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Jul 18, 2002 - Assessment of Genotoxicity of Benzidine and Its Structural Analogues to Human Lymphocytes Using Comet Assay. Ssu-Ching Chen,*,1 ...

TOXICOLOGICAL SCIENCES 72, 283–288 (2003) DOI: 10.1093/toxsci/kfg026 Copyright © 2003 by the Society of Toxicology

Assessment of Genotoxicity of Benzidine and Its Structural Analogues to Human Lymphocytes Using Comet Assay Ssu-Ching Chen,* ,1 Chih-Ming Kao,† Mei-Han Huang,‡ Ming-Kuei Shih,§ Ya-Lei Chen,‡ Shiao-Ping Huang,‡ and Tsan-Zon Liu¶ *Department of Medicine, China Medical College, 91 Hsueh-Shih Rd., Taichung 404, Taiwan; †Institute of Environmental Engineering, National Sun Yat-Sen University, Kaohsiung, Taiwan; ‡Department of Medical Technology, Fooyin University, Kaohsiung, Taiwan; §Department of Food and Beverage Management, National Kaohsiung Hospitality College, Kaohsiung, Taiwan; and ¶Graduate Institute of Medical Biotechnology, Chang Gung University, Taoyan, Taiwan Received July 18, 2002; accepted December 12, 2002

Benzidine has been used for the production of azo dyes, which are widely applied in the textile, paper, and leather industries (Chung et al., 1998; Haley, 1975; Morgan et al., 1994). Benzidine and its analogues can be produced as unwanted by-products via the reduction of azo dyes by intestinal and environmental microorganisms (Chung et al., 1992; Chung and Stevens, 1993). Although the production and use of benzidine have been banned in the United States, this compound is still detected in wastewater effluent discharged from the dye industries as a result of microbial actions (Chung et al., 1998, 2000). Benzidine has been recognized as a human carcinogen (Chung et al., 2000). Numerous studies indicate that occupational exposure to benzidine causes cancer of the bladder in

humans (Choudhary, 1996; Goldwater et al., 1965; Meigs et al., 1986; Piolatto et al., 1991; Shink et al., 1991; You et al., 1990; Zavon et al., 1973). Chung and Cernigilia (1992) reported that benzidine was a major mutagenic moiety of many azo dyes. The mutagenicity of benzidine and its analogues has been determined by many studies using Ames salmonella/microsome assay (Bos et al., 1982; Lazear and Louie, 1978; Prival et al., 1984; Savard and Josephy, 1986). Aromatic amines are widely used as industrial and laboratory reagents. There is a growing interest in examining quantitative structure-activity relationships (QSAR) for the genotoxicity of aromatic amines (You et al., 1993). Most of these QSAR studies used salmonella data as their measure for genotoxicity with the objective of developing predictive models for in vivo toxicity (Debnath et al., 1992; Ford and Griffin, 1992; Ford and Herman, 1992; Kalopissis, 1991). The QSAR of benzidine and its analogues, a series of aromatic amine, had also been established with the Ames test (Chung et al., 2000; Messerly et al., 1987; You et al., 1993). However, the Ames test only detects DNA damage of prokaryotic cells (bacterial cells) caused by genotoxic chemicals, whereas the comet assay (single-cell gel electrophoresis) has been shown to be capable of analyzing DNA damage in many different eukaryotic cells in vitro and in vivo (Rojas et al., 1999). Hence, in this study the comet assay was conducted to determine the genotoxicity of benzidine and its six structural analogues in human lymphocyte, and further establish the QSAR of these chemicals. Moreover, the possibility that genotoxicity of human lymphocytes resulted from benzidine via a free radical-mediated mechanism was also examined in this study.

1 To whom correspondence should be addressed. Fax: 886 –7–5254419. E-mail: [email protected]

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MATERIALS AND METHODS Chemicals. All chemicals were obtained from Sigma Chemical Company (St. Louis, MO). The chemical structures of tested compounds are shown in Figure 1. Tested chemicals were freshly prepared by dissolving in DMSO and were kept in the dark. The final concentration of DMSO was less than 1% of the reaction mixtures.

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Benzidine (BZ) and its six structural analogues (2-aminobiphenyl [2-ABP], 4-aminobiphenyl [4-ABP], 3,3ⴕ-diaminobenzidine [DABZ], 3,3ⴕ-dichlorobenzidine [DCBZ] 3,3ⴕ-dimethoxybenzidine [DEBZ], and 3,3ⴕ-dimthylbenzidine [DMBZ]) were examined for DNA damage in human lymphocytes using the alkaline comet assay. All the tested compounds showed a distinct disparity in their respective DNA-damaging capacities with an order of DABZ > BZ > DCBZ > 2-ABP > DEBZ > 4-ABP > DMBZ when lymphocytes were exposed to these chemicals for 2 h. Results show that the DNA-damaging effects of these compounds had no bearing on some physicochemical parameters including oxidation potentials, the energy differences between the lowest unoccupied molecular orbital and the highest occupied molecular orbital, ionization potentials, dipole moment, and relative partition coefficient. On the other hand, the free radical scavengers, including catalase, SOD, BHT, EDTA, and histidine exerted varying degrees of inhibitory effects on the DNA damage caused by benzidine. This suggests that genotoxicity in lymphocytes caused by benzidine proceeded via a reactive oxygen species (ROS)-mediated mechanism. Key Words: benzidine; comet assay; ROS; scavengers.

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Lymphocyte separation and treatment with tested chemicals. Human lymphocytes were isolated according to the procedures of Sierens et al. (2001) with some modifications. Blood withdrawn from a male donor (healthy nonsmoker, aged 30) was collected into Ficol-Hypague. The samples were then centrifuged at 200 ⫻ g at 25°C for 20 min. The formed lymphocyte forming a layer was directly above the Ficoll-Hypague in the vacutainer. Lymphocytes were then removed, washed with PBS (50 mM, pH 7.4), and then centrifuged for 5 min at 180 ⫻ g. The cell pellets were resuspended in 50 mM PBS solution. The cells were diluted down to a concentration of 2.5 ⫻ 10 5 cells/ml prior to use. Cells were then incubated with chemicals being tested at doses ranging from 0 to 500 ␮M at 37°C for 2 h. Cytotoxicity analysis. The procedures were conducted following the processes described in Yen et al. (2000). A volume of 0.49 ml of cell suspension treated separately with 500-␮M of each tested chemical was mixed with 10 ␮l of 0.4% trypan blue solution. Each chemical was tested for determined of cell viability after 5 min of reaction. The cells were analyzed through microscopic observation to determine the percentage of viability. Comet assay (single-cell gel electrophoresis). The comet assay was performed under alkaline conditions following the method of Singh et al. (1988) with some modifications. Isolated lymphocytes treated with each tested chemical at the indicated doses were incubated in the rotary incubator (37°C, 200 rpm) for 2 h. Conventional microscope slides were dipped into a solution of 85 ␮l of 0.5% of normal melting point agarose (NMP) and 0.5% low melting point agarose (LMP) in PBS (pH 7.4), and allowed to dry on a flat surface at room temperature. Ten ␮l of cell suspension (2.5 ⫻ 10 5 cells/ml) were gently mixed with 75 ␮l of 0.5% (w/v) of LMP in PBS (pH 7.4). Seventy-five ␮l of this suspension was rapidly layered onto the slides precoated with mixtures of 0.5% NMP and 0.5% LMP, and covered with a coverslip. The slides were maintained at 4°C for 5 min, the coverslip was removed, and cells were immersed in a freshly made lysis solution (2.5 M of NaCl, 100 mM Na 2 EDTA, 10 mM Tris and 1% (v/v) of Triton X-100 at pH 10) at 4°C for 10 min. The slides were then placed in a double row in a 260-mm wide horizontal electrophoresis tank containing 0.3 M NaOH and 1 mM Na 2EDTA for 10 min. Thereafter, the electrophoresis (30 V, 300 mA) was conducted for 15 min at 4°C. After the electrophoresis, the slides were then soaked in a cold neutralizing buffer (400 mM of Tris buffer, pH 7.5) at 4°C for 10 min. Slides were

dried in methanol for 5 min, and stored in a low humidity environment before staining with 40 ␮l of PI (2.5 ␮g/ml). Quantification of the comet assay. One hundred comets on each slide were scored visually according to the relative intensity of fluorescence in the tail. An intensity score from class 0 (undamaged) to class 4 (severely damaged) was assigned to each cell, based on the procedures in Visvardis et al. (1997). Thus, the total score for the 100 comets could range from 0 to 400 because the 100 cells were observed individually in each comet assay. Figure 2 illustrates examples of the visual scoring classification for lymphocytes: class 0 (Fig. 2A), class 3 (Fig. 2B), and class 4 (Fig. 2C). Effects of scavengers of ROS (reactive oxygen species) on DNA damage of lymphocytes induced by benzidine. Isolated lymphocytes were mixed with 500 ␮M benzidine and some scavengers (catalase, SOD, BHT, EDTA, and histidine) at indicated doses. Subsequently, these cells were incubated at 37°C for 2 h. Comet assay was then conducted following the procedures described above. Statistical analysis. A nonparametric test (Kruskel-Wallis) was used to evaluate differences in the distribution of DNA damage. For all statistical analyses, a level of 0.05 was used as the lower bound to determine the significance of the variation.

RESULTS

In this study, the cytoxicity of benzidine and its analogues in human lymphocytes was evaluated. The cell viability was greater than 94% when cells were treated with the tested chemicals (except for 4-ABZ) at 500 ␮M and incubated at 37°C for 2 h (Table 1). Although the viability of cells treated with 500 ␮M 4-ABZ was only 77%, the genotoxicity of this chemical could still be detected if a cutoff value of 75% viability (Henderson et al., 1998) is used as the criterion of the positive response. Table 1 summarizes the results of DNA damage in human lymphocytes treated with varying concen-

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FIG. 1. Chemical structures of benzidine and its analogues.

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GENOTOXICITY OF BENZIDINE AND ITS ANALOGUES

compared with other studies, which applied the Ames test for the development of QSAR. Regression line analysis was used to evaluate the correlation between the level of DNA damage and each physicochemical parameter of the tested compounds. However, no significant correlation was observed. Since benzidine caused DNA damage in lymphocytes, the involvement of free radicals in DNA damage caused by benzidine was investigated in this study. Effects of exogenous free radical scavengers on lymphocytes treated with 500-␮M benzidine were analyzed using the comet assay (Table 3). Results show that catalase (the scavenger of H 2O 2) and superoxide dismutase (SOD, the scavenger of superoxide anions) decreased the DNA damage caused by benzidine by 93% and 86%, respectively. Furthermore, denatured catalase and SOD were found to be ineffective on the inhibition of DNA damage.

Chemical DMSO H 2O 2 2-Aminobiphenyl FIG. 2. Comet images of lymphocytes (A–C), illustrating the visual scoring classification; class 0 (A), class 3 (B), and class 4 (C).

trations of tested chemicals at 37°C for 2 h, as measured by comet assay. Results indicate that the positive group (cells pretreated with 1 ␮M H 2O 2) showed maximum levels of DNA damage score (300 ⫾ 28), while the negative control (1% DMSO as solvent for each tested chemical) revealed very low DNA damage score (17 ⫾ 5). At a concentration of 100 ␮M, the tested chemicals, except for DMBZ, exhibited significant DNA damage when compared to the negative control group (p ⬍ 0.05). All 7 tested chemicals revealed the genotoxicity to lymphocytes in a dose-dependent manner. The potencies of DNA damage induced by tested chemicals were, as expressed by the slope (DNA damage score/[␮M]) of the linear portion of the dose-response curve derived from the data of Table 1, shown in Table 2. Results show that the order of severity of DNA damage caused by these tested chemicals was DABZ (0.51) ⬎ BZ (0.46) ⬎ DCBZ (0.33) ⬎ 2-ABZ (0.32) ⬎ DEBZ (0.12) ⬎ 4-ABZ (0.08) ⬎ DMBZ (0.03), respectively. The experimental oxidation potentials, the partition coefficient (K HPLC) and basicity (pKa), the energy difference between the LUMO and HOMO (⌬E), ionization potential (IP), and dipole moment (␮) cited from our previous study (Chung et al., 2000) were applied to establish the model of quantitative structureactivity relationship (QSAR) derived from the comet assay. The application of the comet assay in this study is quite unique

Dose (␮M)

Cell viability (%)

DNA damage score

— 1

97 96

17 ⫾ 5 300 ⫾ 28

100 200 500

ND ND 94

27 ⫾ 16 37 ⫾ 14 172 ⫾ 34

100 200 500

ND ND 74

20 ⫾ 4 19 ⫾ 15 56 ⫾ 22

100 200 500

ND ND 95

52 ⫾ 11 102 ⫾ 25 244 ⫾ 11

100 200 500

ND ND 96

146 ⫾ 26 255 ⫾ 29 300 ⫾ 22

20 40 100

ND ND 97

26 ⫾ 2 46 ⫾ 8 51 ⫾ 1

100 200 500

ND ND 94

19 ⫾ 9 71 ⫾ 11 77 ⫾ 11

100 200 500

ND ND 95

10 ⫾ 6 21 ⫾ 18 29 ⫾ 22

4-Aminobiphenyl

Benzidine

3,3⬘-Diaminobenzidine

3,3⬘-Dichlorobenzidine a

3,3⬘-Dimethoxybenzidine

3,3⬘-Dimethylbenzidine

Note. All chemicals were dissolved in 1% of DMSO (control group). Mean DNA damage scores in arbitrary units (⫾ SD) were calculated from the respective values of at least three treatments (100 cells/slide, duplicate slides/ treatment). ND, not done. a The upper dose was limited to 100 ␮M due to its low solubility in 1% of DMSO.

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TABLE 1 Responses of Lymphocytes to Different Doses of Benzidine and Its Analogues

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TABLE 2 QSAR Parameters for Benzidine and Its Analogues Physicochemical parameters Chemicals

Oxidation potential

K HPLC

pKa

⌬E (electrovolt)

IP (electrovolt)

␮ (Debye)

Potency of DNA damage a

2-Aminobiphenyl 4-Aminobiphenyl Benzidine 3,3⬘-Diaminobenzidine 3,3⬘-Dichlorobenzidine-2HCl 3,3⬘-Dimethoxybenzidine 3,3⬘-Dimethylbenzidine

0.79 0.69 0.36, 0.63 0.48 0.60 0.34, 0.54 0.35, 0.60

0.51 0.54 0 0.20 0.77 0.20 0.30

3.83 4.22 4.80 4.34 2.16 4.20 5.16

5.15 4.54 4.43 3.83 5.09 5.89 4.34

11.68 11.76 10.80 10.18 11.61 11.72 10.43

1.45 1.79 1.86 2.93 0.17 2.17 2.26

0.32 (0.96) b 0.08 (0.94) 0.46 (1.00) 0.51 (0.88) 0.33 (0.89) 0.12 (0.85) 0.03 (0.84)

Note. The physicochemical parameters are cited from Chung et al., 2000. Potency of DNA damage: calculated slopes by linear regression analysis for initial portion of the dose-response curves from Table 1; potency values are arbitrary units/␮M. These values (r) mean the potency of linear regression.

DISCUSSION

Numerous methods have been reported for determining the genotoxicity of benzidine using some biomarkers such as chromosomal aberration (Talaska et al., 1987; You et al., 1993),

TABLE 3 Action of Some Chemicals (the Scavengers of Free Radicals) against DNA Damage Induced by Benzidine Chemical

Dose

DNA damage score

Benzidine Catalase (heated) Catalase SOD (heated) SOD BHT EDTA Histidine

500 ␮M 100 U/ml 100 U/ml 100 U/ml 100 U/ml 0.1 mM 10 mM 15 mM

250 ⫾ 10 168 ⫾ 25 17 ⫾ 2 128 ⫾ 36 33 ⫾ 4 27 ⫾ 4 19 ⫾ 4 10 ⫾ 6

sister-chromatid exchange (Grady et al., 1986; Gupta et al., 1988; Parodi et al., 1983; Willems and de Raat, 1985), micronuclei test (Chiak and Vontorkova, 1987; Mirkova, 1990), and DNA adducts (Martin et al., 1982). However, Betti et al. (1994) reported that cytogenetic methods (chromosomal aberrations, micronuclei and sister-chromatid exchange) are only limited to proliferating cells. Moreover, these methods, used for detecting genotoxic agents, are often tissue- and cell typespecific. Furthermore, the uses of DNA adducts as biomarkers cannot provide any evaluation of the distribution of DNA damage among the individual cells. Conversely, the comet assay is a sensitive, reliable, and rapid method for the detection of DNA double- and singlestrand breaks, alkali-labile sites, and delayed repair-site detection in eukaryotic individual cells (Rojas, 1999). Thus, comet assay was used to detect the DNA damage caused by benzidine and its six structural analogues. In this study, degrees of DNA damage in the lymphocytes, caused by these 7 different chemicals, were observed (Table 1). Among all the tested chemicals, DMBZ caused the least DNA damage in lymphocytes, whereas DABZ was found to be the most potent agent causing DNA damage of lymphocytes. Like the results of the genotoxicity of benzidine and its analogues determined by the Ames test (Chung et al., 2000), different groups attached to benzidine were able to enhance or decrease the genotoxicity of benzidine to lymphocytes in this study. The ranked order of the DNAdamaging capacities of benzidine and its analogues towards lymphocytes was not parallel with the results of the genotoxicity of these chemicals determined by the Ames test. For example, DCBZ exhibited the greatest mutagenicity toward TA 98 with metabolic activation among benzidine and its analogues using the Ames test (Chung et al., 2000), whereas DABZ was the strongest agent among the same chemicals to cause DNA damage of lymphocytes in this study. The addition of a methoxy group to the benzidine molecules decreases the levels of DNA damage (Table 1). On the contrary, the mutagenic potency in DEBZ has been shown to be greater than that

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Results suggest that H 2O 2 and superoxide anions were involved in the benzidine-induced DNA damage. EDTA, an effective chelator for metal ions, inhibited the DNA damage by 92%, indicating that metal ions might play an important role in DNA damage of lymphocytes treated with benzidine. 89% of DNA damage was reversed by the addition of BHT, the chainbreaking scavenger, to the reaction mixtures containing lymphocytes treated with benzidine. This would be able to explain the fact that lipid peroxidation in cells treated with benzidine was inhibited by BHT. Ethanol effects on DNA damage were also evaluated, because it was used as a solvent (0.6%) for BHT. Results show that using ethanol alone would not mitigate the DNA damage (data not shown) that was almost inhibited by histidine, the scavenger of the hydroxyl radical or single oxygen. This suggests that the generation of hydroxyl radical or single oxygen in benzidine-treated lymphocytes might result in the DNA damage of cells.

GENOTOXICITY OF BENZIDINE AND ITS ANALOGUES

structure-activity relationships of these chemicals are still necessary. The generation of the superoxide anion radical was observed during the incubation of benzidine with NADPH-supplemented rat liver microsomes (Manno et al., 1985). Josephy (1986) depicted that the free radical oxidation pathway of benzidine metabolism resulted in the formation of reactive electrophilic species and the attack on DNA to form DNA adducts. The possibility that DNA damage induced by benzidine in lymphocytes resulted from the generation of free radicals was envisaged. All well-known free radical scavengers exerted their inhibitory effects on the levels of DNA damage caused by benzidine. This indicates that the action of ROS generated from benzidine and its analogues would contribute to DNA breakage. Whether DNA damage by other benzidine analogues mediated through a ROS-dependent mechanism awaits further investigation. Furthermore, detailed mechanisms of DNA damage caused by the intrusion of benzidine remain to be elucidated. Results from this study provide us a more fundamental understanding and insight into the effects of benzidine and its analogues on DNA damage in human lymphocytes. Based on these findings, the comet assay is believed to be a more sound technique for the determination of the genotoxicity of toxic chemicals. REFERENCES Ashby, J., Paton, D., Lefevre, P. A., Styles, J. A., and Rose, F. L. (1982). Evaluation of two suggested methods of deactivating organic carcinogens by molecular modification. Carcinogenesis 3, 1277–1282. Betti, C., Davini, T., Giannessi, L., Loprieno, N., and Barale, R. (1994). Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects. Mutat. Res. 307, 323–333. Bos, R. P., Van Dooren, R., de Hurk, E. Y.-V., Gemert, P. J. L., and Henderson, P. T. (1982). Comparison of the mutagenicities of 4-aminobiphenyl and benzidine in Salmonella/microsome, Salmonella/hepatocyte, and host-mediated assays. Mutat. Res. 93, 317–325. Chiak, R., and Vontorkova, M. (1987). Benzidine and 3,3⬘-dichlorobenzidine (DCB) induce micronuclei in the bone marrow and the fetal liver of mice after gavage. Mutagenesis 2, 267–269. Choudhary, G. (1996). Human health perspectives on the environmental exposure to benzidine: A review. Chemosphere 32, 267–291. Chung, K.-T., and Cerniglia, C. E. (1992). Mutagenicity of azo dyes: Structure-activity relationships. Mutat. Res. 277, 201–220. Chung K.-T., Chen, S.-C., Wong, T. Y., Li, Y.-S., Wei, C.-I., and Chou, M. W. (2000). Mutagenicity studies of benzidine and its analogs: Structure-activity relationships. Toxicol. Sci. 56, 351–356. Chung, K.-T., Chen, S.-C., Wong, T. Y., and Wei, C.-I. (1998). Effects of benzidine and benzidine analogues on growth of bacteria including Azotobacter vinelandii. Environ. Toxicol. Chem. 17, 271–275. Chung, K.-T., and Stevens, S. E., Jr. (1993). Degradation of azo dyes by environmental microorganisms and helminthes. Environ. Toxicol. Chem. 2, 2121–2132. Chung, K.-T., Stevens, S. E., Jr., and Cerniglia, C. E. (1992). The reduction of azo dyes by the intestinal microflora. Crit. Rev. Microbiol. 18, 175–190. Debnath, A. K., de Comoadre, R. L., and Hansch, C. (1992). Mutagenicity of

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in benzidine (Chung et al., 2000). Isomers of aminobiphenyl and 2- and 4-aminobiphenyl exhibited different levels of DNA damage to lymphocytes. Results (Table 2) show that 2-aminobiphenyl caused the greater DNA damage in lymphocytes than 4-aminobiphenyl. However, results from our previous study show that the mutagenicity in 4-aminobiphenyl is stronger when compared to that in 2-aminobiphenyl (Chung et al., 2000). The addition of 4 methyl groups to the benzidine molecule 3,3⬘,5,5⬘-tetramethylbenzidine abolishes the mutagenic activity completely, as shown in our previous paper (Chung et al., 2000). The chemical, 3,3⬘-5,5⬘-tetramethylbenzidine is not a carcinogen and is concurrently used as an industrial substituent for benzidine (Ashby et al., 1982). In this study, the genotoxicity of 3,3⬘-5,5⬘-tetramethylbenzidine to lymphocyte was not evaluated, due to its limited solubility in 1% DMSO. Less than 1% of DMSO was used for the chemical dissolution; otherwise, the false induction of DNA damage caused by high concentrations of DMSO was observed (data not shown). Noticeably, most benzidine and its analogues require metabolic activation to become genotoxic in the Ames test (Chung et al., 2000). However, this was not observed in the comet assay in lymphocytes, because they are metabolically competent. Due to the different responses of eukaryotic and prokaryotic cells to genotoxic compounds, the development of a nonbacterial screening assay (e.g., comet assay) is required. In our previous paper (Chung et al., 2000), the structureactivity relationship in benzidine and its analogues, using the Ames test, was established using the comet assay, which made the prediction of genotoxicity more realistic. You et al. (1993) confirmed that mutagenicity of benzidine and its derivatives established in TA 98, TA 98/1,8-DNP 6, and TA 100 strains correlate with the energy of the lowest unoccupied molecular orbital (E LUMO) and pKa values. The pKa of a substituted aromatic amine is influenced by the electron donating/withdrawing ability of its substituent (Chung et al., 2000). Messerly et al. (1987) also reported that the mutagenicities of 3,3⬘disubstituted compounds (dimethoxybenzidine, diaminobenzidine, and dichlorobenzidine) are inversely linearly proportional to their pKa values in TA 98 and TA 100 strains. You et al. (1993) indicated that pKa might influence the mutagenic ability of nitrenium ions, the ultimate products of benzidine metabolism (Josephy, 1986). In our previous study, positive relationships between mutagenicity of benzidine and its analogues and some physicochemical parameters were not observed (Chung et al., 2000). Similarly, this correlation was not observed in this study. Controversial QSAR results on the genotoxicity of benzidine and its analogues by the Ames test have been reported in many studies, although information regarding the QSAR of benzidine and its analogues obtained by comet assay cannot be found. The discrepancy in QSAR might be partly due to the levels of homogeneity in tested compounds and the number of compounds tested. Thus, application of more appropriate calculation models and analysis of more compounds would provide clearer and defensible correlation results. Further

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