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Genotoxicity evaluation of trimethylbenzenes
Mutation Research 412 Ž1998. 299–305
Genotoxicity evaluation of trimethylbenzenes Ewa Janik-Spiechowicz ) , Kalina Wyszynska, Elzbieta Dziubałtowska ´ ˙ Department of Toxicology and Carcinogenesis, The Nofer Institute of Occupational Medicine, PO Box 199, 90-950 Lodz, Poland Received 26 August 1997; revised 25 November 1997; accepted 10 December 1997
Abstract The three trimethyl isomers of benzene Žhemimellitene, 1,2,3-TMB; pseudocumene, 1,2,4-TMB and mesitylene, 1,3,5-TMB. were investigated for different genotoxicity endpoints: in vitro, in the Ames test with Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains in the presence and absence of rat liver S9 metabolic activation; in vivo, in the micronucleus and sister chromatid exchange ŽSCE. tests with bone marrow cells of Imp: Balbrc mice. Only the isomer of benzene with the methyl-group at position 1, 2, 3 was found to have mutagenic effect on S. typhimurium cells. Increase in bacterial reversions was observed in four conventional strains used in this study, but most clearly in TA97a. The mutagenic responses of 1,2,3-TMB with the Salmonella tester strains were observed in the experiments performed in the absence of enzymatic activation. None of the compounds had an influence on the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of mice. However, all the three compounds were observed to have a cytogenetic potential of increasing the SCE level in these cells. Significant responses in SCE induction, compared with the level of those changes in corresponding solvent-administered controls, were obtained at three test doses of 1,2,3-TMB Ž730, 1470, 2200 mgrkg. and 1,2,4-TMB Ž900, 1800, 2700 mgrkg. and at two doses of 1,3,5-TMB Ž1800, 2700 mgrkg.. These data provided a limited evidence for the genotoxic activity of 1,2,3-TMB and inadequate evidence for genotoxic activity of 1,2,4-TMB and 1,3,5-TMB. q 1998 Elsevier Science B.V. Keywords: Hemimellitene; Pseudocumene; Mesitylene; Salmonella typhimurium; Mouse bone marrow
1. Introduction Trimethylbenzenes ŽTMB. are solvents widely used as the components of industrial and commercial dyes and varnishes. In the present study, we evaluated the genotoxic activity of the three trimethyl
)
Corresponding author.
isomers of benzene: hemimellitene, with the methylgroup in the benzene ring in position 1,2,3; pseudocumene, with the methyl-group in position 1,2,4; and mesitylene, with the methyl-group in position 1,3,5 ŽFig. 1.. The following were analysed: reverse mutations from histidine auxotrophy to prototrophy in mutants of Salmonella typhimurium, induction of micronucleated polychromatic erythrocytes and sister chromatid exchange in bone marrow cells of Imp: Balbrc mice. As far as we know there is no
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 2 0 2 - 7
300
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
were found to be 3670 mgrkg Žmale. and 2700 mgrkg Žfemale. for hemimellitene; 5000 mgrkg Žmale., 4100 mgrkg Žfemale. for pseudocumene; 4500 mgrkg Žmale. and 3700 mgrkg Žfemale. for mesitylene, respectively. 2.3. Mutagenicity assay Fig. 1. The structure and the abbreviations of the test trimethylbenzenes.
information about the genotoxicity and structure–activity relationships of trimethylbenzenes. 2. Materials and methods 2.1. Chemicals Fluid hemimellitene, 1,2,3-trimethylbenzene w1,2,3-TMBx ŽCAS No. 526-73-8.; pseudocumene, 1,2,4-trimethylbenzene w1,2,4-TMBx ŽCAS No. 9563-6.; and mesitylene, 1,3,5-trimethylbenzene w1,3,5TMBx ŽCAS No. 108-67-8. were obtained from Fluka. The compounds were diluted in mineral oil Žlight white oil. obtained from Sigma Chemical. Solvent selection was based on findings from the pilot study. We observed that when dimethylsulfoxide ŽDMSO. or ethanol were used for in vitro experiments, the reaction of the compounds appeared to be more toxic for the tester strains of bacteria. 2.2. Toxicity test The concentrations of the compounds used in the in vitro and in vivo studies were based on preliminary experiments. In the mutagenicity assay, the compounds exhibiting the toxic effect Žrecorded as an inhibition of the background lawn of bacteria and as a decrease below 75% in the number of revertants as compared to the control number. were limited in the dose range for testing. In the micronucleus and SCE assay, a range of doses was established according to LD50 determination after Weil w1x. Five different doses of each compound were injected Žsingle intraperitoneal injection. to the groups of mice Žfour micergroup. and animals were observed for 14 days. Death occurred mostly during the first 2 days of the experiment. The LD50 values for the test compounds
The plate incorporation assays using S. typhimurium strains TA97a, TA98, Ta100 and TA102 were performed according to the standard protocol by Ames et al. w2x as revised by Maron and Ames w3x. About 10 h ‘Oxoid’ nutrient broth cultures were used. Rat S9 fraction was prepared from Aroclor 1254-induced male outbred Imp:Lodz rat liver. The protein content of the S9 fraction Ž43.2 mgrcm3 . was determined according to Lowry et al. w4x. The upper limit of the range of S9 levels recommended by Ames, 50 m l per plate, was used in the assays with metabolic activation. The compounds were tested up to the cytotoxic concentrations at least twice Žtwo independent assays. using two plates for each dose level. Three plates were used for the determinations of spontaneous reversion frequency and solvent control, i.e., mineral oil reversion of every bacterial strain. The concentrations of compounds used in these experiments were based on previous studies. Hisq revertant colonies were scored with electronic colony counter Biotran II from New Brunswick Scientific, NJ, after 48 h of incubation at 378C. A chemical was considered to be a mutagen if it induced at least a 2-fold increase in the number of revertants per plate as compared to the revertants per solvent control plate, with accompanying dose–effect relationship in at least one tester strain. 2.4. Micronucleus assay The assays were performed on bone marrow cells from male and female Imp: Balbrc strain mice Žweighing about 23 g, 9 weeks old. according to the standard procedures developed by Schmid w5x. Twodose levels of compounds were injected i.p., with each dose divided into two equal parts and given at a 24-h interval. Two-stage model of the experiments was used. In the first one, 1 ml of mineral oil or tested compound at a dose equal to 80% of LD50 was given to male mice. In the second experiment, the
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
males received dose equivalents of 40 and 80% of LD50 and the females y80% of LD50 . Bone marrow samples were collected in the 30th, 48th and 72nd h after the first injection. From each mouse, four bone marrow smears were made and 1000 polychromatic erythrocytes ŽPCEs. were analysed for the number of micronucleated cells ŽmPCEs.. The ratio of the number of PCEs to the number of normochromatic erythrocytes ŽNCEs. to assess bone marrow cytotoxicity was determined by counting both cell types until the level of 200 NCEs was reached. In the statistical analysis of results, one-way analysis of variance with multiple comparison test by Domanski ´ was used w6x. A chemical was considered genotoxic if it induced a statistically significant and reproducible increase in the number of mPCEs for at least one of the study points. 2.5. Chromatid exchange assay The assays were performed on bone marrow cells from male Imp: Balbrc strain mice Žweighing about 23 g, 9 weeks old., according to the procedures by Allen et al. w7x and the GENE-TOX Program by Latt et al. w8x. 5-bromodeoxyuridine ŽBrdU. tablets were implanted subcutaneously prior to the tested com-
301
pound administration. Four doses of each compound Ž20, 40, 60 and 80% of LD50 ; 5 mouserdose. and the mineral oil as the negative control and mitomycin C as the positive control, were injected i.p. 1 h after BrdU tablet implantation. After 21 h, colchicine Ž3.3 mgrkg; 2 h before the mice were sacrificed. was given by i.p. injection. The preparations from hipotonized and fixed bone marrow cells were stained following the method by Antoshina and Porjadkova w9x. To determine SCE frequency, 50 second division metaphase cells per mouse were analysed. One-way analysis of variance with multiple comparison test by Domanski ´ w6x was used for the statistical estimation of SCE frequency per cell in the treated and control groups of mice.A chemical was considered genotoxic if it induced a significant SCE, compared to the control group, for at least one of the dose levels.
3. Results and discussion In the plate incorporation assays, only hemimellitene showed a positive response in the conventional
Fig. 2. Dose-related increase in the number of Hisq revertants for hemimellitene in S. typhimurium strains.
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
302
Table 1 Reversion of S. typhimurium tester strains with pseudocumene and mesitylene in the presence and absence of S9-mix Compounds
Dose per plate Ž m l or m g.
Mean number of revertants per plate with standard deviation TA97a TA98 TA100
TA102
y S9
qS9
y S9
qS9
y S9
qS9
y S9
qS9
Spontaneous reversion Mineral oil Žsolvent control. Pseudocumene
0 100 1 5 10 20 30
121 " 7 126 " 13 148 " 23 158 " 10 165 " 8 141 " 25 115 " 3
145 " 5 141 " 12 152 " 7 168 " 8 176 " 21 155 " 20 106 " 7
24 " 3 23 " 3 24 " 3 29 " 5 41 " 7 27 " 8 TOX
31 " 3 31 " 5 35 " 4 28 " 1 29 " 4 30 " 3 29 " 6
123 " 71 125 " 41 138 " 15 148 " 18 143 " 9 124 " 7 118 " 4
25 " 4 21 " 10 126 " 62 125 " 5 112 " 4 108 " 3 110 " 4
258 " 6 280 " 12 290 " 33 262 " 16 273 " 20 214 " 8 TOX
294 " 11 315 " 14 279 " 24 276 " 11 276 " 11 236 " 21 TOX
Spontaneous reversion Mineral oil Žsolvent control. Mesitylene
0 100 1 5 10 20 30 40
127 " 15 131 " 10 141 " 13 149 " 29 139 " 17 129 " 13 125 " 8 NT
183 " 6 157 " 19 180 " 26 196 " 16 155 " 30 137 " 29 138 " 20 128 " 11
22 " 4 22 " 4 27 " 3 28 " 5 25 " 2 37 " 5 23 " 5 TOX
30 " 3 32 " 5 31 " 4 35 " 5 31 " 2 39 " 5 28 " 2 31 " 1
138 " 13 143 " 15 143 " 4 152 " 8 140 " 26 154 " 14 130 " 7 TOX
142 " 10 138 " 82 137 " 3 147 " 29 139 " 16 131 " 10 108 " 11 115 " 6
263 " 23 60 " 12 268 " 17 280 " 19 261 " 25 238 " 5 198 " 2 NT
337 " 13 336 " 23 347 " 34 334 " 30 353 " 11 340 " 37 324 " 10 NT
Positive control 4-Nitroquinoline-N-oxide 4-Nitro-o-phenylenediamine Sodium azide 2-Aminofluorene
0.5 3.0 1.5 5.0
613 " 38 243 " 22 128 " 13
334 " 18 680 " 49
21 " 5
696 " 28
786 " 21 138 " 7
771 " 54
287 " 19
520 " 48
TOX s toxic effects Žbackground growth reduced.; NT s not tested.
S. typhimurium strains. The results, presented graphically as the dose–response curves in Fig. 2, indicate that the mutation frequency was elevated in any of the four bacterial strains used in this study. Hemimellitene was found to behave as a reactive mutagen in both the detector strains of base-pair substitution and frameshift mutation, in the absence of metabolic activation. In the presence of S9-mix, a doubling of the control level of histidine reversions at any dose of hemimellitene could not be observed. The TA97a strain was the most sensitive to the mutagenic activity of this compound. The mutagenic responses of the bacterial cells were positive within narrow ranges of the dose. Approximately 5 m lrplate induced about a 2.7-fold ŽTA97a. and about a 2.9fold ŽTA98. increase in the revertant number, compared to the control values, whereas an approximate 10 m lrplate resulted in about a 2.6-fold ŽTA102. and 20 m lrplate about a 3.6-fold ŽTA100. increase in the revertant number.
All the compounds exhibited a toxic effect on all the tester strains, except for TA97a. In the experiments performed both in the presence and absence of S9-mix the highest doses of the compounds: 20 m lrplate for hemimellitene, 30 m lrplate for pseudocumene and 40 m lrplate for mesitylene, produced a decrease in the number of revertants to the level below 75% ŽTable 1.. A similar, negative effect Žpseudocumene, mesitylene. on the Salmonella tester strains was obtained in the experiments in which the compounds were tested using another technique Ždata not shown.. In the preincubation modification of the standard mutagenicity assay Ž20 min at 378C. on S. typhimurium TA98 and TA100 ŽyS9;qS9. mesitylene and pseudocumene Žat doses from 1–20 m lrplate, dissolved in DMSO. induced no gene mutations. All of the three compounds evoked a negative micronucleus response in polychromatic erythrocytes of the bone marrow ŽTable 2.. The intraperitoneal
11
1470 mgrkg 2160 mgrkg 2940 mgrkg
4 12
1 ml 1800 mgrkg 2960 mgrkg 3600 mgrkg 1 ml 2.5 mgrkg
Mineral oil Mesitylene
Distilled water Žsolvent control. Mitomycin C Žpositive control.
12
4
12
4
12
4
4.15 " 0.38)
0.24 " 0.11
0.20 " 0.00
0.23 " 0.10
0.15 " 0.10
0.22 " 0.07
0.17 " 0.06
4.22 " 0.35)
1.25 " 0.15)
0.15 " 0.05
0.14 " 0.05
0.17 " 0.09
0.21 " 0.08 0.17 " 0.09 0.17 " 0.05
0.16 " 0.07
0.20 " 0.08
0.18 " 0.07 0.18 " 0.10 0.16 " 0.8
0.21 " 0.11
0.17 " 0.05
0.22 " 0.10
0.17 " 0.05
72
0.17 " 0.09
0.23 " 0.5
0.22 " 0.09
30
48 0.18 " 0.09
30
48
0.20 " 0.00
0.20 " 0.08
0.18 " 0.05
0.23 " 0.05
0.20 " 0.08
0.20 " 0.08
72
0.22 " 0.05
0.13 " 0.05
0.20 " 0.14
0.71)
0.40)
0.62
1.16
1.18
0.85
0.82
30
48
0.48)
0.33
0.61 0.56
0.74
0.95 1.02
0.72
0.45
0.81
0.14)
1.37
0.42)
0.58
0.68)
1.02
0.62
0.50
72
0.51
0.98
0.90
30
48
0.60
0.60
1.01
0.95
0.84
0.95
0.58
0.85
0.78
72
)Significant difference vs. control at p F 0.05; The data for the groups of male mice treated with 80% LD 50 and mineral oil are expressed as the mean values " S.D. for two-stage experiments. For hemimellitene at the dose of 1470 mgrkg and for pseudocumene at the dose of 4000 mgrkg in 72nd h after administration, only three and seven mice were found to have survived.
24
8 12
23
8 12
1 ml 2000 mgrkg 3280 mgrkg 4000 mgrkg
Mineral oil Pseudocumene
24
8
1 ml
Mineral oil Žsolvent control. Hemimellitene
Female Harvest time Žh .
Male Harvest time Žh .
Female Harvest time Žh .
Male Harvest time Žh .
Ratio of polychromatic to normochromatic erythrocytes
Female
Male
Total dose
Compounds
Number of mice sex % of polychromatic erythrocytes with micronuclei Ž"S.D. .
Table 2 The induction of micronuclei in polychromatic erythrocytes of bone marrow of Imp: Balbrc mice
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305 303
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E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
administration to male and female mice did not result in the induction of micronuclei either at 30, 48 or 72 h after dosing. Pseudocumene and mesitylene revealed toxic properties for the erythrocyte line cells in the bone marrow of male mice. The highest doses of the compounds Ž80% of LD50 . induced a statistically significant decrease in the PCEs to NCEs ratio Žat 72 h the cytotoxicity index for pseudocumene was 0.68, while the corresponding value in mice injected with mineral oil was 0.95; for mesitylene, the corresponding values of the index were 0.40 and 0.42 for the 30th and 72nd h, respectively, compared with 0.61 in the control male mice. However, for hemimellitene the statistical reduction in the PCEs to NCEs ratio was not observed. The dose equivalent of 40% of LD50 Ž1470 mgrkg. was found lethal in one of the 12 mice. The study performed on females using the equivalent of 80% of LD50 for all the tested compounds revealed that the animals survived but at the same time these doses were found not to induce cytotoxicity on bone marrow cells. Hemimellitene, pseudocumene and mesitylene all revealed positive results in the SCE test. Fig. 3
shows the effects of administered doses of all compounds on SCE frequency in mouse bone marrow cells. The genotoxicity of trimethylbenzenes in the mouse bone marrow SCE assay was as follows: pseudocumene) hemimellitene) mesitylene. Compared to the control group of mice, a significantly increased SCE frequency was found for three doses of hemimellitene and pseudocumene and two doses of mesitylene. For hemimellitene the increase in SCE frequency amounted to: 4.14 " 0.17 and 4.73 " 0.24 for the doses of 730 mgrkg and 2200 mgrkg, respectively. For pseudocumene and mesitylene, the SCE increase values were 5.46 " 0.43 and 3.90 " 0.16, respectively for the dose of 900 mgrkg and 6.61 " 0.25; and 4.58 " 0.29, for the 2700 mgrkg dose. In the group of mice which received mineral oil the respective frequencies were as follows: 3.70 " 0.12, 4.29 " 0.13 and 3.58 " 0.18. A characteristic feature of the trimethyl isomers was the toxicity on bone marrow cells. For the highest administered doses of all the compounds, the analysis of SCE induction was impossible owing to the fact that these doses caused either the death of the mice or a dramatic reduction in M2 cells in bone
Fig. 3. Sister chromatid exchanges induced in bone marrow cells of Imp: Balbrc mice.
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
marrow. After the administration of 3600 mgrkg pseudocumene, four mice died and the number of second generation cells of bone marrow from the surviving mouse was insufficient to carry out SCE analysis. The doses of 2940 and 3600 mgrkg for hemimellitene and mesitylene, respectively, were fatal for two animals in each group. In the mice which survived, only M1 cells in bone marrow smears could be observed. From these studies it may be concluded that: Ž1. hemimellitene Ž1,2,3-TMB. is a directly-acting mutagen for S. typhimurium strains TA97a, TA98, TA100 and TA102; Ž2. trimethylbenzenes are incapable of micronucleus induction; and Ž3. trimethylbenzenes are active as SCE-inducing agents.
Acknowledgements This research has been performed under IMP project No. 5.3.The authors would like to thank Mrs. Barbara Pawlak and Mrs. Mariola Włodarek for the technical assistance.
305
References w1x C.S. Weil, Tables for convenient calculation of median effective dose ŽDL 50 or ED50 . and instruction in their use, Biometrics 8 Ž1952. 249–255. w2x B.N. Ames, J. McCann, E. Yamasaki, Methods for detecting carcinogens and mutagens with the Salmonellarmammalianmicrosome mutagenicity test, Mutat. Res. 31 Ž1975. 347–364. w3x D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 Ž1983. 173–215. w4x O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. w5x W. Schmid, The micronucleus test, Mutat. Res. 31 Ž1975. 9–15. w6x C. Domanski, Non-Parametric Statistical Test, National Medi´ cal Publishing House, Warsaw, 1979 Žin Polish.. w7x J.W. Allen, G. Shuler, R.W. Mendes, S.A. Latt, A simplified technique for in vivo analysis of sister-chromatid exchanges using 5-bromodeoxyuridine tablets, Cytogenet. Cell Genet. 18 Ž1977. 231–237. w8x S.A. Latt, J. Allen, S.E. Bloom, A. Carrano, E. Falke, D. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield, S. Wolff, Sister chromatid exchange: a report of GENE-TOX program, Mutat. Res. 87 Ž1981. 17–62. w9x M.M. Antoshina, N.A. Porjadkova, A technique for differential staining of sister chromatids without using fluorochrome, Citol. Genet. 4 Ž1978. 349–352.
Genotoxicity evaluation of trimethylbenzenes Ewa Janik-Spiechowicz ) , Kalina Wyszynska, Elzbieta Dziubałtowska ´ ˙ Department of Toxicology and Carcinogenesis, The Nofer Institute of Occupational Medicine, PO Box 199, 90-950 Lodz, Poland Received 26 August 1997; revised 25 November 1997; accepted 10 December 1997
Abstract The three trimethyl isomers of benzene Žhemimellitene, 1,2,3-TMB; pseudocumene, 1,2,4-TMB and mesitylene, 1,3,5-TMB. were investigated for different genotoxicity endpoints: in vitro, in the Ames test with Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains in the presence and absence of rat liver S9 metabolic activation; in vivo, in the micronucleus and sister chromatid exchange ŽSCE. tests with bone marrow cells of Imp: Balbrc mice. Only the isomer of benzene with the methyl-group at position 1, 2, 3 was found to have mutagenic effect on S. typhimurium cells. Increase in bacterial reversions was observed in four conventional strains used in this study, but most clearly in TA97a. The mutagenic responses of 1,2,3-TMB with the Salmonella tester strains were observed in the experiments performed in the absence of enzymatic activation. None of the compounds had an influence on the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of mice. However, all the three compounds were observed to have a cytogenetic potential of increasing the SCE level in these cells. Significant responses in SCE induction, compared with the level of those changes in corresponding solvent-administered controls, were obtained at three test doses of 1,2,3-TMB Ž730, 1470, 2200 mgrkg. and 1,2,4-TMB Ž900, 1800, 2700 mgrkg. and at two doses of 1,3,5-TMB Ž1800, 2700 mgrkg.. These data provided a limited evidence for the genotoxic activity of 1,2,3-TMB and inadequate evidence for genotoxic activity of 1,2,4-TMB and 1,3,5-TMB. q 1998 Elsevier Science B.V. Keywords: Hemimellitene; Pseudocumene; Mesitylene; Salmonella typhimurium; Mouse bone marrow
1. Introduction Trimethylbenzenes ŽTMB. are solvents widely used as the components of industrial and commercial dyes and varnishes. In the present study, we evaluated the genotoxic activity of the three trimethyl
)
Corresponding author.
isomers of benzene: hemimellitene, with the methylgroup in the benzene ring in position 1,2,3; pseudocumene, with the methyl-group in position 1,2,4; and mesitylene, with the methyl-group in position 1,3,5 ŽFig. 1.. The following were analysed: reverse mutations from histidine auxotrophy to prototrophy in mutants of Salmonella typhimurium, induction of micronucleated polychromatic erythrocytes and sister chromatid exchange in bone marrow cells of Imp: Balbrc mice. As far as we know there is no
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 2 0 2 - 7
300
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
were found to be 3670 mgrkg Žmale. and 2700 mgrkg Žfemale. for hemimellitene; 5000 mgrkg Žmale., 4100 mgrkg Žfemale. for pseudocumene; 4500 mgrkg Žmale. and 3700 mgrkg Žfemale. for mesitylene, respectively. 2.3. Mutagenicity assay Fig. 1. The structure and the abbreviations of the test trimethylbenzenes.
information about the genotoxicity and structure–activity relationships of trimethylbenzenes. 2. Materials and methods 2.1. Chemicals Fluid hemimellitene, 1,2,3-trimethylbenzene w1,2,3-TMBx ŽCAS No. 526-73-8.; pseudocumene, 1,2,4-trimethylbenzene w1,2,4-TMBx ŽCAS No. 9563-6.; and mesitylene, 1,3,5-trimethylbenzene w1,3,5TMBx ŽCAS No. 108-67-8. were obtained from Fluka. The compounds were diluted in mineral oil Žlight white oil. obtained from Sigma Chemical. Solvent selection was based on findings from the pilot study. We observed that when dimethylsulfoxide ŽDMSO. or ethanol were used for in vitro experiments, the reaction of the compounds appeared to be more toxic for the tester strains of bacteria. 2.2. Toxicity test The concentrations of the compounds used in the in vitro and in vivo studies were based on preliminary experiments. In the mutagenicity assay, the compounds exhibiting the toxic effect Žrecorded as an inhibition of the background lawn of bacteria and as a decrease below 75% in the number of revertants as compared to the control number. were limited in the dose range for testing. In the micronucleus and SCE assay, a range of doses was established according to LD50 determination after Weil w1x. Five different doses of each compound were injected Žsingle intraperitoneal injection. to the groups of mice Žfour micergroup. and animals were observed for 14 days. Death occurred mostly during the first 2 days of the experiment. The LD50 values for the test compounds
The plate incorporation assays using S. typhimurium strains TA97a, TA98, Ta100 and TA102 were performed according to the standard protocol by Ames et al. w2x as revised by Maron and Ames w3x. About 10 h ‘Oxoid’ nutrient broth cultures were used. Rat S9 fraction was prepared from Aroclor 1254-induced male outbred Imp:Lodz rat liver. The protein content of the S9 fraction Ž43.2 mgrcm3 . was determined according to Lowry et al. w4x. The upper limit of the range of S9 levels recommended by Ames, 50 m l per plate, was used in the assays with metabolic activation. The compounds were tested up to the cytotoxic concentrations at least twice Žtwo independent assays. using two plates for each dose level. Three plates were used for the determinations of spontaneous reversion frequency and solvent control, i.e., mineral oil reversion of every bacterial strain. The concentrations of compounds used in these experiments were based on previous studies. Hisq revertant colonies were scored with electronic colony counter Biotran II from New Brunswick Scientific, NJ, after 48 h of incubation at 378C. A chemical was considered to be a mutagen if it induced at least a 2-fold increase in the number of revertants per plate as compared to the revertants per solvent control plate, with accompanying dose–effect relationship in at least one tester strain. 2.4. Micronucleus assay The assays were performed on bone marrow cells from male and female Imp: Balbrc strain mice Žweighing about 23 g, 9 weeks old. according to the standard procedures developed by Schmid w5x. Twodose levels of compounds were injected i.p., with each dose divided into two equal parts and given at a 24-h interval. Two-stage model of the experiments was used. In the first one, 1 ml of mineral oil or tested compound at a dose equal to 80% of LD50 was given to male mice. In the second experiment, the
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
males received dose equivalents of 40 and 80% of LD50 and the females y80% of LD50 . Bone marrow samples were collected in the 30th, 48th and 72nd h after the first injection. From each mouse, four bone marrow smears were made and 1000 polychromatic erythrocytes ŽPCEs. were analysed for the number of micronucleated cells ŽmPCEs.. The ratio of the number of PCEs to the number of normochromatic erythrocytes ŽNCEs. to assess bone marrow cytotoxicity was determined by counting both cell types until the level of 200 NCEs was reached. In the statistical analysis of results, one-way analysis of variance with multiple comparison test by Domanski ´ was used w6x. A chemical was considered genotoxic if it induced a statistically significant and reproducible increase in the number of mPCEs for at least one of the study points. 2.5. Chromatid exchange assay The assays were performed on bone marrow cells from male Imp: Balbrc strain mice Žweighing about 23 g, 9 weeks old., according to the procedures by Allen et al. w7x and the GENE-TOX Program by Latt et al. w8x. 5-bromodeoxyuridine ŽBrdU. tablets were implanted subcutaneously prior to the tested com-
301
pound administration. Four doses of each compound Ž20, 40, 60 and 80% of LD50 ; 5 mouserdose. and the mineral oil as the negative control and mitomycin C as the positive control, were injected i.p. 1 h after BrdU tablet implantation. After 21 h, colchicine Ž3.3 mgrkg; 2 h before the mice were sacrificed. was given by i.p. injection. The preparations from hipotonized and fixed bone marrow cells were stained following the method by Antoshina and Porjadkova w9x. To determine SCE frequency, 50 second division metaphase cells per mouse were analysed. One-way analysis of variance with multiple comparison test by Domanski ´ w6x was used for the statistical estimation of SCE frequency per cell in the treated and control groups of mice.A chemical was considered genotoxic if it induced a significant SCE, compared to the control group, for at least one of the dose levels.
3. Results and discussion In the plate incorporation assays, only hemimellitene showed a positive response in the conventional
Fig. 2. Dose-related increase in the number of Hisq revertants for hemimellitene in S. typhimurium strains.
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
302
Table 1 Reversion of S. typhimurium tester strains with pseudocumene and mesitylene in the presence and absence of S9-mix Compounds
Dose per plate Ž m l or m g.
Mean number of revertants per plate with standard deviation TA97a TA98 TA100
TA102
y S9
qS9
y S9
qS9
y S9
qS9
y S9
qS9
Spontaneous reversion Mineral oil Žsolvent control. Pseudocumene
0 100 1 5 10 20 30
121 " 7 126 " 13 148 " 23 158 " 10 165 " 8 141 " 25 115 " 3
145 " 5 141 " 12 152 " 7 168 " 8 176 " 21 155 " 20 106 " 7
24 " 3 23 " 3 24 " 3 29 " 5 41 " 7 27 " 8 TOX
31 " 3 31 " 5 35 " 4 28 " 1 29 " 4 30 " 3 29 " 6
123 " 71 125 " 41 138 " 15 148 " 18 143 " 9 124 " 7 118 " 4
25 " 4 21 " 10 126 " 62 125 " 5 112 " 4 108 " 3 110 " 4
258 " 6 280 " 12 290 " 33 262 " 16 273 " 20 214 " 8 TOX
294 " 11 315 " 14 279 " 24 276 " 11 276 " 11 236 " 21 TOX
Spontaneous reversion Mineral oil Žsolvent control. Mesitylene
0 100 1 5 10 20 30 40
127 " 15 131 " 10 141 " 13 149 " 29 139 " 17 129 " 13 125 " 8 NT
183 " 6 157 " 19 180 " 26 196 " 16 155 " 30 137 " 29 138 " 20 128 " 11
22 " 4 22 " 4 27 " 3 28 " 5 25 " 2 37 " 5 23 " 5 TOX
30 " 3 32 " 5 31 " 4 35 " 5 31 " 2 39 " 5 28 " 2 31 " 1
138 " 13 143 " 15 143 " 4 152 " 8 140 " 26 154 " 14 130 " 7 TOX
142 " 10 138 " 82 137 " 3 147 " 29 139 " 16 131 " 10 108 " 11 115 " 6
263 " 23 60 " 12 268 " 17 280 " 19 261 " 25 238 " 5 198 " 2 NT
337 " 13 336 " 23 347 " 34 334 " 30 353 " 11 340 " 37 324 " 10 NT
Positive control 4-Nitroquinoline-N-oxide 4-Nitro-o-phenylenediamine Sodium azide 2-Aminofluorene
0.5 3.0 1.5 5.0
613 " 38 243 " 22 128 " 13
334 " 18 680 " 49
21 " 5
696 " 28
786 " 21 138 " 7
771 " 54
287 " 19
520 " 48
TOX s toxic effects Žbackground growth reduced.; NT s not tested.
S. typhimurium strains. The results, presented graphically as the dose–response curves in Fig. 2, indicate that the mutation frequency was elevated in any of the four bacterial strains used in this study. Hemimellitene was found to behave as a reactive mutagen in both the detector strains of base-pair substitution and frameshift mutation, in the absence of metabolic activation. In the presence of S9-mix, a doubling of the control level of histidine reversions at any dose of hemimellitene could not be observed. The TA97a strain was the most sensitive to the mutagenic activity of this compound. The mutagenic responses of the bacterial cells were positive within narrow ranges of the dose. Approximately 5 m lrplate induced about a 2.7-fold ŽTA97a. and about a 2.9fold ŽTA98. increase in the revertant number, compared to the control values, whereas an approximate 10 m lrplate resulted in about a 2.6-fold ŽTA102. and 20 m lrplate about a 3.6-fold ŽTA100. increase in the revertant number.
All the compounds exhibited a toxic effect on all the tester strains, except for TA97a. In the experiments performed both in the presence and absence of S9-mix the highest doses of the compounds: 20 m lrplate for hemimellitene, 30 m lrplate for pseudocumene and 40 m lrplate for mesitylene, produced a decrease in the number of revertants to the level below 75% ŽTable 1.. A similar, negative effect Žpseudocumene, mesitylene. on the Salmonella tester strains was obtained in the experiments in which the compounds were tested using another technique Ždata not shown.. In the preincubation modification of the standard mutagenicity assay Ž20 min at 378C. on S. typhimurium TA98 and TA100 ŽyS9;qS9. mesitylene and pseudocumene Žat doses from 1–20 m lrplate, dissolved in DMSO. induced no gene mutations. All of the three compounds evoked a negative micronucleus response in polychromatic erythrocytes of the bone marrow ŽTable 2.. The intraperitoneal
11
1470 mgrkg 2160 mgrkg 2940 mgrkg
4 12
1 ml 1800 mgrkg 2960 mgrkg 3600 mgrkg 1 ml 2.5 mgrkg
Mineral oil Mesitylene
Distilled water Žsolvent control. Mitomycin C Žpositive control.
12
4
12
4
12
4
4.15 " 0.38)
0.24 " 0.11
0.20 " 0.00
0.23 " 0.10
0.15 " 0.10
0.22 " 0.07
0.17 " 0.06
4.22 " 0.35)
1.25 " 0.15)
0.15 " 0.05
0.14 " 0.05
0.17 " 0.09
0.21 " 0.08 0.17 " 0.09 0.17 " 0.05
0.16 " 0.07
0.20 " 0.08
0.18 " 0.07 0.18 " 0.10 0.16 " 0.8
0.21 " 0.11
0.17 " 0.05
0.22 " 0.10
0.17 " 0.05
72
0.17 " 0.09
0.23 " 0.5
0.22 " 0.09
30
48 0.18 " 0.09
30
48
0.20 " 0.00
0.20 " 0.08
0.18 " 0.05
0.23 " 0.05
0.20 " 0.08
0.20 " 0.08
72
0.22 " 0.05
0.13 " 0.05
0.20 " 0.14
0.71)
0.40)
0.62
1.16
1.18
0.85
0.82
30
48
0.48)
0.33
0.61 0.56
0.74
0.95 1.02
0.72
0.45
0.81
0.14)
1.37
0.42)
0.58
0.68)
1.02
0.62
0.50
72
0.51
0.98
0.90
30
48
0.60
0.60
1.01
0.95
0.84
0.95
0.58
0.85
0.78
72
)Significant difference vs. control at p F 0.05; The data for the groups of male mice treated with 80% LD 50 and mineral oil are expressed as the mean values " S.D. for two-stage experiments. For hemimellitene at the dose of 1470 mgrkg and for pseudocumene at the dose of 4000 mgrkg in 72nd h after administration, only three and seven mice were found to have survived.
24
8 12
23
8 12
1 ml 2000 mgrkg 3280 mgrkg 4000 mgrkg
Mineral oil Pseudocumene
24
8
1 ml
Mineral oil Žsolvent control. Hemimellitene
Female Harvest time Žh .
Male Harvest time Žh .
Female Harvest time Žh .
Male Harvest time Žh .
Ratio of polychromatic to normochromatic erythrocytes
Female
Male
Total dose
Compounds
Number of mice sex % of polychromatic erythrocytes with micronuclei Ž"S.D. .
Table 2 The induction of micronuclei in polychromatic erythrocytes of bone marrow of Imp: Balbrc mice
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305 303
304
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
administration to male and female mice did not result in the induction of micronuclei either at 30, 48 or 72 h after dosing. Pseudocumene and mesitylene revealed toxic properties for the erythrocyte line cells in the bone marrow of male mice. The highest doses of the compounds Ž80% of LD50 . induced a statistically significant decrease in the PCEs to NCEs ratio Žat 72 h the cytotoxicity index for pseudocumene was 0.68, while the corresponding value in mice injected with mineral oil was 0.95; for mesitylene, the corresponding values of the index were 0.40 and 0.42 for the 30th and 72nd h, respectively, compared with 0.61 in the control male mice. However, for hemimellitene the statistical reduction in the PCEs to NCEs ratio was not observed. The dose equivalent of 40% of LD50 Ž1470 mgrkg. was found lethal in one of the 12 mice. The study performed on females using the equivalent of 80% of LD50 for all the tested compounds revealed that the animals survived but at the same time these doses were found not to induce cytotoxicity on bone marrow cells. Hemimellitene, pseudocumene and mesitylene all revealed positive results in the SCE test. Fig. 3
shows the effects of administered doses of all compounds on SCE frequency in mouse bone marrow cells. The genotoxicity of trimethylbenzenes in the mouse bone marrow SCE assay was as follows: pseudocumene) hemimellitene) mesitylene. Compared to the control group of mice, a significantly increased SCE frequency was found for three doses of hemimellitene and pseudocumene and two doses of mesitylene. For hemimellitene the increase in SCE frequency amounted to: 4.14 " 0.17 and 4.73 " 0.24 for the doses of 730 mgrkg and 2200 mgrkg, respectively. For pseudocumene and mesitylene, the SCE increase values were 5.46 " 0.43 and 3.90 " 0.16, respectively for the dose of 900 mgrkg and 6.61 " 0.25; and 4.58 " 0.29, for the 2700 mgrkg dose. In the group of mice which received mineral oil the respective frequencies were as follows: 3.70 " 0.12, 4.29 " 0.13 and 3.58 " 0.18. A characteristic feature of the trimethyl isomers was the toxicity on bone marrow cells. For the highest administered doses of all the compounds, the analysis of SCE induction was impossible owing to the fact that these doses caused either the death of the mice or a dramatic reduction in M2 cells in bone
Fig. 3. Sister chromatid exchanges induced in bone marrow cells of Imp: Balbrc mice.
E. Janik-Spiechowicz et al.r Mutation Research 412 (1998) 299–305
marrow. After the administration of 3600 mgrkg pseudocumene, four mice died and the number of second generation cells of bone marrow from the surviving mouse was insufficient to carry out SCE analysis. The doses of 2940 and 3600 mgrkg for hemimellitene and mesitylene, respectively, were fatal for two animals in each group. In the mice which survived, only M1 cells in bone marrow smears could be observed. From these studies it may be concluded that: Ž1. hemimellitene Ž1,2,3-TMB. is a directly-acting mutagen for S. typhimurium strains TA97a, TA98, TA100 and TA102; Ž2. trimethylbenzenes are incapable of micronucleus induction; and Ž3. trimethylbenzenes are active as SCE-inducing agents.
Acknowledgements This research has been performed under IMP project No. 5.3.The authors would like to thank Mrs. Barbara Pawlak and Mrs. Mariola Włodarek for the technical assistance.
305
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