* 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
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ABSTRACT |
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Key Words: benzidine; comet assay; ROS; scavengers.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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-µM of each tested chemical was mixed with 10 µl 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 µl 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 µl of cell suspension (2.5 x 105 cells/ml) were gently mixed with 75 µl of 0.5% (w/v) of LMP in PBS (pH 7.4). Seventy-five µl 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 Na2 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 Na2EDTA 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 µl of PI (2.5 µg/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
).
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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.
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RESULTS |
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DISCUSSION |
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Conversely, the comet assay is a sensitive, reliable, and rapid method for the detection of DNA double- and single-strand 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 DNA-damaging 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 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 structure-activity 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-DNP6, and TA 100 strains correlate with the energy of the lowest unoccupied molecular orbital (ELUMO) 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 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.
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NOTES |
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