, 1986. w23x S. S¸ ardas¸, B. S¸ ener, A.E. Karakaya, High performance liquid chromatography analysis of hair-dye ingredients, J. Fac. Pharm. Gazi 2 Ž1985. 51–57. w24x J.D. Tucker, A. Auletta, M.C. Cimino, K.L. Dearfield, D. Jacobson-Kram, R.R. Tice, A.V. Carrano, Sister-chromatid exchange: second report of the Gene-Tox program, Mutation Res. 297 Ž1993. 101–180. w25x N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 Ž1988. 184–191. w26x D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutation Res. 339 Ž1995. 37–59. w27x N.P. Singh, R.R. Tice, R.E. Stephens, E.L. Schneider, A microgel electrophoresis technique for the direct quantitation of DNA damage and repair in individual fibroblasts cultured on microscope slides, Mutation Res. 252 Ž1991. 289–296. w28x S. S¸ ardas¸, Microgel electrophoresis assay Žcomet assay. for detecting DNA damage in individual cells, J. Fac. Pharm. Gazi 13 Ž1996. 65–73. w29x L.E. Kier, D.J. Brusick, A.E. Auletta, E.S. Von Halle, M.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Prival, T.K. Rao, V. Ray, The Salmonella typhimuriumrmammalian microsomal assay: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res. 168 Ž1986. 69–240. w30x P. Perry, S. Wolff, New Giemsa method for differential staining of sister chromatids, Nature 251 Ž1974. 156–158. w31x B. Rydberg, K.J. Johanson, Estimation of DNA strand breaks
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in single mammalian cells, in: P.C. Hanawalt, E.C. Friedberg, C.F. Fox ŽEds.. DNA Repair Mechanisms, Academic Press, New York, 1978, pp. 465–468. ¨ O. Ostling, K.J. Johanson, Microelectrophoretic study of radiation induced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun. 123 Ž1984. 291–293. M. Donald, A. Zimmer, Atlas of Clinical Laboratory Procedures, 1, The Blakiston Division McGraw-Hill Co., New York, 1967, p. 62. E. Yamasaki, B. Ames, Concentration of mutagens from urine by adsorption with the nonpolar resin XAD-2: Cigarette smokers have mutagenic urine?, Proc. Natl. Acad. Sci. USA 74 Ž1977. 3555–3559. R.P. Bos, A.O. Leenaars, J.L.G. Theuws, P.T. Henderson, Mutagenicity of urine of nurses handling cytostatic drugs, influence of smoking, Int. Arch. Occup. Environ. Health 50 Ž1982. 359–369. D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutation Res. 113 Ž1983. 173–215. S. Venitt, C. Crofton-Sleigh, K. Briggs, Monitoring exposures of nursing and pharmacy personnel to cytotoxic drugs: Urinary mutation assays and urinary platinum as markers of absorption, Lancet i Ž1984. 74–77. T.M. Ong, W.C.R. Mukhtar, E. Zeiger, Differential effects of cytochrome P450 induced on promutagen activation capabilities and enzymatic activities of S9 from rat liver, J. Environ. Pathol. Toxicol. 4 Ž1980. 55–65. P. Venier, E. Clonfero, D. Cottica, C. Gava, M. Zordan, L. Pozzoli, A.G. Levis, Mutagenic activity and polycyclic aromatic hydrocarbon levels in urine of workers exposed to coal tar pitch volatiles in an anode plant, Carcinogenesis 6 Ž1985. 749–752. A. Petria, Lecture Notes on Medical Statistics, Blackwell Scientific, Oxford, 1978. S. S¸ ardas¸, S. Gok, ¨ A.E. Karakaya, Increased frequency of sister chromatid exchanges in the peripheral lymphocytes of cigarette smokers, Toxicol. In Vitro 5 Ž1991. 263–265. S. S¸ ardas¸, B. Karahalil, D. Akyol, A.E. Karakaya, The effect of smoking on sister chromatid exchange rate of newborn infants born to smoking mothers, Mutation Res. 341 Ž1995. 249–258. S. S¸ ardas¸, D. Walker, D. Akyol, A.E. Karakaya, Assessment of smoking-induced DNA damage in lymphocytes of smoking mothers of newborn infants using the alkaline single-cell gel electrophoresis technique, Mutation Res. 335 Ž1995. 213–217. S. S¸ ardas¸, S. Gok, ¨ A.E. Karakaya, Sister chromatid exchanges in the lymphocytes of nurses handling antineoplastic drugs, Toxicol. Lett. 55 Ž1991. 311–315. S. S¸ ardas¸, H. Cuhruk, A.E. Karakaya, Y. Atakurt, Sister chromatid exchanges in operating room personnel, Mutation Res. 279 Ž1992. 117–120. S. S¸ ardas¸, A.E. Karakaya, Y. Furtun, Sister chromatid exchanges in workers from car-painting workshops, Int. Arch. Occup. Environ. Health 66 Ž1994. 33–35. N.Y. Kado, C. Manson, E. Eisenstadt, D.P.H. Hsieh, The kinetic of mutagen excretion in the urine of cigarette smokers, Mutation Res. 157 Ž1985. 227–233.
Genotoxicity studies on professional hair colorists exposed to oxidation hair dyes S. S¸ ardas¸ ) , N. Aygun, ¨ A.E. Karakaya Gazi UniÕersity, Faculty of Pharmacy, Department of Toxicology, 06330 Ankara, Turkey Received 2 January 1997; revised 6 March 1997; accepted 18 July 1997
Abstract The cytogenic repercussions of occupational exposure to oxidation hair dyes were assessed by using three assays in professional hair colorists. The assays were sister chromatid exchanges ŽSCE. in circulating lymphocytes to evaluate the interchange of DNA replication products at apparently homologous chromosomal loci, single cell gel electrophoretic ŽSCGE. assay to detect the presence of DNA strand breaksralkali-labile damage, and the Ames assay using Salmonella typhimurium strain TA98 to detect the urine mutagenicity. The ability of these assays to detect genetic damage caused by oxidation hair dyes in man compared with closely matched controls produced the following findings. Ži. The SCE assay could not detect the mutagenic effect in lymphocytes of exposed subjects from whom complete data were obtained. However, subjects Žcontrols and exposed. with a history of smoking had slightly increased SCEs than the non-smokers in both groups. Žii. The extent of DNA migration ŽSCGE assay. did not distinguish between the samples in either the exposed or control subjects. Like the SCE results, the exposed and control smoker subjects showed a greater proportion of damaged lymphocytes with apparent migration of DNA. Žiii. No clear differences in the mutagenic activity of the urine samples were observed between the exposed and control subjects. But, pooling exposed and controls together, a positive and significant variation in the urinary mutagenic effect was observed with the number of cigarettes smoked per day. q 1997 Elsevier Science B.V. Keywords: Sister chromatid exchange; Single cell gel electrophoresis; Comet assay; Ames assay; Oxidation hair dye; Occupational exposure
1. Introduction The most common dyeing systems for coloring the hair are the permanent Žoxidation. hair dyes, lasting for several months. They are usually compounded of 7 to 12 aromatic substances. For additional substances and a sophisticated discussion of hair dye chemistry, the reader is referred to Marzulli and Green w1x. There is evidence that many hair dye constituents Žincluding certain phenylenediamines, )
Corresponding author.
nitrophenylenediamines, diaminotoluenes and diaminoanisoles. are mutagenic, and some are carcinogenic to rodents w2–6x. Marketed hair dyes were first reported to be mutagenic in connection with a biochemistry class experiment conducted by Dr. Bruce Ames at the University of California, Berkeley w7x. The mutagenicity of some hair dyes has also been demonstrated in mammalian cells in culture w8–10x. These compounds readily penetrate the human skin, and some have been detected in the urine of people who have dyed their hair under normal conditions of use w2,11–13x. The skin penetration capacity of 2,4-
1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 3 5 - 6
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diaminotoluene Ž2,4-TDA. became a matter of public interest following newspaper publicity in 1970, and this hair dye ingredient was removed from hair dye formulations voluntarily by some manufacturers. Considerable controversy concerning the safety of hair dyes led the United States to mandate a cancer warning label on hair dyes containing 2,4-TDA and diazo dyes w14x. General provisions for cosmetic products, including hair dyes, are compelled in the Council Directive of the European Economic Community ŽEEC. in 1976 on the Approximation of the Laws of the Member States Relating to Cosmetic Products w15x, which is amended constantly. Therefore diazo dyes were gradually banned, withdrawn or severely restricted in different countries in different years. The cosmetic regulations in Norway, Finland and Turkey are closely modelled on the EEC Directive and in 1992 under the EEC Cosmetics Directive, diazo dyes were also banned in Turkey w16x. Since the numerous individual chemicals used in hair colorants have varied over time, data on humans or animals in mutagenicity studies differ depending on the year of publication. Some authors ŽBurnett et al. w17x, Kirkland et al. w18x, Turanitz et al. w19x, Hofer et al. w20x, Babish et al. w21x. failed to show mutagens detectable in a sensitive microbial system or in sister chromatid exchanges frequencies between pre-dyeing samples due to authority over cosmetics. In the latest report in series of Office of Population Censuses and Surveys w22x covering the years 1979–80 and 1982–83, male hairdressers and barbers in England and Wales were reported to have had increased mortality from cancers at all sites at ages 15–64 Ž45 deaths, 21.6 expected. and from lung cancer Ž21 deaths, 7.9 expected.. Single female hairdressers also showed a significant excess of cancers at all sites at ages 15–64. Hairdressers may also be exposed to volatile solvents, propellants and aerosols from hair sprays and other care products. Most of the hair dyes sold on the Turkish market are oxidation-type, and we have previously reported the ingredients analyzed by HPLC and were able to detect 2,4-TDA w23x; the range of detectable concentration in the urine of professional hairdressers, who usually did not wear gloves when applying hair dyes, was 16–67 mgrl w13x.
As a follow-up to our above studies, the objective of this study was to apply the following three monitoring methodologies simultaneously to a group of professional hair colorists applying marketed oxidation dyes after new regulatory aspects in Turkey, and to unexposed controls: Ži. sister chromatid exchange ŽSCE. analysis on human peripheral blood lymphocytes which allows the estimation of DNA damage produced by mutagens and carcinogens w24x; Žii. the single cell gel electrophoresis ŽSCGE. technique, which is a promising tool for estimation of DNA damage at the individual cell level in in vivo or in vitro studies w25–28x; Žiii. Mutagenicity assessment of urine by the Ames test w29x, which represents an indication of the absorbtion, metabolism and excretion of the potential mutagens by the body. The role of several possible confounders was taken into account.
2. Materials and methods 2.1. Subjects The study consisted of 15 professional male hair colorists selected from the busiest hairdressing saloons for dye application in Ankara Turkey. Their ages ranged between 17 and 43 years and were in the profession for 2–25 years. They were applying different mixtures of permanent Žoxidation. dyes every day, in order to achieve the required color balance of their female customers. None of them were wearing gloves during 2–10 applications a day ŽFig. 1 shows the cracked and dyed hands of an applier., and 6 of
Fig. 1. Cracked and dyed hands of a hair dye applier.
S. S¸ardas¸ et al.r Mutation Research 394 (1997) 153–161
them were smokers. The controls were closely matched by age, smoking habits and were male office employees. At the end of the week monitored, total urine for 500 ml was collected on Friday and at the time of urine delivery, 6 ml of peripheral venous blood was drawn, into heparinized tubes. Blood samples were processed immediately after collection; urine was filtered, and stored at y208C until assayed. 2.2. SCE analysis 0.8 ml heparinized whole blood were added within the same day of sampling to 10 ml TC 199 Medium ŽSeromed. supplemented with 20% fetal calf serum ŽSeromed ., 1% PHA ŽSigma ., 25 m M 5bromodeoxyuridine ŽSigma. and 100 U penicillin Ž10 mgrml.. Cultures were incubated in the dark for 72 h at 378C. The cells were exposed to 5 = 10y7 colchicine for the final 2 h. Lymphocytes were collected, fixed and air-dried, and stained with the fluorescence plus the Giemsa technique of Perry and Wolff w30x. SCEs were analyzed in 20 cells containing 46 chromosomes in each preparation, and the mean SCE frequency was calculated as SCEs per cell of each subject. SCEs were scored blind by two independent investigators. 2.3. SCGE assay In order to isolate peripheral blood lymphocytes from heparinized whole blood samples, 5 ml of blood was carefully layered over 8 ml lymphocyte separation medium in a Sterilin Universal ŽBibby Sterilin. and centrifuged at 2000 = g for 15 min. After the plasma layer was removed and saved, the buffy layer was carefully removed and the cells were washed with TC 199 medium and then collected by 10 min centrifugation at 1000 = g. Lymphocytes were resuspended at approximately 10 7rml in TC 199 medium with 20% vrv plasma and 10% vrv dimethylsulfoxide ŽDMSO.. Lymphocytes were transferred to microfuge tubes and stored at y808C. 2.3.1. Slide preparation Slides were prepared in duplicate. 120 ml normal 0.5% agarose in PBS was layered on to a precleaned microscope slides, immediately covered with a cov-
155
erslip and allowed to solidify. 1500–10 5 cells were mixed with 75 ml of 0.5% low-melting-point agarose in PBS at 378C, the coverslip removed and the mixture added to the slide. The coverslip was replaced and the slide placed on ice for 5 min. After solidification of the agarose the coverslip was gently removed, a top layer of 75 ml low-melting-point agarose added, the coverslip replaced and the slide returned to ice. Once the top layer had solidified, the coverslip was removed and the slide gently immersed in cold lyzing solution Ž2.5 M NaCl, 100 mM EDTA, 1% N-lauroylsarcosine, 10 mM Tris– HCl, pH 10 to which 1% Triton X-100 and 10% DMSO had been added fresh.. The slides were left at 48C for at least 1 h. 2.3.2. Electrophoresis and staining The slides were removed from the lyzing solution and placed close together in a horizontal gel electrophoresis tank ŽPharmacia, GNA 100. near the anode. It was filled with fresh electrophoresis buffer Ž300 mM NaOH, 1 mM EDTA, pH 13. to a level of 0.25 cm above the slides which were then left to soak for 40 min in the alkali. Electrophoresis was carried out for 20 min at 19 V 300 mA using a consort E 425 electrophoresis power supply. All the above steps were carried out under dimmed yellow light to avoid any additional DNA damage occurring. After electrophoresis, the slides were gently removed from the tank and neutralizing buffer Ž0.4 M Tris– HCl, pH 7.5. added drop-wise to the slides three times, allowing the slides to sit for 5 min each time. Excess liquid was blotted from each slide and the DNA was stained with 50 ml ethidium bromide Ž40 mgrml.. A clean coverslip was then placed over the slide. Slides were stored in a closed container at 48C and analyzed within 24 h, as gel dehydration over longer storage times led to deterioration in slide quality. 2.3.3. Image analysis Slides were examined at 200 = magnification under a fluorescent microscope ŽZeiss. equipped with an excitation filter of 546 nm and a barrier filter of 590 nm. Fifty-five cells per sample, selected at random, were analyzed under constant sensitivity. For each cell, the length of migration of any damaged DNA Žtotal length of head and tail minus nuclear
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diameter. was determined w31,32,25x. Quantitation was carried out by two independent investigators using the photographs of comets. 2.4. Urine mutagenicity analysis Frozen urine samples of 15 professionalists and 10 closely matching controls were thawed at room temperature and filtered through a Whatman ŽNo. 1. filter. A small volume was reserved for creatinine measurement w33x. Polymeric adsorbent bead XAD-2 was pretreated by three washes with reagent grade acetone, four washes with methanol, and ten washes with distilled water. Urine was passed successively through the XAD-2 at a rate of 4 mlrmin. Beads were washed with five volumes of distilled water to remove residual histidine and bound material was eluted from the individual columns with 15 volumes of acetone solvent. Solvent was removed by low heat under a steam of nitrogen. The XAD-bound residues were dissolved in DMSO, and concentrates were stored at y208C until assayed for mutagens w34–37x. 2.4.1. Bacterial tester strain and S9 Salmonella typhimurium strain TA98 was obtained from Dr. B.N. Ames ŽBiochemistry Department, University of California, Berkeley. for which Ames reported positive results with hair dyes and smoking w7x. The strain was stored and tested before use for the maintenance of characteristics and for the sensitivity to standard mutagens as described by Maron and Ames w36x. Microsomal enzyme fraction ŽS9. was prepared from 5-week-old rats treated with Na-phenobarbital plus b-naphthoflavone combination w38x. 2.4.2. Test procedure Bacteria were inoculated in to 50 ml of nutrient broth ŽOxoid. and grown overnight at 378C. The assays contained 100 ml of S9 and urine concentrate in DMSO. 130 units of b-glucuronidase were added to each 100 ml of urine. The mixture was incubated at 378C for 20 min, then 2.0 ml of top agar containing biotin and histidine were added and the mixture was poured onto the standard Ames plates for the selection of histidine ŽHisq. revertants w36x. The mutagenic activity of the urine samples tested was expressed as R srR c , which is the ratio between the
number of revertant colonies counted in the treated plates, and the number of spontaneous revertants in the presence of solvent and S9 mix only. The mutagenic activity was also corrected for the creatinine content of the urine sample tested, according to the formula R s y R crCr, where R s is the number of revertants in the sample, R c that in solvent control, and Cr the amount of creatinine Žg. in the urine sample w39x. 2.5. Statistical eÕaluation SCE results were evaluated by using one- or two-way analyses of variance ŽANOVA Program. and multiple regression analyses using a straight-line model ŽNew regression program. to examine the degree of linear relationship between variables. Urine mutagenicity statistics were based on distribution-free Mann–Whitney U test and matched pairs were analyzed with equivalent distribution-free Wilcoxon’s test for paired samples w40x.
3. Results and discussion Table 1 shows individual information and the mean SCE and the SCGE frequency for professional hair colorists and controls. As a whole group the mean SCE frequency was 4.05 " 1.2 among the exposed and 4.78 " 0.24 among the unexposed, the difference being not statistically significant. An observed tendency for positive variation of SCGE with exposure to oxidation hair dyes and controls also did not reach statistical significance. The whole mean length of DNA migration was 1.17 mm in exposed and 1.49 mm in controls Ž R 2 s 0.33, p s 0.14 and R 2 s 0.37, p s 0.10, respectively.. However, the results obtained by SCGE and the mean SCE frequency observed in professional colorists and controls seem to show similar trends with measures of DNA damage increasing with the frequency and history of smoking ŽFig. 2 and Fig. 3 show the SCE and the grade of damage ŽSCGE., respectively, in a professional hair colorist.. It can be clearly seen in Table 1 that mean SCE frequencies in smokers in exposed group Ž4.69 " 1.03. and controls Ž5.81 " 0.46. were higher than SCE frequencies of nonsmokers in both exposed Ž3.42 " 0.32. and control
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Table 1 SCGE and SCE frequency in lymphocytes of professional hair colorists and controls
Exposed group Smokers 1 2 3 4 5 Mean " SD Non-smokers 6 7 8 9 10 11 12 13 Mean " SD Control group Smokers 1 2 3 4 5 Mean " SD Non-smokers 6 7 8 9 10 11 12 13 Mean " SD
Age
Cigarettes smokedrday
Employment Žyears.
Length of DNA migration Žmm.
Mean DNA migration Ž55 cells.
Mean SCErcell Ž20 cells.
26 17 18 43 19
10 ) 10 ) 10 ) 20 10 )
7 4 4.5 25 6
1.5 2.3 1.9 2.8 1.7 2.04 " 0.51
0.027 0.042 0.035 0.051 0.030
4.15 4.60 4.0 1 6.51 4.22 4.69 " 1.03
17 21 21 19 17 18 19 30
– – – – – – – –
3.5 6 7 5 4 2 2 14
– – 1.3 – – – 1.2 – 0.31 " 0.57
– – 0.023 – – – 0.021 –
3.00 3.55 3.10 3.50 3.25 4.01 3.30 3.66 3.42 " 0.32
29 19 20 45 21
10 ) 20 10 ) 10 ) 10 )
– – – – –
2.2 2.9 2.0 3.1 2.4 2.52 " 0.46
0.040 0.052 0.036 0.056 0.043
5.66 6.49 5.20 5.84 5.89 5.81 " 0.46
18 20 23 22 20 18 20 27
– – – – – – – –
– – – – – – – –
1.4 – 1.2 – – – – 1.2 0.47 " 0.65
0.025 – 0.021 – – – – 0.021
4.30 3.26 4.22 4.14 4.38 3.74 3.50 4.00 3.94 " 0.40
groups Ž3.94 " 0.40.. Also, the length of DNA migration in smokers in both exposed Ž2.04 mm. and controls Ž2.52 mm. were higher than the SCGE evaluations of non-smokers exposed Ž0.31 mm. and controls Ž0.47 mm.. The results expressed in Table 1 are for 13 professionals, since two of the blood samples were clotted and the atttempt to analyze those were unsuccessful. However, there was no
problem with their urine samples; therefore the following mutagenic activities are expressed for all 15 professionals. All testing was performed in triplicate using Salmonella strain TA98rS9 fraction from rats; induced Na-phenobarbital, b-glucuronidase and creatinine amounts of all subjects are shown in Table 2. The mean number of revertant colonies" SD for the TA98 strain in hair dye professionals with the addi-
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Fig. 3. Grade of damage ŽSCGE. in smoker professional hair dyer. Fig. 2. SCE frequency of a smoker professional hair dyer.
tion of S9 mix were 3.80 " 8.23, and for controls 1.67 " 0.98. Without S9 mix the means were 1.50 " 2.26 for hair dye professionals and 1.24 " 0.81 for controls. The mutagenic activities in the exposed groups do not differ significantly from their own controls Ž p ) 0.05.. However, the revertant colony counts in the urine of smokers in exposed hairdressers Ž8.42 " 12.10, 2.96 " 3.65. and controls Ž2.41 " 0.94, 1.53 " 1.25. were higher than non-smoker exposed Ž0.72 " 0.67, 0.54 " 0.68. and non-smoker controls Ž1.17 " 0.69, 1.05 " 0.36. with or without the presence of S9 mix, respectively. Our results show that no mutagenic activity on S. typhimurium strain TA98 was detectable in the urine of exposed subjects, which may be attributed to the occupational exposure to the oxidation hair dyes. Our results also indicate a noticeable and variable excretion of mutagens active on strain TA98 in the urine of smokers, independently of occupational exposure. Fig. 4 shows a S. typhimurium TA98 plate of a smoker exposed professional hair dyer. Therefore, professional hair colorists do not receive significant systemic doses of mutation with the
present formulations that could be detected by SCE, SCGE or Ames test. However, the only significant differences observed in this study were those between smokers and non-smokers; this was found in exposed professionals as well as controls, using three different tests. The evaluation of the performance of SCE and SCGE assay in the detection of the in vivo damage caused by smoking was similar to a number of previous studies w41–43x and significant increases in the mean frequency of SCE have also been reported in various occupational exposed and smoker subjects compared with their smoker controls w44– 46x. Ames assay also appears to be good choice for monitoring smokers, as also confirmed by Kado et al. w47x, Ames et. al. w7x and many others. There are no published data on hair dyes in humans as yet using the recently developed SCGE technique. Existing data on humans by Kirkland et al. w18x. SCE was studied in the peripheral lymphocytes of a small group of volunteers comprising 13 women and one man immediately before and 6 h and 7 days after one normal application of hair dyes, all of which were mutagenic to S. typhimurium TA 1538rTA 98, and no consistent increase in the number of SCE per cell was recorded. Hofer et al. w20x
Notes to Table 2: Whole mean of exposed subjects Žmean " SD. s 3.80 " 8.23 ŽS9 q .; 1.50 " 2.26 ŽS9 y .. Whole mean of controls subjects Žmean " SD. s 1.67 " 0.98 ŽS9 q .; 1.24 " 0.81 ŽS9 y .. b-G, b-glucuronidase; NM, non-mutagenic. a R srR c s Mutagenic activity expressed as the ratio between the number of mutants produced by the sample and the number of spontaneous mutants in the solvent control. b R s y R crCr s Corrected mutagenic activity expressed as the ratio between net induced revertants and grams of creatinine in the sample Žsee Section 2..
Non-smokers 5 6 7 8 9 10 Mean " SD
Control group Smokers 1 2 3 4 Mean " SD
Non-smokers 7 8 9 10 11 12 13 14 15 Mean " SD
Exposed group Smokers 1 2 3 4 5 6 Mean " SD
– – – – – –
10–20 10 ) 10 ) 10 )
– – – – – – – – –
10–20 10 ) 10 ) 10 ) 20 10 )
Cigarettes smokedr day
510 460 530 520 505 510
500 570 510 390
540 504 490 540 470 530 500 546 510
524 480 490 510 520 490
Urine volume Žml.
6.63 5.98 6.89 6.76 6.57 6.63
6.50 7.41 6.63 5.07
7.02 6.55 6.37 7.02 6.11 6.89 6.50 7.08 6.63
6.81 6.24 6.37 6.63 6.76 6.37
b -G Ž m l.
0.2243 0.1746 0.0358 0.1179 0.2310 0.1114
0.1207 0.0472 0.1255 0.1988
0.1022 0.1442 0.1642 0.1076 0.1952 0.0876 0.0934 0.1498 0.0593
0.1225 0.0369 0.1873 0.0818 0.0848 0.1276
Creatinine amount Žgr100 ml. Cr
9 57 29 36 26 12
50 72 43 52
4 3 5 45 4 24 27 5 33
374 24 27 21 558 16
S9 Žq .
16 45 35 29 18 21
10 72 24 34
13 1 2 11 6 4 11 32 53
176 2 47 29 94 4
S9 Žy .
Rs
29 28 29 19 19 23
29 19 23 23
26 25 20 26 26 20 19 25 25
22 20 26 19 19 20
S9 Žq .
Salmonella typhimurium TA98
Table 2 Mutagenic activity of urinary extracts from hair dye exposed subjects on S. typhimurium TA98 " S9 mix
29 29 29 22 22 23
29 22 23 23
26 23 15 26 26 15 19 23 23
18 15 26 19 22 15
S9 Žy .
Rc
0.31 1.97 1.00 1.89 1.37 0.52 1.17 " 0.69
1.72 3.79 1.87 2.26 2.41 " 0.94
0.15 0.12 0.25 1.73 0.15 1.20 1.42 0.20 1.32 0.72 " 0.67
17.00 1.20 1.04 1.10 29.40 0.80 8.42 " 12.10
S9 Žq .
0.55 1.55 1.20 1.31 0.81 0.91 1.05 " 0.36
0.34 3.27 1.04 1.48 1.53 " 1.25
0.50 0.04 0.13 0.42 0.23 0.27 0.58 0.39 2.30 0.54 " 0.68
9.78 0.13 1.80 1.53 4.27 0.27 2.96 " 3.65
S9 Žy .
R srR c a
Mutagenic activity
NM 160.00 28.00 144.00 30.00 NM
174.00 1122.00 159.00 146.00
NM NM NM 177.00 NM 46.00 86.00 NM 135.00
2873.00 108.00 5.34 24.45 6356.00 NM
S9 Žq .
NM 92.00 168.00 59.00 NM NM
NM 1059.00 8.00 55.00
NM NM NM NM NM NM NM 60.00 506.00
1290.00 NM 112.00 122.00 849.00 NM
S9 Žy .
R s y R crCr b
Corrected mutagenic activity
S. S¸ardas¸ et al.r Mutation Research 394 (1997) 153–161 159
160
S. S¸ardas¸ et al.r Mutation Research 394 (1997) 153–161
by the consumer, and we believe that the results reported here by three different tests ŽSCE, SCGE and Ames assay. have special significance since they provide information on smoker subjects, but the slow penetration and release of currently permitted dyes from the skin into the systemic circulation seems unlikely to have significant detectable genotoxic effects in occupational exposure. References Fig. 4. S. typhimurium TA98 plate of a smoker professional hair dyer.
studied chromosomal aberrations in lymphocytes from 6 women and 4 men who volunteered to have their hair dyed 13 times at intervals of 3 to 6 weeks with commercial preparations, and no difference was observed between the control and treated groups. In conjunction with this study, Turanitz et al. determined the SCE in blood samples taken before the first exposure, after a sham dyeing and after the first three and the last three actual dyeing procedures. No evidence was found of any effect of repeated hair dyeing on the frequency of SCE w19x. In a study in the USA, mutagenicity was determined in urine specimens collected prior and during 24 h period immediately after application of several hair coloring products containing high levels of dyes and dye intermediates w17x. Concentrated ŽXAD-2 resin. urine samples did not increase the number of reverse mutations in S. typhimurium TA 1538 in the presence of exogenous metabolic system from rat liver ŽS9.. Another study was conducted in New York State, USA on cosmetologists who were occupationally exposed to a wide range of chemicals including hair dyes, and a control group of dental personnel w21x. Urine samples were tested for mutagenicity in S. typhimurium TA 100 in the presence and absence of S9. In the presence of S9, there was no difference between the groups, but without S9 the frequency of mutagenic urine samples was 15% higher among cosmetologists than dental personnel. In conclusion, the available studies relate to exposures that occurred at different years, during which period there were changes in both the types and quantities of products used by the hairdressers and
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