direct-black-3 and benzidine

The objective of this study was to evaluate the effects of pretreatment of ethanol (EtOH) and phenobarbital (PB), which are known to affect the metabolism of xenobiotics, in the formation of hemoglobin adducts in rats administered with benzidine (BZ) and Direct Black 38 (DB38). The experimental rats were divided into BZ and DB38 groups; each group was subdivided into control, EtOH, and PB groups. Blood samples were separated into hemoglobin and plasma immediately after obtaining and basic hydrolysis was done to convert the adducts into aromatic amines. Hydrolyzed BZ, monoacetylbenzidine (MABZ), and 4-aminobiphenyl (4ABP) were separated by reversed-phase liquid chromatography without derivatization. Then, quantitative analyses were performed using a high performance liquid chromatograph equipped with an electrochemical detector. The amount of metabolites was expressed in the hemoglobin binding index (HBI). As a result, the formations of hemoglobin in BZ-, MABZ-, and 4ABP-HBI of BZ-EtOH and BZ-PB groups were increased compared with those of BZ-control group. In DB38 group, all of HBIs except for BZ-HBI were increased more than those of DB38-control group regardless of the pretreatment. These results are attributable to the fact that EtOH and PB induced N-hydroxylation is related to the formation of hemoglobin adducts. They indicate that EtOH not only increases the adduct formation by inducing N-hydroxylation but also induces N-acetylation. PB induced N-hydroxylation and increased the adduct formation in BZ group, but decreased the adduct formation in DB38 group due to decreasing azo reduction. These results suggest that the effects of EtOH or PB should be considered in biochemical monitoring of BZ and DB38 for the assessment of intermittent exposure to BZ and DB38.

Topics: Acetylation; Animals; Azo Compounds; Benzidines; Chromatography, High Pressure Liquid; Coloring Agents; Electrochemistry; Ethanol; Hemoglobins; Hydroxylation; Male; Phenobarbital; Rats; Rats, Sprague-Dawley

Direct black 38 (DB38) dye is a well-established toxic and carcinogenic compound. Present investigation reports isolation of an Enterococcus gallinarum strain capable of decolorizing and degrading it. Changes in toxicity and mutagenicity of DB38 and its metabolites were also determined using a battery of carefully selected tests (cytotoxicity, respiration inhibition test and Ames test). Toxicity assays were carried out on E. gallinarum itself as this also gave information about suitability of this strain for the dye decolorization operation. The strain was found to reduce both toxicity and mutagenicity of DB38 metabolites. Benzidine and 4-aminobiphenyl (4-ABP) were identified as the DB38 metabolites, responsible for its toxic and mutagenic properties, by HPLC-MS analysis. Further degradation of benzidine and 4-ABP was found to result in the decrease in toxicity and mutagenicity.

Topics: Aminobiphenyl Compounds; Azo Compounds; Benzidines; Biodegradation, Environmental; Chromatography, High Pressure Liquid; Coloring Agents; Enterococcus; Inactivation, Metabolic; Mass Spectrometry; Mutagenicity Tests; Salmonella typhimurium

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.

Topics: 3,3'-Dichlorobenzidine; Aminobiphenyl Compounds; Animals; Azo Compounds; Benzidines; Carcinogens; DNA Replication; Liver; Mutagens; Rats

The role of the rat intestinal flora in the azo reduction of some benzidine-based dyes was studied in vitro and in vivo. The formation of benzidine was measured after anaerobic incubation of direct black 38, direct blue 6 and direct brown 95 in the presence of caecal bacteria in vitro. Benzidine was absorbed from the intestinal tract much better than the parent compounds. Oral administration of direct black 38 or direct brown 95 to Wistar rats results in the urinary excretion of mutagens. After oral administration of these dyes to germ-free Wistar rats no mutagenicity was observed in urine. The present results show that after oral administration, reduction by the intestinal flora should be considered as the first essential step in the biotoxification of benzidine-based dyes.

Topics: Administration, Oral; Animals; Azo Compounds; Benzidines; Biological Transport; Chromatography, High Pressure Liquid; Intestinal Mucosa; Male; Mutagens; Rats; Rats, Inbred Strains

Benzidine-based azo dyes are proven mutagens and have been linked to bladder cancer. Previous studies have indicated that their initial reduction is the result of the azo reductase activity of the intestinal microbiota. Metabolism of the benzidine-based dye Direct Black 38 was examined by using a semicontinuous culture system that simulates the lumen of the human large intestine. The system was inoculated with freshly voided feces, and an active flora was maintained as evidenced by volatile fatty acid and gas production. Within 7 days after exposure to the dye, the following metabolites were isolated and identified by gas chromatography-mass spectrometry:benzidine, 4-aminobiphenyl, monoacetylbenzidine, and acetylaminobiphenyl. Benzidine reached its peak level after 24 h, accounting for 39.1% of the added dye. Its level began to decline, and by day 7 the predominant metabolite was acetylaminobiphenyl, which accounted for 51.1% of the parent compound. Formation of the deaminated and N-acetylated analogs of benzidine, which have enhanced mutagenicity and lipophilicity, previously has not been attributed to the intestinal microbiota.

Topics: Aminobiphenyl Compounds; Azo Compounds; Bacteria, Anaerobic; Benzidines; Biotransformation; Chromatography, Gas; Chromatography, Ion Exchange; Feces; Fermentation; Humans; Mutagens; Oxidation-Reduction; Time Factors

Topics: Animals; Azo Compounds; Bacteria; Benzidines; Coloring Agents; Feces; Haplorhini; Humans; Intestines; Kinetics; Rats; Species Specificity; Structure-Activity Relationship

Studies have established that Direct Black 38 and two other benzidine-based dyes are carcinogenic. The carcinogenic effect has been generally considered attributable to the metabolic release of benzidine from Direct Black 38 and similar dyes. However, Ames tests indicated that when Direct Black 38 is reduced with sodium dithionate it is more mutagenic than can be accounted for by complete release of all the benzidine present in the dye molecule. While most dyes are not mutagenic when tested with S-9, a series of benzidine congener dyes were all found to be mutagenic with either TA 98 or TA 100 strains, if the dyes were first reduced with sodium dithionate. Unreduced dyes were not mutagenic. Neither anaerobic conditions nor addition of riboflavin induced mutagenicity of these dyes under the condition of our experiments.

Topics: Anaerobiosis; Animals; Azo Compounds; Benzidines; Carcinogens; Coloring Agents; Dithionite; Mutagenicity Tests; Mutagens; Rats; Salmonella typhimurium