Induction of SCEs in CHL cells by dichlorobiphenyl derivative water pollutants, 2-phenylbenzotriazole (PBTA) congeners and river water concentrates.

We recently identified dichlorobiphenyl (DCB) derivatives and 2-phenylbenzotriazole (PBTA) congeners as major mutagenic constituents of the waters of the Waka River and the Yodo River system in Japan, respectively. In this study we examined sister chromatid exchange (SCE) induction by two dichlorobiphenyl derivatives, 3,3'-dichlorobenzidine (DCB, 4,4'-diamino-3,3'-dichlorobiphenyl) and 4,4'-diamino-3,3'-dichloro-5-nitrobiphenyl (5-nitro-DCB); three PBTA congeners, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1), 2-[2-(acetylamino)-4-[N-(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), and 2-[2-(acetylamino)amino]-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6); and water concentrates from the Waka River in Chinese hamster lung (CHL) cells. Concentration-dependent induction of SCE was found for all DCBs and PBTAs examined in the presence of S9 mix, and statistically significant increases of SCEs were detected at 2 microg per ml of medium or higher concentrations. SCE induction of MeIQx was examined to compare genotoxic activities of these water pollutants. According to the results, a ranking of the SCE-inducing potency of these compounds is the following: 5-nitro-DCB approximately MeIQx>PBTA6>PBTA-1 approximately PBTA-2>DCB. Water samples collected at a site at the Waka River showed concentration-related increases in SCEs at 6.25-18.75 ml-equivalent of river water per ml of medium with S9 mix. The concentrations of 5-nitro-DCB and DCB in the river water samples were from 2.5 to 19.4 ng/l and from 4100 to 18,900 ng/l, respectively. However, these chemicals showed only small contribution to SCE induction by the Waka River water.

Evaluation of urinary mutagenicity in azo dye manufacture workers.

OBJECTIVES: The aim of the study was to evaluate urinary mutagenicity in workers employed in a major chemical plant located near Rouen (France) that produces dichlorobenzidine and azo dyes. MATERIALS AND METHODS: Samples were obtained from 47 male workers aged 38.9+/-11.3 years (range, 21-58 years), mean duration of employment 9.0+/-8.7 years (range, 1-32 years) for urinary mutagenicity determination with use of the Ames fluctuation test (strains TA 98 and TA 100 with and without metabolic activation) and gas chromatography/mass spectrometry. To assess occupational exposure of workers, urine samples were collected in two series. First, initial just after a one-month holiday (non-exposure). Second, four months later during regular occupational activity. During the same periods, workers completed a questionnaire, which sought information concerning their working conditions, non-occupational factors, and personal habits. RESULTS: Of the total 47 samples tested, 3 (6%) collected just after a one-month holiday and 6 (12%) samples collected during regular occupational activity were positive in at least one mutagenicity assay. Dichlorobenzidine traces ranging from 1.6 to 8.9 ppb were detected in 4 (8%) samples of the exposed as well as in 4 (8%) samples of non-exposed workers. No significant differences between biological and analytical responses obtained in the non-exposure period and after occupational exposure were observed, however, 5 (11%) workers in this group presented urinary mutagenicity that could be related to occupational exposure. CONCLUSIONS: The study suggests that some industrial hygiene problems, revealed in the analysis of questionnaire responses and confirmed by our evaluation, could be undoubtedly and easily solved to improve working conditions of the employees.

Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species.

4-Aminobyphenyl (4-Ab), benzidine (Bz), and Bz congeners were evaluated for their ability to induce genotoxicity through an oxidative mechanism. The mutagenicity of these compounds was tested in the presence and absence of Aroclor 1254-induced rat S9 mix using Salmonella typhimurium tester strain TA102, which is sensitive to agents producing reactive oxygen species (ROS). In the presence of S9, 4-Ab, Bz, N-acetyl-benzidine, and 3,3-dimethoxybenzidine were strongly mutagenic in TA102, whereas, 3,3,5,5-tetra-methylbenzidine, 3,3-dimethylbenzidine (O-tolidine), and N,N-diacetylbenzidine were not mutagenic. In addition, 3,3-dichlorobenzidine and 4,4-dinitro-2-biphenylamine were directly mutagenic in TA102. Incorporation of the free radical and metal scavengers, catalase, superoxide dismutase (SOD), butylated hydroxytolune (BHT), and ethylenediamine tetraacetic acid (EDTA) reduced the mutagenic responses of 4-Ab and Bz, whereas heat-inactivated catalase and SOD had no effect. 4-Ab and Bz also induced lipid peroxidation in the presence of S9 mix as shown using the thiobarbituric acid reactive substances assay. The results of this study indicate that 4-Ab and Bz induce mutations through the induction of ROS.

Sorption of benzidine and 3,3'-dichlorobenzidine to lake sediments. 1. Conceptualization and development of a multiparameter model.

Aromatic amines, such as benzidine and 3,3'-dichlorobenzidine (DCB), are part of the dyes and pigments manufacturing process. The prolonged use of these carcinogenic chemicals in the past generation has introduced a significant amount of contamination to the environment. Their persistency in several mediums has sparked a number of studies in an attempt to develop predictive tools of their fate and transport in the environment. In this study, benzidine and DCB batch isotherms were developed and evaluated. The sediment samples were variable in composition, ranging from sandy to silty-clay sediment samples. The batch isotherms were then analyzed using high-performance liquid chromatography. Subsequently, a multiparameter model (MPM) that accounted for partitioning, covalent bonding, and cation exchange was developed and tested in an effort to understand the various mechanisms. Results proved the proposed model to be effective in predicting sorption of aromatic amines to lake sediments. The findings suggest that the MPM can provide a better understanding of the sorption process of aromatic amines than more conventional models.

Light-induced mutagenicity in Salmonella TA102 and genotoxicity/cytotoxicity in human T-cells by 3,3'-dichlorobenzidine: a chemical used in the manufacture of dyes and pigments and in tattoo inks.

DCB, 3,3'-dichlorobenzidine, is used primarily as an intermediate in the manufacture of diarylide yellow or azo red pigments for printing ink, textile, paint, and plastics. It is also used in tattoo inks. In this article, we investigate light-induced toxicity of DCB in both bacteria and human Jurkat T-cells. DCB itself is not toxic or mutagenic to Salmonella typhimurium TA102, but is photomutagenic at concentrations as low as 2 microM and phototoxic at concentrations >100 microM when bacteria are exposed to DCB and light at the same time (1.2 J/cm2 of UVA and 2.1 J/cm2 of visible light). Furthermore, DCB is both photocytotoxic and photogenotoxic to human Jurkat T-cells. Under a light irradiation dose of 2.3 J/cm2 of UVA and 4.2 J/cm2 of visible light, it causes the Jurkat T-cells to become nonviable in a DCB dose-dependent manner and the nonviable cells reaches 60% at DCB concentrations higher than 50 microM. At the same time, DNA fragmentation is observed for cells exposed to both DCB and light, determined by single cell gel electrophoresis (alkaline comet assay). As much as 5% (average) DNA fragmentation was observed when exposed to 200 microM DCB and light irradiation. This suggests that DCB can penetrate the cell membrane and enter the cell. Upon light activation, DCB in the cells can cause various cellular damages, leading to nonviable Jurkat T-cells. It appears, the nonviable cells are not caused solely by fragmentation of cellular DNA, but by other damages such as to proteins and cell membranes, or DNA alkylation. Therefore, persons exposed to DCB through environmental contamination or through tattoo piercing using DCB-containing inks must not only concern about its toxicity without exposing to light, but also its phototoxicity.

Determination of hemoglobin adducts formed in rats exposed orally with 3,3'-dichlorobenzidine by GC/MS-SIM.

3,3'-dichlorobenzidine (DCB) can be metabolically N-acetylated and/or N-oxidized, and can form hemoglobin adducts. Gas chromatography/mass spectrometry-selected ion monitoring detection mode (GC/MS-SIM) could be a good analytical method to detect them. 4-Aminobiphenyl and phenanthrene-d10 were used as internal standards, and standard metabolites of DCB were synthesized from DCB. Pyridine is a promoter and acetic acid is a controller in the acetylation of DCB during titrating with acetyl chloride. After washing with acetone, the purity of N-acetyl DCB and N,N'-diacetyl DCB were 98.72% and 98.82%, respectively. The maximum detection limits (MDLs) by GC/MS-SIM were 0.5 microg/L in DNA adduct and 1.0 ng/g in hemoglobin for DCB and N-acetyl DCB. The base peaks of their fragmentation pattern were 252 m/z at the peak of DCB, 252 m/z and 294 m/z at the peak of N-acetyl DCB, and 252 m/z, 294 m/z, and 336 m/z at the peak of N,N'-diacetyl DCB. This analytical method was applied to determine hemoglobin adducts formed in young female Sprague-Dawley rats orally exposed with 20, 30, and 40 mg DCB/kg/day for three weeks. Two adducts were detectable by GC/MS-SIM after alkaline hydrolysis of hemoglobin samples and extraction. The structure of these adducts could be assigned to DCB and N-acetyl DCB by co-chromatography with the synthetic standards. After the first week of treatment, the total amount of hemoglobin adducts determined was 837.5 approximately 2501 ng/g hemoglobin. The adduct levels were increased up to 1203.3 approximately 2605.4 ng/g after the second week, and slightly decreased after the third week. The ratio of DCB and N-acetyl DCB was nearly similar in all treatment groups at the third week, such as 4.28 approximately 4.78. Three different treatments (20, 30, and 40 mg DCB/kg) of rats resulted in dose-proportional increases in the total and DCB amount of hemoglobin adducted formed for three weeks. The relative contribution of DCB and N-acetyl DCB to the total hemoglobin adduct level was strongly dose dependent. The results show that GC/ MS-SIM is suitable for the biological monitoring of humans exposed to DCB or DCB-containing products.

Using base-specific Salmonella tester strains to characterize the types of mutation induced by benzidine and benzidine congeners after reductive metabolism.

Although benzidine (Bz), 4-aminobiphenyl (ABP), 3,3'-dichlorobenzidine HCl (DCBz), 3,3'-dimethylbenzidine (DMBz), 3,3'-dimethoxybenzidine (DMOBz) and the benzidine congener-based dye trypan blue (TB) produce primarily frameshift mutations in Salmonella typhimurium, the base-substitution strain TA100 also responds to these compounds when S9 is present. Performing DNA sequence analysis, other investigators have shown that ABP induces frameshift, base-pair and complex mutations. Also, it was found that an uninduced hamster liver S9 preparation with glucose-6-phosphate dehydrogenase, FMN, NADH and four times glucose 6-phosphate gave a stronger mutagenic response than the conventional plate incorporation with rat S9 activation mixture for all the compounds tested. Using the base-specific tester strains of S. typhimurium (TA7001-TA7006) with the above reductive metabolic activation system, we surveyed these compounds for the ability to produce specific base-pair substitutions after reductive metabolism. Bz was weakly mutagenic in TA7005 (0.04 revertants/microg). ABP was mutagenic in TA7002 (1.4 revertants/microg), TA7004 (0.6 revertants/microg), TA7005 (2.98 revertants/microg) and TA7006 (0.4 revertants/microg). DCBz was weakly mutagenic in TA7004 (0.01 revertants/microg). It was concluded that benzidine induced some CG->AT transversions in addition to frameshift mutations. ABP induced TA->AT, CG->AT, and CG->GC transversions as well as GC->AT transitions. DCBz induced only GC->AT transitions. Because DMBz, DMOBz and TB were not mutagenic in this base-substitution mutagen detection system, their mutagenic activity was attributed strictly to frameshift mechanisms.

Determination of dichlorobenzidine-hemoglobin adducts by GC/MS-NCI.

A method based on gas chromatography/mass spectrometry-negative ion chemical ionization detection (GC/MS-NCI) was developed for the determination of 3,3'-dichlorobenzidine (DCB)-hemoglobin adducts. Adducts were released from hemoglobin by mild alkaline hydrolysis and determined by GC/MS-NCI after extraction and derivatization with heptafluorobutyric anhydride (HFBA). 2,2'-DCB was used as internal standard and the recovery of the diarylamine derivatives in the overall procedure was 65-88%. The limit of detection attained was below 0.1 ng/g hemoglobin for DCB as well as for the metabolite N-acetyl-3,3'-dichlorobenzidine (acDCB). The method was shown to be linear up to 150 ng/g hemoglobin. In the NCI mass spectra of the HFB derivatives the dominant ion is (M-HF)-. Due to the presence of two chlorines in the diarylamines, the characteristic ratio of 1.5 for m/z 624 to 626 (for diHFB-DCB and diHFB-2,2'-DCB) and m/z 470 to 472 (for HFB-acDCB) can be observed and used for identification. The method was applied to the determination of DCB-hemoglobin adducts formed in young female Wistar rats after treatment for 4 weeks with 0.006%, 0.0012% or 0.00024% DCB via the drinking water. Two adducts were detectable by GC/MS-NCI after alkaline hydrolysis of hemoglobin samples, extraction and derivatization. The structure of these adducts could be assigned to DCB and acDCB by co-chromatography with the synthetic standards and by the presence of the characteristic ion (M-HF)-. Assessment of the time dependence of hemoglobin adduct formation during subchronic treatment with DCB revealed an increase in adduct levels during weeks 1-3. After this time adduct levels essentially remained constant. In hemoglobin samples isolated from animals treated for 4 weeks with DCB a dose-proportional increase in the total amount DCB- and acDCB-hemoglobin adducts from 8.1 ng DCB/g hemoglobin at 0.3 mg/kg body weight per day (0.00024% in drinking water) to 159.9 ng DCB/g hemoglobin at 5.8 mg/kg body weight per day (0.006% in drinking water) was observed. The ratio of the DCB adduct to the acDCB adduct was strongly dose dependent. At low DCB doses the acDCB- and DCB adducts were formed at similar levels, whereas at high DCB doses the DCB adduct was predominant.

ortho-Substituent effects on the in vitro and in vivo genotoxicity of benzidine derivatives.

Benzidine and its 3,3'-diamino, 3,3'-dimethyl, 3,3'-dimethoxy, 3,3'-difluoro, 3,3'-dichloro, 3,3'-dibromo, 3,3'-dicarbomethoxy and 3,3'-dinitro derivatives together with 2-nitrobenzidine and 3-nitrobenzidine were compared for their in vitro and in vivo genotoxicity. Relative mutagenicity was established with Salmonella strains TA98, TA98/1,8-DNP6 and TA100 with and without S9 activation. All the derivatives in the presence of S9 were more mutagenic than benzidine with 3,3'-dinitro- and 3-nitro-benzidine having the greatest mutagenicity. Mutagenicity in all 3 strains with S9 activation could be correlated to electron-withdrawing ability of substituent groups, as measured by the basicity of the amines. This correlation was explained on the basis that electron-withdrawing groups could favor the stability of the mutagenic intermediate N-hydroxylamine and also enhance the reactivity of the ultimate mutagenic species, the nitrenium ion. Mutagenicity was also correlated to the energy of the lowest unoccupied molecular orbitals (ELUMO). Hydrophobicity was found to have very limited effect on the relative mutagenicity of our benzidine derivatives. The in vivo endpoint was chromosomal aberrations in the bone-marrow cells of mice following intraperitoneal administration of benzidine and its derivatives. In contrast to the in vitro results, while all the amines were genotoxic in vivo, only the 3-nitro derivative had a significant increase in toxicity over benzidine.

Hydroperoxidase I catalyzes peroxidative activation of 3,3'-dichlorobenzidine to a mutagen in Salmonella typhimurium.

Dichlorobenzidine can be peroxidatively activated in Salmonella typhimurium Ames tester strains. Mutagenicity is observed when an S. typhimurium strain which is sensitive to frame-shift mutagens is incubated with dichlorobenzidine and hydrogen peroxide. In this paper, we show that the bacterial enzyme, hydroperoxidase I, is responsible for much of this activation. We constructed isogenic tester strains which lack hydroperoxidase I or II, due to Tn10 insertions in the structural genes encoding these proteins. Hydrogen peroxide-dependent mutagenicity of dichlorobenzidine was measured in each strain. A tester strain lacking hydroperoxidase I activity was much less sensitive than was the parent strain. When hydroperoxidase I activity was restored in this strain, via a plasmid-borne copy of the gene encoding the Escherichia coli protein, sensitivity to peroxide-dependent dichlorobenzidine mutagenicity was enhanced.

Dichlorobenzidine-DNA binding catalyzed by peroxidative activation in Salmonella typhimurium.

Peroxidative oxidation of dichlorobenzidine in vitro results in covalent binding to exogenous DNA. In a modified Ames assay, mutagenicity is observed in S. typhimurium strain TA98 following the incubation of dichlorobenzidine, bacteria, and hydrogen peroxide. In this paper, we demonstrate that [14C]dichlorobenzidine becomes covalently bound to S. typhimurium macromolecules, including DNA, when exogenous hydrogen peroxide is supplied. We compared the levels of binding in a pair of otherwise isogenic strains with wild-type (oxyR+) versus constitutive (oxyR1) expression of the hydrogen peroxide stress-induced regulon. Binding was approximately twofold higher in TA4124 (oxyR1) than in TA4123 (oxyR+). Bacterial hydroperoxidases may catalyze the activation of dichlorobenzidine to mutagenic and DNA binding species in this system.

UDS activity in the rat liver of the human carcinogens benzidine and 4-aminobiphenyl, and the rodent carcinogens 3,3'-dichlorobenzidine and direct black 38.

The activity of benzidine and three structurally related carcinogens in an in vivo/in vitro rat liver assay for unscheduled DNA synthesis is described. The first three of these chemicals, benzidine, 4-aminobiphenyl (4AB) and Direct Black 38 have been reported as positive in this assay by other investigators, albeit the data reported for 4AB were limited. The fourth compound, 3,3'-dichlorobenzidine has not been studied in this assay before. Each compound gave a clear positive response under conditions of routine testing.

Peroxidative activation of 3,3'-dichlorobenzidine to mutagenic products in the Salmonella typhimurium test.

The direct and H2O2-dependent mutagenicity of 3,3'-dichlorobenzidine (DCB) were compared in Salmonella tester strains TA98, TA98/1,8-DNP6, TA100 and TA102 using the Ames test. DCB exhibited both direct and H2O2-dependent mutagenicity to both tester strains TA98 and TA98/1,8-DNP6. This H2O2-dependent mutagenicity of DCB was prevented by horseradish peroxidase. DCB, in contrast to its effects in tester strains TA98, was not mutagenic to TA100 and TA102 either directly or in the presence of H2O2. These results suggest that mechanisms, perhaps enzymes endogenous to tester strains TA98, may play a role in the activation of DCB.

Benzidine and 3,3'-dichlorobenzidine (DCB) induce micronuclei in the bone marrow and the fetal liver of mice after gavage.

Single oral dose of benzidine (300 mg/kg) and DCB (1000 mg/kg) to male ICR mice elicited positive response in the bone marrow micronucleus test. In the transplacental micronucleus test, the compounds were administered to pregnant females in the same manner. A significant increase in the frequency of micronuclei occurred in the fetal liver, but not in the bone marrow of mothers. The relative values of bone marrow and transplacental micronucleus tests for the prediction of carcinogenicity of these compounds is discussed.