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Sister chromatid exchanges of human peripheral blood lymphocytes induced by N,N-diethylaniline in vitro
Mutation Research 395 Ž1997. 151–157
Sister chromatid exchanges of human peripheral blood lymphocytes induced by N,N-diethylaniline in vitro Qing Li ) , Masayasu Minami Department of Hygiene and Public Health, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan Received 16 June 1997; revised 1 September 1997; accepted 1 September 1997
Abstract N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes. To evaluate its genotoxicity, we examined whether it can induce sister chromatid exchanges ŽSCEs. in human lymphocytes. We found that N,N-diethylaniline significantly increased the frequency of SCEs both in the absence and presence of S-9 mix. The SCEs from cultures treated by N,N-diethylaniline in the presence of S-9 mix displayed a marked increase which was about 5-fold greater than the control. ANOVA analyses indicated that there is a dose–response relationship between doses of N,N-diethylaniline and the frequency of SCEs, especially in the presence of S-9 mix. The results suggested that N,N-diethylaniline has genotoxicity. q 1997 Elsevier Science B.V. Keywords: Sister chromatid exchange; N,N-Diethylaniline; S-9 mix; Human lymphocyte; Genotoxicity
1. Introduction N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes w1x. It has been reported that N,N-diethylaniline was negative in Ames tests using Salmonella typhimurium TA 100, TA 98 and other strains in the absence or presence of S-9 mix, but weakly positive using TA 98 in the presence of norharman and S-9 mix w2x. On the other hand, Yoshimi et al. w3x found that N,N-diethylaniline was negative in an unscheduled DNA synthesis ŽUDS. test using rat hepatocytes. It is well known that sister chromatid exchanges ŽSCEs. can be used to evaluate
) Corresponding author. Tel.: q81 3 38222131 Ž5259.; Fax: q81 3 56853065; E-mail: [email protected]
the genotoxicity of chemicals w4–8x. To clarify its genotoxicity, we examined the frequency of SCEs in human peripheral blood lymphocytes treated with N,N-diethylaniline in vitro in the presence or absence of S-9 mix, a metabolic activation reagent.
2. Materials and methods 2.1. Reagents Because N,N-diethylaniline ŽCAS 91-66-7. is rather insoluble in water, and is not convenient for in vitro SCEs testing, we used N,N-diethylaniline–HCl, which was purchased from Wako Pure Chemical Industries, Osaka, Japan. S-9 mix ŽS-9rco-factor-C set. was purchased from Oriental Yeast Co., Tokyo,
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 6 2 - 9
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Japan. The S-9 mix in 6.7 ml contains 2 ml S-9 Ž30%. and 4.7 ml co-factor-C Ž70%., which consist of 1.34 ml of 20 mM HEPES buffer ŽpH 7.2., 0.67 ml of 50 mM MgCl 2 , 330 mM KCl, 50 mM glucose-6-phosphate, 40 mM NADPŽNa 2 . and distilled water, respectively, and was freshly prepared before each use. RPMI 1640 was from Nissui Pharmaceutical, Tokyo, Japan. Fetal bovine serum ŽFBS. was purchased from Cell Culture Laboratories, Cleveland, OH, USA, and heat-inactivated at 568C for 30 min prior to use. Glutamine, 2-mercaptoethanol Ž2ME., colcemid, Hoechst 33258 and 5X-bromodeoxyuridine ŽBrdU. were purchased from Sigma, St. Louis, MO, USA. Phytohemagglutinin-M ŽPHAM. was purchased from Gibco BRL, NY, USA. w 3 HxThymidine was purchased from ICN Biomedicals, Irvine, CA, USA. 2.2. Cytotoxicity test of N,N-diethylaniline–HCl and S-9 mix using human lymphocytes [9] The nonspecific cytotoxicity of N,N-diethylaniline–HCl and S-9 mix using human lymphocytes was examined to determine the optimal concentrations for conducting in vitro SCEs testing, where N,N-diethylaniline does not inhibit the proliferation of lymphocytes, but can induce SCEs. Lymphocytes were separated from human peripheral blood of 4 healthy subjects, then plated into flat-bottomed 96well microplates in triplicate at a density of 5 = 10 5 cellsrwell in RPMI 1640 containing 10% FBS and 5 = 10y5 M 2-ME and 3% PHA-M, and incubated in 5% CO 2 at 378C for 24 h. Then N,N-diethylaniline–HCl at 0, 5, 10, 25, 50, 100, 200, 500, 1000 and 2500 ppm ŽFig. 1A. and S-9 mix at 0%, 5%, 10%, 17%, 20% and 30% ŽFig. 1B. were added to the wells in triplicate and cultured in a total volume of 200 mlrwell at 378C in 5% CO 2 . The cells were pulsed with 0.6 mCirwell of w 3 HxThymidine Ž22.2 kBq. for the final 24 h and harvested 72 h after culture initiation. The level of w 3 HxThymidine incorporation was determined in a liquid scintillation counter. For the 1 h treatment with S-9 mix, the lymphocytes were treated with S-9 mix at 0%, 10%, 15% and 20% at 378C for 1 h. The cells were collected after centrifugation, and the pellets were washed twice with prewarmed RPMI 1640 medium
Fig. 1. Cytotoxicity of N,N-diethylaniline–HCl Župper. and S-9 mix Žmiddle. in 48 h in vitro treatment and S-9 mix in 1 h in vitro treatment Žlower. on the blastogenesis of human lymphocytes stimulated by PHA-M. Data are presented as means"SEM Ž ns 4.. The result in the absence of N,N-diethylaniline–HCl Ž0 ppm. was 61866.7"1319.4.
to remove the S-9 mix. Then the cells were re-incubated for 72 h as for the 48 h treatment. 2.3. N,N-Diethylaniline–HCl-induced SCEs in Õitro without S-9 mix in 48 h treatment [4–6] Samples of 0.5–0.6 ml of human peripheral blood obtained from 4 healthy donors Ž2 males and 2 females; 25–33 years old. were added to 5 ml RPMI 1640 containing 15% FBS, 5 = 10y5 M 2-ME and 3% PHA-M in a flask, and incubated in 5% CO 2 at 378C for 24 h. Then, BrdU at 30 mM and N,N-diethylaniline–HCl at a final concentration of 0, 10 or 100 ppm were added to the cultures and the cultures were re-incubated for 48–50 h in the dark. Colcemid treatment was conducted at a final concentration of 0.15 mgrml, 2.5–3 h before harvesting the cells. After harvesting the cultures, hypo-osmotic treatment was conducted with 0.075 M KCl at 378C. After
Q. Li, M. Minamir Mutation Research 395 (1997) 151–157
centrifugation, the pellets were fixed three times with a fixative consisting of three parts methanol and one part glacial acetic acid at room temperature, then the cells in the fixative were stored at 48C for 48–72 h. Slides were prepared and stained by Hoechst 33258 at 5 mgrml and 4% Giemsa. Frequencies of SCEs were evaluated by scoring at least 50 cells in second-division metaphase for each subject at each dose and the SCEs were indicated as SCEsrcell.
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medium to remove N,N-diethylaniline–HCl and S-9 mix. Then the cells were added to 5 ml RPMI 1640 containing 15% FBS, 5 = 10y5 M 2-ME, 3% PHA-M and BrdU at 30 mM in a flask. Each culture was further incubated for 48–50 h in 5% CO 2 at 378C in the dark, and treated with colcemid at 2.5–3 h before harvesting. Then the cells were collected and slides were prepared as described above. 2.5. Statistical analyses
2.4. N,N-Diethylaniline–HCl-induced SCEs in Õitro with and without S-9 mix in 1 h treatment [7,8] In the metabolic activation experiments, samples of 0.5–0.6 ml of human peripheral blood obtained from the 4 healthy donors were added to 5 ml RPMI 1640 containing 15% FBS, 5 = 10y5 M 2-ME and 3% PHA-M in a flask and incubated in 5% CO 2 at 378C for 24 h. Then, the cultures were harvested and treated with N,N-diethylaniline–HCl at a final concentration of 0, 100, 200 or 500 ppm in the presence or absence of 20% S-9 mix at 378C for 1 h. The cells were collected after centrifugation, and the pellets were washed twice with prewarmed RPMI 1640
Statistical analyses were performed using ANOVA and t-test. A p-value of - 0.05 was regarded as significant.
3. Results 3.1. Optimal concentrations of N,N-diethylaniline– HCl and S-9 mix for in Õitro SCEs As shown in Fig. 1A, treatment of 1000 ppm or more of N,N-diethylaniline–HCl for 48 h in vitro had inhibitory effects on the lymphocyte transforma-
Fig. 2. Frequency of SCEs induced by N,N-diethylaniline–HCl without S-9 mix in 48 h in vitro treatment, A: averaged SCEs based on counted cell numbers pooled from 4 subjects; B: the averages of SCEs of the individual values are indicated. The numbers in A indicate counted cell numbers pooled from 4 subjects. Sub1 ŽM. and Sub2 ŽM. in B mean data from subjects 1 and 2, both of whom are males; Sub3 ŽF. and Sub4 ŽF. mean data from subjects 3 and 4, both females. Data are presented as means" SEM. ) ) p - 0.01, significantly different from control; a p - 0.01, significantly different from 10 ppm by unpaired t-test. Two-way ANOVA: Fdose s 38.03, p s 0.0004; Findividual s 3.89, p s 0.074.
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Fig. 3. Frequency of SCEs induced by N,N-diethylaniline–HCl without S-9 mix in 1 h in vitro treatment. A: averaged SCEs based on counted cell numbers pooled from 4 subjects; B: the averages of SCEs of the individual values are indicated. The numbers in A indicate counted cell numbers pooled from 4 subjects. Sub1 ŽM. and Sub2 ŽM. in B mean data from subjects 1 and 2, both of whom are males; Sub3 ŽF. and Sub4 ŽF. mean data from subjects 3 and 4, both females. Data are presented as means" SEM. ) ) p - 0.01, significantly different from control; a p - 0.01, significantly different from 100 ppm; $ p - 0.01, significantly different from 200 ppm by unpaired t-test. Two-way ANOVA: Fdose s 81.72, p s 8 = 10y7 ; Findividual s 29.39, p s 6 = 10y5 .
Fig. 4. Frequency of SCEs induced by N,N-diethylaniline–HCl with 20% S-9 mix in 1 h in vitro treatment. A: averaged SCEs based on counted cell numbers pooled from 4 subjects; B: the averages of SCEs of the individual values are indicated. The numbers in A indicate counted cell numbers pooled from 4 subjects. Sub1 ŽM. and Sub2 ŽM. in B mean data from subjects 1 and 2, both of whom are males; Sub3 ŽF. and Sub4 ŽF. mean data from subjects 3 and 4, both females. Data are presented as means" SEM. ) ) p - 0.01, significantly different from control; a p - 0.01, significantly different from 100 ppm; $ p - 0.01, significantly different from 200 ppm by unpaired t-test. Two-way ANOVA: Fdose s 121.03, p s 1 = 10y7 ; Findividual s 2.73, p s 0.106.
Q. Li, M. Minamir Mutation Research 395 (1997) 151–157
tion response to PHA-M. Based on these results, 0, 10 and 100 ppm of N,N-diethylaniline–HCl were selected for in vitro SCEs testing of 48 h treatment without S-9 mix. As shown in Fig. 1B, treatment of 5% or more of S-9 mix for 48 h in vitro had inhibitory effects on the lymphocyte transformation response to PHA-M. Therefore, we did not use the S-9 mix in culture during the 48 h treatment. However, the S-9 mix at 20% for 1 h in vitro treatment was not cytotoxic to lymphocytes ŽFig. 1C.. Thus, 0, 100, 200 and 500 ppm of N,N-diethylaniline–HCl were selected for in vitro SCEs in the 1 h treatment in the presence or absence of 20% S-9 mix. 3.2. N,N-Diethylaniline–HCl induced SCEs in Õitro without S-9 mix in 48 h treatment As shown in Fig. 2, frequencies of SCEs in the cultures treated with N,N-diethylaniline–HCl without S-9 mix were significantly greater than in the control culture. There were significant differences in the frequency of SCEs between 0 and 10 or 100 ppm, and between 10 and 100 ppm Žall p - 0.01; Fig. 2A.. ANOVA analysis indicated that the dose of N,N-diethylaniline–HCl significantly affected the frequency of SCEs Ž F s 38.03; p s 0.0004., but the difference between individuals was not significant ŽFig. 2B..
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aniline–HCl significantly increased to about 5-fold the control level ŽS-9 mix only.. There were significant differences in the frequency of SCEs between the control and 100, 200 or 500 ppm, respectively Žall p - 0.01.. There were also significant differences in frequency of SCEs between 100 and 200 or 500, and between 200 and 500 ppm, respectively Žall p - 0.01; Fig. 4A.. ANOVA analysis indicated that there was a dose–response relationship between the frequency of SCEs and the dose of N,N-diethylaniline–HCl Ž F s 121.03; p s 1 = 10y7 ., but the difference between individuals was not significant ŽFig. 4B.. Differences in the frequency of SCEs between cultures with and without S-9 mix at doses of 100, 200 and 500 ppm were also significant Žall p - 0.01; Fig. 3A and Fig. 4A.. Fig. 5 shows a cell that was treated with N,N-diethylaniline–HCl at 500 ppm in the presence of S-9 mix for 1 h in vitro. The S-9 mix seems to convert
3.3. N,N-Diethylaniline–HCl induced SCEs in Õitro with and without S-9 mix in 1 h treatment As shown in Fig. 3, frequencies of SCEs in the cultures treated with N,N-diethylaniline–HCl without S-9 mix were significantly greater than that in the control culture. There were significant differences in the frequency of SCEs between the control and 100, 200 or 500 ppm, respectively Žall p - 0.01.. There were also significant differences in the frequency of SCEs between 100 and 500, and between 200 and 500 ppm Žall p - 0.01; Fig. 3A.. ANOVA analysis indicated that the dose of N,N-diethylaniline–HCl significantly affected SCEs Ž F s 81.72; p s 8 = 10y7 ., and the difference between individuals was significant Ž F s 29.39; p s 6 = 10y5 ; Fig. 3B.. In the presence of S-9 mix ŽFig. 4., the frequency of SCEs in the cultures treated with N,N-diethyl-
Fig. 5. Photograph of SCEs induced by N,N-diethylaniline–HCl at 500 ppm in the presence of 20% S-9 mix by 1 h in vitro treatment Ž10=100..
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N,N-diethylaniline into a stronger form that is a very effective inducer of SCEs.
4. Discussion The present results demonstrated that N,N-diethylaniline increased the frequency of SCEs with or without S-9 mix. When S-9 mix was added to the culture, SCEs significantly increased by about 3–4fold at 200 and 500 ppm of N,N-diethylaniline, suggesting that N,N-diethylaniline has strong genotoxicity. There was also a significant dose–response relationship between dose of N,N-diethylaniline and the frequency of SCEs ŽFig. 4.. The frequency of SCEs induced by N,N-diethylaniline with S-9 mix was as great as that using positive controls, e.g. ethyl methanesulfonate ŽEMS., which does not require activation, and cyclophosphamide which requires activation w8x. We found that enzymes present in the S-9 mix can convert N,N-diethylaniline from a relatively weak in vitro agent into a very strong inducer of SCEs. Gorrod et al. w10x reported that N,N-diethylaniline could be metabolized to mono-N-deethylkylation and N-oxidation compounds by rabbit liver microsomes in vitro. We speculate that N,N-diethylaniline can be converted to an alkylating agent through N-deethylkylation metabolized by the S-9 mix, which partially contributes to increases in the frequency of SCEs. It has been reported that alkylating agents such as EMS and mitomycin C w4,11x can induce increases in frequency of SCEs. However, further study is needed to confirm whether alkylating agents can be formed by treating N,N-diethylaniline in vitro with the S-9 mix. In the absence of S-9 mix, two possible mechanisms for the increased frequency of SCEs in N,N-diethylaniline-treated cultures compared to controls are: Ži. genotoxicity of N,N-diethylaniline itself; and Žii. metabolic activity of human lymphocytes. As shown in Fig. 2A and Fig. 3A, there was no significant difference in SCEs between 48 h and 1 h in vitro treatments at 100 ppm of N,N-diethylaniline without S-9 mix, suggesting that the period of treatment in vitro did not affect the frequency of SCEs in the absence of S-9 mix in the present study. The difference between individuals was not significant in 48 h treatment without S-9 mix Ž F s 3.89;
p s 0.074; Fig. 2B. and in 1 h treatment with S-9 mix Ž F s 2.73; p s 0.106; Fig. 4B., respectively, but the difference between individuals was significant in 1 h treatment without S-9 mix Ž F s 29.39; p s 6 = 10y5 ; Fig. 3B.. Differences between individuals have been reported previously w12x. However, as shown in Fig. 2B, Fig. 3B and Fig. 4B, despite significant differences between individuals in 1 h treatment without S-9 mix ŽFig. 3B., trends of dose-dependent increases of SCEs were similar among the 4 subjects, suggesting that all 4 people contribute equally to the results. We could find only two reports concerning genotoxicity of N,N-diethylaniline w2,3x. One reported that N,N-diethylaniline was negative in Ames tests using Salmonella typhimurium TA 100, TA 98 and other strains in the absence or presence of S-9 mix, but weakly positive using TA 98 in the presence of norharman and S-9 w2x. The other reported that N,Ndiethylaniline was negative in rat hepatocytes for the induction of DNA repair in vitro w3x. Our results indicated that N,N-diethylaniline is a strong genotoxic chemical. The differences between our results and others can be explained as follows: Ži. we used N,N-diethylaniline–HCl instead of N,N-diethylaniline, and the higher water-solubility of N,N-diethylaniline–HCl may improve its metabolism by the S-9 mix in vitro; Žii. differences in the methodology among SCEs, the Ames test w2x and the UDS test w3x yielded different results ŽDhillon and Dhillon w7x reported that norethisterone acetate induced positive reactions in SCEs in human lymphocytes, but negative results by the Ames test in the absence or presence of S-9 mix.; and Žiii. we used S-9 mix to metabolize N,N-diethylaniline, whereas Yoshimi et al. w3x did not use a metabolic activation system in the UDS test. In conclusion, N,N-diethylaniline had strong genotoxicity in vitro, which remains to be confirmed in vivo.
Acknowledgements We are grateful to Dr. H. Inagaki ŽDepartment of Hygiene and Public Health, Nippon Medical School. for helpful discussions.
Q. Li, M. Minamir Mutation Research 395 (1997) 151–157
References w7x w1x M.L. Richardson, D296: N,N-diethylaniline, in: M.L. Richardson ŽEd.., The Dictionary of Substances and their Effects, Vol. 2, The Royal Society of Chemistry, Cambridge, 1993, pp. 447–448. w2x H. Shimizu, N. Takemura, Mutagenicity of some aniline derivatives, in: R.R. Orford, J.W. Cowell, G.G. Jamieson, E.J. Love ŽEds.., Occupational Health in Chemical Industry, Medichem, Edmonton, AB, 1983, pp. 497–506. w3x N. Yoshimi, S. Sugie, H. Iwata, K. Niwa, H. Mori, C. Hashida, H. Shimizu, The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes, Mutation Res. 206 Ž1988. 183–191. w4x P. Perry, H.J. Evans, Cytological detection of mutagencarcinogen exposure by sister chromatid exchange, Nature 258 Ž1975. 121–125. w5x R.R. Tice, B. Lambert, K. Morimoto, A. Hollaender, A review of the international symposium on sister-chromatid exchanges: Twenty-five years of experimental research, Environ. Mutagen. 6 Ž1984. 737–752. w6x T. Rajah, Y.R. Ahuja, In vivo genotoxic effects of smoking
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w12x
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and occupational lead exposure in printing press workers, Toxicol. Lett. 76 Ž1995. 71–75. V.S. Dhillon, I.K. Dhillon, Genotoxicity evaluation of norethisterone acetate, Mutation Res. 367 Ž1996. 1–10. D.G. Stetka, S. Wolff, Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens II. In vitro test for compounds requiring metabolic activation, Mutation Res. 41 Ž1976. 343–350. Q. Li, Z.Y. Wang, H. Inagaki, Y.J. Li, M. Minami, Evaluation of contact sensitivity to formaldehyde and tetramethylthiuram monosulfide using a modified lymphocyte transformation test, Toxicology 104 Ž1995. 17–23. J.W. Gorrod, D.J. Temple, A.H. Beckett, The N- and alphaC-oxidation of N,N-dialkylanilines by rabbit liver microsomes in vitro, Xenobiotica 9 Ž1979. 17–25. M. Ohtsuru, Y. Ishii, S. Tanaka, H. Higashi, G. Kosaki, Sister chromatid exchanges in lymphocytes of cancer patients receiving mitomycin C treatment, Cancer Res. 40 Ž1980. 477–480. E.H. Park, J.K. Yung, D.H. Byun, J.Y. Lee, J.S. Lee, Baseline frequency of sister-chromatid exchanges in 142 persons of the general Korean population, Mutation Res. 268 Ž1992. 239–246.
Sister chromatid exchanges of human peripheral blood lymphocytes induced by N,N-diethylaniline in vitro Qing Li ) , Masayasu Minami Department of Hygiene and Public Health, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan Received 16 June 1997; revised 1 September 1997; accepted 1 September 1997
Abstract N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes. To evaluate its genotoxicity, we examined whether it can induce sister chromatid exchanges ŽSCEs. in human lymphocytes. We found that N,N-diethylaniline significantly increased the frequency of SCEs both in the absence and presence of S-9 mix. The SCEs from cultures treated by N,N-diethylaniline in the presence of S-9 mix displayed a marked increase which was about 5-fold greater than the control. ANOVA analyses indicated that there is a dose–response relationship between doses of N,N-diethylaniline and the frequency of SCEs, especially in the presence of S-9 mix. The results suggested that N,N-diethylaniline has genotoxicity. q 1997 Elsevier Science B.V. Keywords: Sister chromatid exchange; N,N-Diethylaniline; S-9 mix; Human lymphocyte; Genotoxicity
1. Introduction N,N-Diethylaniline is a reagent used in organic synthesis and is an important intermediate in the manufacturing of dyes w1x. It has been reported that N,N-diethylaniline was negative in Ames tests using Salmonella typhimurium TA 100, TA 98 and other strains in the absence or presence of S-9 mix, but weakly positive using TA 98 in the presence of norharman and S-9 mix w2x. On the other hand, Yoshimi et al. w3x found that N,N-diethylaniline was negative in an unscheduled DNA synthesis ŽUDS. test using rat hepatocytes. It is well known that sister chromatid exchanges ŽSCEs. can be used to evaluate
) Corresponding author. Tel.: q81 3 38222131 Ž5259.; Fax: q81 3 56853065; E-mail: [email protected]
the genotoxicity of chemicals w4–8x. To clarify its genotoxicity, we examined the frequency of SCEs in human peripheral blood lymphocytes treated with N,N-diethylaniline in vitro in the presence or absence of S-9 mix, a metabolic activation reagent.
2. Materials and methods 2.1. Reagents Because N,N-diethylaniline ŽCAS 91-66-7. is rather insoluble in water, and is not convenient for in vitro SCEs testing, we used N,N-diethylaniline–HCl, which was purchased from Wako Pure Chemical Industries, Osaka, Japan. S-9 mix ŽS-9rco-factor-C set. was purchased from Oriental Yeast Co., Tokyo,
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 6 2 - 9
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Japan. The S-9 mix in 6.7 ml contains 2 ml S-9 Ž30%. and 4.7 ml co-factor-C Ž70%., which consist of 1.34 ml of 20 mM HEPES buffer ŽpH 7.2., 0.67 ml of 50 mM MgCl 2 , 330 mM KCl, 50 mM glucose-6-phosphate, 40 mM NADPŽNa 2 . and distilled water, respectively, and was freshly prepared before each use. RPMI 1640 was from Nissui Pharmaceutical, Tokyo, Japan. Fetal bovine serum ŽFBS. was purchased from Cell Culture Laboratories, Cleveland, OH, USA, and heat-inactivated at 568C for 30 min prior to use. Glutamine, 2-mercaptoethanol Ž2ME., colcemid, Hoechst 33258 and 5X-bromodeoxyuridine ŽBrdU. were purchased from Sigma, St. Louis, MO, USA. Phytohemagglutinin-M ŽPHAM. was purchased from Gibco BRL, NY, USA. w 3 HxThymidine was purchased from ICN Biomedicals, Irvine, CA, USA. 2.2. Cytotoxicity test of N,N-diethylaniline–HCl and S-9 mix using human lymphocytes [9] The nonspecific cytotoxicity of N,N-diethylaniline–HCl and S-9 mix using human lymphocytes was examined to determine the optimal concentrations for conducting in vitro SCEs testing, where N,N-diethylaniline does not inhibit the proliferation of lymphocytes, but can induce SCEs. Lymphocytes were separated from human peripheral blood of 4 healthy subjects, then plated into flat-bottomed 96well microplates in triplicate at a density of 5 = 10 5 cellsrwell in RPMI 1640 containing 10% FBS and 5 = 10y5 M 2-ME and 3% PHA-M, and incubated in 5% CO 2 at 378C for 24 h. Then N,N-diethylaniline–HCl at 0, 5, 10, 25, 50, 100, 200, 500, 1000 and 2500 ppm ŽFig. 1A. and S-9 mix at 0%, 5%, 10%, 17%, 20% and 30% ŽFig. 1B. were added to the wells in triplicate and cultured in a total volume of 200 mlrwell at 378C in 5% CO 2 . The cells were pulsed with 0.6 mCirwell of w 3 HxThymidine Ž22.2 kBq. for the final 24 h and harvested 72 h after culture initiation. The level of w 3 HxThymidine incorporation was determined in a liquid scintillation counter. For the 1 h treatment with S-9 mix, the lymphocytes were treated with S-9 mix at 0%, 10%, 15% and 20% at 378C for 1 h. The cells were collected after centrifugation, and the pellets were washed twice with prewarmed RPMI 1640 medium
Fig. 1. Cytotoxicity of N,N-diethylaniline–HCl Župper. and S-9 mix Žmiddle. in 48 h in vitro treatment and S-9 mix in 1 h in vitro treatment Žlower. on the blastogenesis of human lymphocytes stimulated by PHA-M. Data are presented as means"SEM Ž ns 4.. The result in the absence of N,N-diethylaniline–HCl Ž0 ppm. was 61866.7"1319.4.
to remove the S-9 mix. Then the cells were re-incubated for 72 h as for the 48 h treatment. 2.3. N,N-Diethylaniline–HCl-induced SCEs in Õitro without S-9 mix in 48 h treatment [4–6] Samples of 0.5–0.6 ml of human peripheral blood obtained from 4 healthy donors Ž2 males and 2 females; 25–33 years old. were added to 5 ml RPMI 1640 containing 15% FBS, 5 = 10y5 M 2-ME and 3% PHA-M in a flask, and incubated in 5% CO 2 at 378C for 24 h. Then, BrdU at 30 mM and N,N-diethylaniline–HCl at a final concentration of 0, 10 or 100 ppm were added to the cultures and the cultures were re-incubated for 48–50 h in the dark. Colcemid treatment was conducted at a final concentration of 0.15 mgrml, 2.5–3 h before harvesting the cells. After harvesting the cultures, hypo-osmotic treatment was conducted with 0.075 M KCl at 378C. After
Q. Li, M. Minamir Mutation Research 395 (1997) 151–157
centrifugation, the pellets were fixed three times with a fixative consisting of three parts methanol and one part glacial acetic acid at room temperature, then the cells in the fixative were stored at 48C for 48–72 h. Slides were prepared and stained by Hoechst 33258 at 5 mgrml and 4% Giemsa. Frequencies of SCEs were evaluated by scoring at least 50 cells in second-division metaphase for each subject at each dose and the SCEs were indicated as SCEsrcell.
153
medium to remove N,N-diethylaniline–HCl and S-9 mix. Then the cells were added to 5 ml RPMI 1640 containing 15% FBS, 5 = 10y5 M 2-ME, 3% PHA-M and BrdU at 30 mM in a flask. Each culture was further incubated for 48–50 h in 5% CO 2 at 378C in the dark, and treated with colcemid at 2.5–3 h before harvesting. Then the cells were collected and slides were prepared as described above. 2.5. Statistical analyses
2.4. N,N-Diethylaniline–HCl-induced SCEs in Õitro with and without S-9 mix in 1 h treatment [7,8] In the metabolic activation experiments, samples of 0.5–0.6 ml of human peripheral blood obtained from the 4 healthy donors were added to 5 ml RPMI 1640 containing 15% FBS, 5 = 10y5 M 2-ME and 3% PHA-M in a flask and incubated in 5% CO 2 at 378C for 24 h. Then, the cultures were harvested and treated with N,N-diethylaniline–HCl at a final concentration of 0, 100, 200 or 500 ppm in the presence or absence of 20% S-9 mix at 378C for 1 h. The cells were collected after centrifugation, and the pellets were washed twice with prewarmed RPMI 1640
Statistical analyses were performed using ANOVA and t-test. A p-value of - 0.05 was regarded as significant.
3. Results 3.1. Optimal concentrations of N,N-diethylaniline– HCl and S-9 mix for in Õitro SCEs As shown in Fig. 1A, treatment of 1000 ppm or more of N,N-diethylaniline–HCl for 48 h in vitro had inhibitory effects on the lymphocyte transforma-
Fig. 2. Frequency of SCEs induced by N,N-diethylaniline–HCl without S-9 mix in 48 h in vitro treatment, A: averaged SCEs based on counted cell numbers pooled from 4 subjects; B: the averages of SCEs of the individual values are indicated. The numbers in A indicate counted cell numbers pooled from 4 subjects. Sub1 ŽM. and Sub2 ŽM. in B mean data from subjects 1 and 2, both of whom are males; Sub3 ŽF. and Sub4 ŽF. mean data from subjects 3 and 4, both females. Data are presented as means" SEM. ) ) p - 0.01, significantly different from control; a p - 0.01, significantly different from 10 ppm by unpaired t-test. Two-way ANOVA: Fdose s 38.03, p s 0.0004; Findividual s 3.89, p s 0.074.
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Fig. 3. Frequency of SCEs induced by N,N-diethylaniline–HCl without S-9 mix in 1 h in vitro treatment. A: averaged SCEs based on counted cell numbers pooled from 4 subjects; B: the averages of SCEs of the individual values are indicated. The numbers in A indicate counted cell numbers pooled from 4 subjects. Sub1 ŽM. and Sub2 ŽM. in B mean data from subjects 1 and 2, both of whom are males; Sub3 ŽF. and Sub4 ŽF. mean data from subjects 3 and 4, both females. Data are presented as means" SEM. ) ) p - 0.01, significantly different from control; a p - 0.01, significantly different from 100 ppm; $ p - 0.01, significantly different from 200 ppm by unpaired t-test. Two-way ANOVA: Fdose s 81.72, p s 8 = 10y7 ; Findividual s 29.39, p s 6 = 10y5 .
Fig. 4. Frequency of SCEs induced by N,N-diethylaniline–HCl with 20% S-9 mix in 1 h in vitro treatment. A: averaged SCEs based on counted cell numbers pooled from 4 subjects; B: the averages of SCEs of the individual values are indicated. The numbers in A indicate counted cell numbers pooled from 4 subjects. Sub1 ŽM. and Sub2 ŽM. in B mean data from subjects 1 and 2, both of whom are males; Sub3 ŽF. and Sub4 ŽF. mean data from subjects 3 and 4, both females. Data are presented as means" SEM. ) ) p - 0.01, significantly different from control; a p - 0.01, significantly different from 100 ppm; $ p - 0.01, significantly different from 200 ppm by unpaired t-test. Two-way ANOVA: Fdose s 121.03, p s 1 = 10y7 ; Findividual s 2.73, p s 0.106.
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tion response to PHA-M. Based on these results, 0, 10 and 100 ppm of N,N-diethylaniline–HCl were selected for in vitro SCEs testing of 48 h treatment without S-9 mix. As shown in Fig. 1B, treatment of 5% or more of S-9 mix for 48 h in vitro had inhibitory effects on the lymphocyte transformation response to PHA-M. Therefore, we did not use the S-9 mix in culture during the 48 h treatment. However, the S-9 mix at 20% for 1 h in vitro treatment was not cytotoxic to lymphocytes ŽFig. 1C.. Thus, 0, 100, 200 and 500 ppm of N,N-diethylaniline–HCl were selected for in vitro SCEs in the 1 h treatment in the presence or absence of 20% S-9 mix. 3.2. N,N-Diethylaniline–HCl induced SCEs in Õitro without S-9 mix in 48 h treatment As shown in Fig. 2, frequencies of SCEs in the cultures treated with N,N-diethylaniline–HCl without S-9 mix were significantly greater than in the control culture. There were significant differences in the frequency of SCEs between 0 and 10 or 100 ppm, and between 10 and 100 ppm Žall p - 0.01; Fig. 2A.. ANOVA analysis indicated that the dose of N,N-diethylaniline–HCl significantly affected the frequency of SCEs Ž F s 38.03; p s 0.0004., but the difference between individuals was not significant ŽFig. 2B..
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aniline–HCl significantly increased to about 5-fold the control level ŽS-9 mix only.. There were significant differences in the frequency of SCEs between the control and 100, 200 or 500 ppm, respectively Žall p - 0.01.. There were also significant differences in frequency of SCEs between 100 and 200 or 500, and between 200 and 500 ppm, respectively Žall p - 0.01; Fig. 4A.. ANOVA analysis indicated that there was a dose–response relationship between the frequency of SCEs and the dose of N,N-diethylaniline–HCl Ž F s 121.03; p s 1 = 10y7 ., but the difference between individuals was not significant ŽFig. 4B.. Differences in the frequency of SCEs between cultures with and without S-9 mix at doses of 100, 200 and 500 ppm were also significant Žall p - 0.01; Fig. 3A and Fig. 4A.. Fig. 5 shows a cell that was treated with N,N-diethylaniline–HCl at 500 ppm in the presence of S-9 mix for 1 h in vitro. The S-9 mix seems to convert
3.3. N,N-Diethylaniline–HCl induced SCEs in Õitro with and without S-9 mix in 1 h treatment As shown in Fig. 3, frequencies of SCEs in the cultures treated with N,N-diethylaniline–HCl without S-9 mix were significantly greater than that in the control culture. There were significant differences in the frequency of SCEs between the control and 100, 200 or 500 ppm, respectively Žall p - 0.01.. There were also significant differences in the frequency of SCEs between 100 and 500, and between 200 and 500 ppm Žall p - 0.01; Fig. 3A.. ANOVA analysis indicated that the dose of N,N-diethylaniline–HCl significantly affected SCEs Ž F s 81.72; p s 8 = 10y7 ., and the difference between individuals was significant Ž F s 29.39; p s 6 = 10y5 ; Fig. 3B.. In the presence of S-9 mix ŽFig. 4., the frequency of SCEs in the cultures treated with N,N-diethyl-
Fig. 5. Photograph of SCEs induced by N,N-diethylaniline–HCl at 500 ppm in the presence of 20% S-9 mix by 1 h in vitro treatment Ž10=100..
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N,N-diethylaniline into a stronger form that is a very effective inducer of SCEs.
4. Discussion The present results demonstrated that N,N-diethylaniline increased the frequency of SCEs with or without S-9 mix. When S-9 mix was added to the culture, SCEs significantly increased by about 3–4fold at 200 and 500 ppm of N,N-diethylaniline, suggesting that N,N-diethylaniline has strong genotoxicity. There was also a significant dose–response relationship between dose of N,N-diethylaniline and the frequency of SCEs ŽFig. 4.. The frequency of SCEs induced by N,N-diethylaniline with S-9 mix was as great as that using positive controls, e.g. ethyl methanesulfonate ŽEMS., which does not require activation, and cyclophosphamide which requires activation w8x. We found that enzymes present in the S-9 mix can convert N,N-diethylaniline from a relatively weak in vitro agent into a very strong inducer of SCEs. Gorrod et al. w10x reported that N,N-diethylaniline could be metabolized to mono-N-deethylkylation and N-oxidation compounds by rabbit liver microsomes in vitro. We speculate that N,N-diethylaniline can be converted to an alkylating agent through N-deethylkylation metabolized by the S-9 mix, which partially contributes to increases in the frequency of SCEs. It has been reported that alkylating agents such as EMS and mitomycin C w4,11x can induce increases in frequency of SCEs. However, further study is needed to confirm whether alkylating agents can be formed by treating N,N-diethylaniline in vitro with the S-9 mix. In the absence of S-9 mix, two possible mechanisms for the increased frequency of SCEs in N,N-diethylaniline-treated cultures compared to controls are: Ži. genotoxicity of N,N-diethylaniline itself; and Žii. metabolic activity of human lymphocytes. As shown in Fig. 2A and Fig. 3A, there was no significant difference in SCEs between 48 h and 1 h in vitro treatments at 100 ppm of N,N-diethylaniline without S-9 mix, suggesting that the period of treatment in vitro did not affect the frequency of SCEs in the absence of S-9 mix in the present study. The difference between individuals was not significant in 48 h treatment without S-9 mix Ž F s 3.89;
p s 0.074; Fig. 2B. and in 1 h treatment with S-9 mix Ž F s 2.73; p s 0.106; Fig. 4B., respectively, but the difference between individuals was significant in 1 h treatment without S-9 mix Ž F s 29.39; p s 6 = 10y5 ; Fig. 3B.. Differences between individuals have been reported previously w12x. However, as shown in Fig. 2B, Fig. 3B and Fig. 4B, despite significant differences between individuals in 1 h treatment without S-9 mix ŽFig. 3B., trends of dose-dependent increases of SCEs were similar among the 4 subjects, suggesting that all 4 people contribute equally to the results. We could find only two reports concerning genotoxicity of N,N-diethylaniline w2,3x. One reported that N,N-diethylaniline was negative in Ames tests using Salmonella typhimurium TA 100, TA 98 and other strains in the absence or presence of S-9 mix, but weakly positive using TA 98 in the presence of norharman and S-9 w2x. The other reported that N,Ndiethylaniline was negative in rat hepatocytes for the induction of DNA repair in vitro w3x. Our results indicated that N,N-diethylaniline is a strong genotoxic chemical. The differences between our results and others can be explained as follows: Ži. we used N,N-diethylaniline–HCl instead of N,N-diethylaniline, and the higher water-solubility of N,N-diethylaniline–HCl may improve its metabolism by the S-9 mix in vitro; Žii. differences in the methodology among SCEs, the Ames test w2x and the UDS test w3x yielded different results ŽDhillon and Dhillon w7x reported that norethisterone acetate induced positive reactions in SCEs in human lymphocytes, but negative results by the Ames test in the absence or presence of S-9 mix.; and Žiii. we used S-9 mix to metabolize N,N-diethylaniline, whereas Yoshimi et al. w3x did not use a metabolic activation system in the UDS test. In conclusion, N,N-diethylaniline had strong genotoxicity in vitro, which remains to be confirmed in vivo.
Acknowledgements We are grateful to Dr. H. Inagaki ŽDepartment of Hygiene and Public Health, Nippon Medical School. for helpful discussions.
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