DNA Damage Induced by 3,3'-Dimethoxybenzidine in Liver and Urinary Bladder Cells of Rats and Humans

A. Martelli, L. Robbiano, R. Carrozzino, C. Porta Puglia, F. Mattioli, M. Angiola and G. Brambilla1

Department of Internal Medicine, Division of Clinical Pharmacology and Toxicology, University of Genoa, Viale Benedetto XV, 2, I-16132 Genoa, Italy

Received June 1, 1999; accepted August 19, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3,3'-Dimethoxybenzidine (DMB), a congener of benzidine used in the dye industry and previously found to be carcinogenic in rats, was evaluated for its genotoxic activity in primary cultures of rat and human hepatocytes and of cells from human urinary bladder mucosa, as well as in liver and bladder mucosa of intact rats. A similar modest dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic DMB concentrations ranging from 56 to 180 µM. Replicating rat hepatocytes displayed a modest increase in the frequency of micronucleated cells after a 48-h exposure to 100 and 180 µM concentrations. In primary cultures of human urinary bladder mucosa cells exposed for 20 h to 100 and 180 µM DMB, the Comet assay revealed a clear-cut increase of DNA fragmentation. In rats given one-half LD50 of DMB as a single oral dose, the GSH level was reduced in both the liver and urinary bladder mucosa, whereas DNA fragmentation was detected only in the bladder mucosa. Taken as a whole, these results suggest that DMB should be considered a potentially genotoxic chemical in both rats and humans; the selective effect on the rat urinary bladder might be the consequence of pharmacokinetic behavior.

Key Words: 3–3'-dimethoxybenzidine; DNA fragmentation; micronucleus test; rat; human; hepatocytes; bladder cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3,3'-Dimethoxybenzidine (DMB; o-dianisidine), a congener of benzidine also employed in the dye industry, is classified by the International Agency for Research on Cancer, on the basis of sufficient evidence for carcinogenicity to rodents (IARC, 1987a), as being among those chemicals possibly carcinogenic to humans. Evidence for this is still inadequate. Following oral administration, DMB produced tumors in rats at various sites, including the bladder and the liver (IARC, 1974; Morgan et al., 1994Go). In vitro, DMB has been found to be mutagenic to bacteria in the presence of metabolic activation, and to induce sister chromatid exchanges in Chinese hamster cells and DNA repair synthesis in primary rat hepatocytes (IARC, 1987). You et al. (1993) confirmed the mutagenicity of DMB in the Salmonella typhimurium strain TA98 with S9 activation, and observed a significant increase of chromosomal aberrations in the bone-marrow cells of mice given a single ip injection of this amine. No data are available on genetic and related effects of DMB in humans, but it has been found in the urine of exposed workers (IARC, 1974). No epidemiological data on the occurrence of cancer in workers exposed to DMB alone appear in the literature, but it should be considered that exposure to this chemical and related amines such as benzidine has been strongly associated with the occurrence of urinary bladder cancer in man (IARC, 1987a). Moreover, it should be taken into account that DMB is used as an intermediate in the production of several azo-dyes, and that studies on the metabolism and disposition of dyes derived from DMB have established that, following ingestion, the azo linkages of these compounds are reduced, probably by bacteria in the intestine, to release the parent amine (Morgan et al., 1994Go).

In this report, we present the results of experiments carried out to evaluate the capability of DMB to induce:

The rationale for the choice of the above-mentioned assays was based not only on the absence in the literature of studies performed on DMB, but also on the following strategy: the induction of DNA fragmentation in primary rat and human hepatocytes was investigated to verify possible interspecies differences in the metabolic activation of DMB, and the evaluation of the same endpoint in primary cultures from human urinary bladder mucosa in order to establish whether metabolic activation of this chemical also takes place in the tissue that might be the target of its carcinogenic activity. The micronucleus test in primary rat hepatocytes was suggested by the knowledge that some genotoxic chemicals which do not cause DNA lesions (Nicolini et al., 1982Go; Sina et al., 1983Go) induce chromosomal damage (Ishidate et al., 1988Go; Mavournin et al., 1990Go) and vice versa. The induction of DNA fragmentation in liver and urinary bladder mucosa of intact rats was examined to assess in vivo the possible influence of pharmacokinetic events (absorption, distribution and elimination) not reproducible in vitro.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and animals.
3,3'-Dimethoxybenzidine (CAS no. 119–90–4), collagenase type IV, Williams' medium E (WME), Eagle's minimal essential medium (MEM), diethylmaleate, epidermal growth factor (EGF), and N-nitrosodimethylamine (NDMA) were purchased from Sigma Chimica (Milan, Italy). Insulin and fetal bovine serum were from Boehringer Mannheim Italia (Milan, Italy). All other chemicals were reagent grade.

Male Sprague-Dawley rats (200–250 g; about 2 months old) were purchased from Harlan Nossan (Correzzana, Italy) and allowed to acclimate in our animal facility for 1 week before use. They were housed in an air-conditioned room (22 ± 2°C, relative humidity 50 ± 10%) with a 12-h light/dark cycle, kept on bedding chips (Harlan Nossan), and had free access to tap water and rat chow (TRM, Harlan Nossan).

In Vitro Studies
Cytotoxicity and DNA-damaging activity.
Rat hepatocytes were isolated by collagenase perfusion as described by Williams (1977). The percentage of viable cells, as measured by the trypan blue-exclusion method, ranged from 80 to 90%. Fresh human liver was obtained from discarded surgical material during the course of prescribed surgery. Donor HH122 was a 46-year-old male and donor HH130 a 70-year-old female; both of them underwent surgery for metastases of colon carcinoma.

Hepatocytes were isolated from apparently healthy tissue as described by Strom et al. (1982). The proportion of viable cells after perfusion was 84% in donor HH122, and 91% in donor HH 130. Isolated rat or human hepatocytes were suspended in WME supplemented with 10% fetal bovine serum and gentamicin (50 µg/ml). Aliquots of these suspensions were plated in 60-mm, uncoated dishes (2 x 106 cells/dish) for the DNA fragmentation assay and in 35-mm dishes coated with rat tail collagen (1 x 106 cells/dish) for determination of cytotoxicity. After an attachment period of 3 h at 37°C in an atmosphere of 95% air-5% CO2, hepatocytes were washed and incubated for 20 h with serial concentrations of the test compound in serum-free WME. The medium containing DMB was freshly prepared from a stock solution of dimethylsulfoxide (DMSO); the maximum DMSO concentration (0.5%) was present in control cultures. NDMA was directly dissolved in the medium. At the end of treatment, cells were immediately assayed for cytotoxicity by trypan blue exclusion, and for DNA fragmentation by the alkaline technique, as previously reported (Brambilla et al., 1989Go). This technique measures the rate at which DNA single strands, released in alkali, are able to pass through a filter, and the kinetics of elution provides a sensitive measure of the frequency of DNA single-strand breaks and alkali-labile sites (Kohn et al., 1976Go). The DNA content of the 10 eluted fractions, and that remaining on the filter, were determined by the microfluorimetric procedure described by Cesarone et al. (1979). The DNA elution rate constant, K (ml–1), was calculated from the equation K = (-ln FR)/V, were FR is the fraction of DNA retained on the filter and V is the eluting volume (13 ml). As a first approximation, K is directly proportional to the frequency of DNA breaks (Kohn et al., 1976Go). Data are expressed both as percentage of DNA eluted from the filter and as relative elution rate (Kt/Kc), where Kt is the elution rate constant of DNA from treated cultures and Kc the elution rate constant of DNA from control cultures. Because the rate of DNA alkaline elution can be affected by variations in buffer composition, pH and temperature, the ratio Kt/Kc was calculated using values of Kt and Kc from the same run. Values of Kt/Kc from different runs were then averaged. Statistical analysis was performed by using the nonparametric Wilcoxon 2-sample 2-tailed test (Rümke and De Jonge, 1964Go).

Fresh human urinary bladder tissue was obtained from discarded surgical material during the course of prescribed surgery. Donors 1, 2, 3, and 4 were males of ages ranging from 55 to 60 years; donor 5 was a 64-year-old female; all of them underwent surgery for tumors of the urinary bladder. A fragment of apparently healthy tissue was incubated for 20 h with DMB in serum-free WME. At the end of treatment the epithelial mucosa of the bladder was carefully stripped mechanically from the underlying muscle layer and minced into pieces of about 1-mm3. The fragments were incubated in 20 ml of 0.125% trypsin/0.05% EDTA in Hanks balanced salt solution (HBSS) at 37°C with gentle stirring. After 30 min, tissue pieces were allowed to settle 3–5 min and the incubation medium was aspirated and discarded, as it contained mostly red blood cells. Fresh trypsin/EDTA solution was added and the fragments were incubated for an additional 30 min. After settling of tissue pieces, disaggregated epithelial mucosa cells were present in the supernatant. Trypsin in the aspirate was inactivated by adding 10 ml of bovine serum. Residual tissue was trypsinized further for an additional 30 min, the solution aspirated, and the enzyme inactivated as above. The cell suspensions were then pooled, centrifuged at 1000 rpm for 5 min; the cell pellet resuspendend in an adequate aliquot of WME, and immediately assayed for cytotoxicity by trypan-blue exclusion and for DNA fragmentation by the Comet assay, as described by Singh et al. (1988). We used this assay instead of alkaline elution because of the low number of cells harvested from the fragments of urinary bladder. Ten µl of cellular suspension (>10,000 cells) were mixed with 75 µl of low-melting-point agarose at 37°C and then added to normal-melting-point, agarose-coated microscope slides. The slides were immersed in cold lysing solution for 1 h, and then placed in an electrophoresis tray with an alkaline solution (300 mM NaOH, 1 mM Na2EDTA; pH 13) for 20 min to allow DNA to unwind. Electrophoresis was conducted at room temperature for 20 min at 25 V and 300 mA. The slides were washed, stained with ethidium bromide, and examined at 400x magnification using a fluorescence microscope. Images of 50 randomly chosen cells from 2 slides were examined for each donor, and the extent of DNA damage was quantified by measuring the tail length and the tail moment with computerized image analysis (Hellmann et al., 1995Go)

Micronucleus assay.
The procedure employed for determination of micronuclei induction in primary cultures of replicating rat hepatocytes was essentially that described by Hwang et al. (1993). Hepatocytes, isolated as described in the preceding paragraph, were suspended in MEM (0.4 mM Ca++) supplemented with 10% fetal bovine serum, non-essential amino acids, and 50 µg/ml gentamicin, and plated in 60-mm dishes coated with rat tail collagen (104 cells/cm2). At the end of a 3-h attachment interval, the medium was removed and cultures were re-fed with serum-free medium supplemented with insulin (10–7 M), dimethylsulfoxide (2%), EGF (20 ng/ml), and serial concentrations of the test compound. After a 48-h incubation, the cultures were washed and re-fed with medium supplemented only with insulin (10–7 M) and EGF (20 ng/ml). Incubation was stopped 48 h later by washing the cultures with cold phosphate-buffered saline (PBS); hepatocytes were then exposed for 7 min to the hypotonic shock solution (0.01 M KCl) and fixed with methanol:formalin:acetic acid (85:10:5). Micronucleated cells were scored after staining with Feulgen, at 1200x magnification.

In Vivo Studies
DNA fragmentation in the rat liver and urinary bladder mucosa.
Rats were fasted overnight before treatment and given, by gastric intubation, 960 mg/kg of DMB (1/2 LD50) in 0.01 ml/g body weight distilled water with 0.5% carboxymethylcellulose as suspending agent. The controls received an equal volume of the vehicle. Rats were sacrificed for the evaluation of DNA fragmentation 4 h after treatment. Liver and urinary bladder were quickly removed and then separately processed as follows in Merchant's solution (0.14 M NaCl, 1.47 mM KH2PO4, 2.7 mM KCl, 8.0 mM Na2HPO4, 0.53 mM Na2EDTA; pH 7.4) in order to obtain cell suspensions. The liver was briefly minced and then homogenized using a loosely fitting Potter-Elvehjem homogenizer; after sedimentation of the large tissue fragments, single cells remaining in the supernatant were pelleted at 50 x g for 4 min. This procedure, routinely used to obtain viable liver cells for the alkaline elution assay (Brambilla et al., 1978Go), is similar to that used by Sasaki et al., (1997) for the Comet assay. The collection of bladder epithelial cells was performed as described by Morimoto et al. (1989). In brief, the bladder was everted, ligated, and inflated with cold Merchant's solution, and the epithelium was collected by scraping gently with the edge of a cover slip. Both liver and bladder cells were finally resuspended in a suitable volume of Merchant's solution and counted in a hemocytometer. The fraction of viable cells, determined with the trypan-blue-exclusion method, was, in each animal and for both organs, higher than 80%. Due to the low number of cells harvested from the urinary bladder, the Comet assay was used to evaluate DNA fragmentation. The procedure used was the same as described for the in vitro assays. For the comparison of the pooled data between control and each dose level, we used ANOVA followed by Dunnet's test.

The GSH content of the liver and of the urinary bladder mucosa were determined in 0.1-ml deproteinized, cytosol containing 5% trichloroacetic acid, according to Ellmann (1959), with 2 ml 0.6 mM 5,5'-dithio-bis (2-nitrobenzoic acid) and 0.9 ml 0.2 M Tris–HCl buffer. Diethylmaleate, administered ip at a dose of 700 mg/kg, was used as positive control of GSH depletion.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity and DNA-damaging activity.
A preliminary cytotoxicity assay provided evidence that a 20-h exposure to DMB concentrations, ranging from 56 to 180 µM, produced a modest but dose-dependent reduction in the fraction of viable trypan blue-excluding primary rat hepatocytes. At 180 µM, the highest concentration soluble in culture medium, the average fraction of viable cells was 83% of that observed in controls.

Data provided by the DNA damage/alkaline elution assay performed in primary rat hepatocytes (Table 1Go) indicate that a 20-h exposure to the same DMB concentrations produced a modest but statistically significant and dose-dependent increase of DNA elution rate, i.e., of the frequency of DNA single-strand breaks and/or alkali-labile sites. Under the same experimental conditions, DMB induced a similar dose-dependent degree of DNA fragmentation in primary cultures from a male and a female human donor (Table 2Go). NDMA (5 mM), used as positive control, elicited the expected amount of DNA fragmentation in both rat and human hepatocytes, thus demonstrating their metabolic competence.


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TABLE 1 DNA Fragmentation in Primary Cultures of Rat Hepatocytes after 20-h Exposure to DMB
 

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TABLE 2 DNA Fragmentation in Primary Cultures of Human Hepatocytes after 20-h Exposure to DMB.
 
Data provided by the DNA damage/Comet assay performed in primary cultures of cells from human urinary bladder mucosa exposed to DMB for 20 h are listed in Table 3Go. Testing only in human cells, and the use of the Comet assay were due to the difficulty in obtaining a sufficient number of cells from the rat urinary bladder and to the low yields of cells isolated from the fragments of human bladder, respectively. Migration was measured for 50 nuclei for donor and dose-point. In primary cultures from 4 male and 1 female donors, 180 µM DMB consistently produced a clear-cut increase of both tail length and tail moment, indicative of DNA fragmentation. At the 100 µM concentration, tested only in cultures from two donors, DNA damage was of similar degree.


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TABLE 3 Damage of Nuclear DNA in Primary Cultures of Human Urinary Bladder Mucosa Exposed to DMB for 20 h
 
Micronucleus assay.
Induction of micronuclei after a 48-h exposure to 56, 100, or 180 µM DMB was investigated in primary rat hepatocytes stimulated to proliferate with low Ca++ concentration, insulin, and EGF. Data listed in Table 4Go show that a statistically significant increase in the frequency of micronucleated hepatocytes was detected in only 1 of 3 independent experiments and only at the highest concentration tested. However, it appears from the pooled data that the effect was dose-dependent, and that the average frequency of micronucleated hepatocytes was 4.9-fold higher than in controls at 180 µM DMB. NDMA, used as positive control, induced the expected positive response.


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TABLE 4 Frequencies of Micronucleated Cells in Primary Cultures of Replicating Rat Hepatocytes after 48-h Exposure to DMB
 
DNA fragmentation in vivo.
None of the rats treated with the single oral dose of 960 mg/kg DMB died or showed marked signs of toxicity. Table 5Go shows, as indexes of DNA damage, both tail length and tail moment of DNA migration of nuclei from the liver and urinary bladder mucosa of each rat, as well as the means obtained from the pooled data. Migration was measured for 50 nuclei per rat and the statistical analysis was performed using the pooled 150 nuclei of the 3 rats. The treatment with DMB was associated with a statistically significant increase in the migration of DNA from the urinary bladder mucosa, whereas DNA damage was absent in the liver. In 3 DMB-treated rats GSH levels were reduced, as compared to controls, of 44 ± 5% in the liver and of 28 ± 6% in the urinary bladder mucosa. Diethylmaleate, used as positive control, reduced GSH level 90% in the liver and 60% in the urinary bladder.


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TABLE 5 Damage of Nuclear DNA from Liver and Urinary Bladder Mucosa of Rats Given a Single Oral Dose of DMB
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies on DMB genotoxicity were, up to now, essentially limited to the evaluation of mutagenic activity in bacteria and of clastogenic activity in systems studied in vitro and in vivo. No data are available on genetic and related effects of DMB in human cells. The results of the present study add some useful information on the genotoxic activity of DMB in metabolically competent rat and human cells, and suggest that, in the intact rat, the tissue specificity of DNA lesions might be dependent on pharmacokinetic events absent from in vitro systems.

Following its chronic oral administration at doses ranging from a few mg to more than 100 mg/kg/day, DMB was found to produce in rats a dose-dependent development of tumors at various sites (IARC, 1974; Morgan et al., 1994Go): skin, Zymbal's gland, oral cavity epithelium, liver, bladder, preputial glands, and intestine of both males and females. Like benzidine, DMB is not carcinogenic prior to metabolic activation, and this is consistent with the report that it is a bacterial mutagen only when activated by a liver enzyme preparation (IARC, 1987). Rodgers et al. (1983) have shown that DMB is rapidly metabolized in the rat via N-acetylation, hydroxylation, O-demethylation, and glucuronidation, and that, of the eight metabolites identified in urine and bile, N-acetyl-DMB is the most potent bacterial mutagen.

Our results show that DMB induces, in primary cultures of rat hepatocytes, a dose-dependent degree of DNA fragmentation. The observed parallel increase in the frequency of micronucleated hepatocytes, which is an index of chromosome breaks or whole chromosome loss, indicates the promutagenic character of DNA lesions. These results are consistent with the carcinogenic activity of DMB for the rat liver and confirm that this benzidine congener is activated by hepatocytes to reactive species and acts as initiator in the process of liver carcinogenesis.

In the intact rat, a single oral dose of DMB equal to half the LD50 was found to induce DNA fragmentation in the urinary bladder mucosa but not in the liver. The limit of this assay is that only one dose and one time between treatment and sacrifice were investigated, but it is known that relative potency in tests for genotoxicity is not a useful indicator of carcinogenic potency (IARC, 1997). The absence of effect in the liver, which is in contrast with the genotoxic effect observed in primary hepatocytes, cannot be explained at present. However, it is worth noting that the same discrepancy was observed in studies performed on DMB-induced DNA repair synthesis that was found to occur in primary rat hepatocytes exposed to 0.5–1 mM concentrations (Probst et al., 1981Go). It did not occur in hepatocytes from rats given oral doses ranging from 50 to 1000 mg/kg (Madle et al., 1994Go). The presence, in the same rats, of DNA fragmentation in the urinary bladder mucosa may be alternatively interpreted as follows: DMB is metabolically activated in the liver, but its reactive species attain higher concentrations in urine during the elimination phase; it is activated in cells of urinary bladder mucosa; both the above mechanisms take place.

Results obtained in primary cultures from liver and urinary bladder mucosa from human donors show, for the first time, that DMB causes DNA fragmentation in both these cell types, but to a more marked extent in the latter. With respect to these findings, first, it should be considered that the human tissue was derived from liver containing metastases and bladder containing primary tumors. Even if the fragments used in our assays were apparently normal, they might be not entirely representative of normal liver and bladder, and biochemical alterations might be present in spite of normal morphology. However, in the genotoxicity assay performed on primary hepatocytes from 68 human donors (Martelli, 1997Go), and on bladder cells from a limited number of donors (unpublished results), we consistently observed that the degree of DNA damage induced by a test chemical did not depend on the pathological condition of the organ, nor did it vary significantly in extent in cultures from different donors. In addition, NDMA testing was consistently positive in human hepatocytes. Second, it should be considered that, due to the difficulty of obtaining samples of human urinary bladder, and to the limited number of cells harvested from each fragment, our data on the DNA-damaging activity of DMB for these cells do not define a dose-response curve. However, the similar degree of DNA fragmentation observed at 100 and 180 µM DMB suggests that DNA damage might occur also at lower concentrations. Even with the caution recommended by these considerations, our findings should be interpreted as indicating that human hepatocytes and, to a more marked extent, urinary bladder mucosa cells biotranform DMB into reactive species capable of damaging DNA. The observed genotoxic effects, although relevant to the knowledge of the possible mechanism(s) of DMB carcinogenicity, do not necessarily indicate that DMB is carcinogenic to humans. They should be interpreted solely as suggesting the need for epidemiological studies aimed at assessing whether exposure to DMB is associated, as has already been established for benzidine (IARC, 1987), with an increased cancer risk, the bladder being the preferential target.


    ACKNOWLEDGMENTS
 
This research was supported by a grant of the MURST (Italy) targeted project "New Assessment Approaches in Toxicology" (1996), and by the funds granted by Genoa University (1997).


    NOTES
 
1 To whom correspondence should be addressed. Fax: +39–010–353–8232. E-mail: farmdimi{at}unige.it. Back


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 DISCUSSION
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