• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.9 no.2
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• pp.100-109
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• 1999

This study evaluates the pretreatment for analysis of benzidine metabolites in urine by measuring the recovery rates according to the temperature and periods of storage of the urine. By the solid phas e extraction, the recovery rates of basic hydrolysis are benzidine 67.4 %, monoacetylbenzidine 105.1 %, and diacetylbenzidine 115.8 %, respectively. By the liquid extraction, the recovery rates of back-extraction into 0.1 M perchloric acid are benzidine 105.7%, monoacetylbenzidine 94.2 %, diacetylbenzidine 72.8 %, respectively. The difference of the recovery rates between the back-extraction into 0.1 M HCl and 0.1 M perchloic acid after basic hydrolysis are 101 % and 98.8 %, respectively. When the recovery rates of the urinary s amples of pH 3, pH 7, pH 12 at $25^{\circ}C$ and $-76^{\circ}C$ are compared for four weeks, there are no differences according to the temperature and the periods of storage. The above results show that the solid phase extraction and back-extraction by 0.1 M perchloric acid after basic hydrolys is are suitable for the analysis of benzidine metabolites. There are no difference of the recovery rates of the urinary samples stored at $25^{\circ}C$ and $-76^{\circ}C$ at pH 3, pH 7, pH 12, respectively for 28 days.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.10 no.2
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• pp.124-139
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• 2000

This study was performed to investigate monoacetylbenzidine(MABZ) and benzidine(BZ) hemoglobin adducts among workers who worked at benzidine based dye manufacturing company, and exposed by benzidine and benzidine based dye. The hemoglobin adducts were compared with work environment assessment result for evaluating the usefulness of biological monitoring. The mean BZ hemoglobin adducts among the first synthesis worker's hemoglobin adducts were $40.69{\mu}gBZ/g$ Hb and those of dry and packing workers were $22.14{\mu}gBZ/g$ Hb. The mean of MABZ hemoglobin adducts among 1st synthesis workers were $255.84{\mu}gMABZ/g$ Hb, dispersion worker's hemoglobin adducts were $76.17{\mu}gMABZ/g$ Hb and synthesis worker's hemoglogin adducts were $28.66{\mu}gMABZ/g$ Hb. Work environment assessment results during past 3 years were $0.0065mg/m^3$ and $0.5659mg/m^3$ of benzidine based dye concentration in ambient air of drying and packing only. Dye producing process was categorized by the possibility of exposure to benzidine and benzidine based dye. BZ and MABZ hemoglobin adducts were $19.55{\mu}gBZ/g$ Hb, $119.80{\mu}gMABZ/g$ Hb among workers who exposed by benzidine dihydrochloride and $16.32{\mu}gBZ/g$ Hb, $316.56{\mu}gMABZ/g$ Hb among workers who exposed by benzidine based dye. BZ hemoglobin adducts were not detected among control group and MABZ hemoglobin adducts were $5.33{\mu}gMABZ/g$ Hb. The differences between control and other exposed group was statistically significant. But there was no statistically significant differences between benzidine dihydrochloride exposed process and benzidine based dye exposed group. BZ and MABZ hemoglobin adducts were $2.23{\mu}gBZ/g$ Hb, $76.17{\mu}gMABZ/g$ Hb and $3.46{\mu}gBZ/g$ Hb, $21.33{\mu}gMABZ/g$ Hb. So hemoglobin adducts of MABZ were 5 ~ 30 time higher than those of BZ(P<0.003). Above results indicate that work environment assessment didn't detected benzidine and benzidine based dye in ambient air but biological monitoring detected those of hemoglobin adducts. Two group's hemoglobin adducts exposed benzidine dihydrochloride and benzidine based dye were high level but wasn't statistically significant and those were not detected in control group.

• Journal of Preventive Medicine and Public Health
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• v.35 no.3
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• pp.229-235
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• 2002

Objectives : To evaluate the effects on the formation of benzidine-hemoglobin, and benzidine metabolite-hemoglobin adducts, caused by pretreatment with the known xenobiotic metabolism effectors, ethanol and phenobarbital, in rats administered Direct Black 38 dye. Methods : The experimental rats were divided into three groups: a control group, an ethanol group and a phenobarbital group. Rats were pretreated with ethanol (1g/kg) or phenobarbital (80mg/kg) 24 hours prior to the oral administration of Direct Black 38 (0.5mmol/kg), with the control group being administered the same amount of distilled water. Blood samples were obtained from the vena cava of 5 rats from each group prior to, and at 30 min, 3h, 5h, 9h, 12h, 24h, 48h, 72h, 96h, and 144h following the oral administration of Direct Black 38. Directly after sampling the blood was separated into hemoglobin and plasma, with the adducts being converted into aromatic amines by basic hydrolysis. Hydrolyzed benzidiene, monoacetylbenzidine and 4-aminobiphenyl were analyzed by reverse-phase liquid chromatography with an electrochemical detector, The quantitative amount of the metabolites was expressed by the hemoglobin binding index (HBI). Results : In the ethanol group, benzidine-, monoacetylbenzidine-, and 4-aminobiphenyl-HBI were increased to a greater extent than those in the control group. These results were attributed to the ethanol inducing N-hydrgxylation, which is related to the formation of the hemoglobin adduct, In the phenobarbital group, all the HBIs, with the exception of the benzidine-HBI, were increased to a greater extent than those of the control group. These results were attributed to the phenobarbital inducing N-hydroxylation related to the formation of the hemoglobin adduct. The N-acetylation ratio was only increased with the phenobarbital pretreatment due to the lower benzidine-HBI of the phenobarbital group compared to these of the control and ethanol groups. The N-acetylation ratios for all groups were higher than f for the duration of the experimental period. Although the azo reduction was unaffected by the ethanol, it was inhibited by the phenobarbital, The ratio of the benzidine-HBI in the phenobarbital group was lower than those of the ethanol the control groups for the entire experiment. Conclusion : Our results indicate that both ethanol and phenobarbital increase the formation of adducts by the induction of N-hydroxylation, but also induced N-acetylation. Phenobarbital decreased the formation of benzidine-HBI due to the decrease of the azo reduction. These results suggest that the effects or ethanol and phenobarbital need to be considered in the biochemical monitoring of Direct Black 38.

• YAKHAK HOEJI
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• v.44 no.5
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• pp.384-390
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• 2000

Metabolism study of the dye, benzidine, was performed by gas chromatography-mass selective detector (GC/MSD) in the urine of rats orally administered 100 mg/kg benzidine. Urine samples were collected in metabolic cages for 0-24, 24-48, and 48-72 hrs. Ten ml of the urine was extracted with XAD-2 resin and the XAD-2 column was eluted with methanol. After evaporation, benzidine and its metabolites were extracted with diethyl ether (for non-conjugated fraction). For conjugated metabolites, $\beta$-glucu-ronidase was added to the aqueous layer that was incubated for 1 hr at 5$0^{\circ}C$ and the aqueous layer was extracted as in non-conjugated fraction. Aliquot of trimethylsilylated derivatives was applied to the GC/MSD. The mutagenicity of benzidine and its acetylated metabolites was tested by histidine/reversion assay. Five metabolites observed and confirmed either by electron impact and chemical ionization modes of the GC/MSD, or authentic compounds were monoacetyl-, diacetyl-, hydroxyacetyl-, hydroxydiacetyl-, and hydroxy-benzidine. Monoacetyl-benzidine was more potent than benzidine in histidine/reversion assay. This data indicates that monoacetylation of benzidine may be one of the metabolites produced in metabolic activation process.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.11 no.2
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• pp.118-125
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• 2001

Recently, biochemical analysis using hemoglobin adduct is frequently performed to evaluate the exposure to chemical carcinogens. However, data on the effect of co-exposure with other chemicals on hemoglobin adduct formation are seldom provided. The objective of this study is to evaluate the effects of pretreatment of ethanol(EtOH) and phenobarbital(PB), which are known to affect metabolism of xenobiotics, on the formation of hemoglobin adducts in the rats(Sprague-Dawley) administered benzidine(BZ). The experimental rats were divided into control, EtOH, and P8 groups. Rats were pretreated with EtOH or PB 24 hours before the oral administration of BZ. Blood sampling was taken before the administration of the chemicals and 0.5, 3, 6, 9, 12, 24, 48, 72, 96, and 144 hours after the administration of the BZ in 5 rats each. The blood was separated into hemoglobin and plasma immediately after taking the blood samples, and the adducts were undergone basic hydrolysis to convert them into aromatic amines. Hydrolyzed BZ, monoacetylbenzidine (MABZ), and 4-aminobiphenyl(4ABP) were separated by reversed-phase liquid chromatography without derivatization, and quantitative analyses of them were performed by a highperformance liquid chromatograph equipped with electrochemical detector. The quantitative amount of the metabolites was expressed by hemoglobin binding index(HBI), BZ-, MABZ-, and 4ABP-HBI of EtOH and PB groups were increased more than those of control group. These results are attributable to the fact that EtOH and PB induced N-hydroxylation related to the hemoglobin adduct formation. The ratio of N-acetylation (viz, MABZ-HBI/BZ-HBI) showed no significant difference between EtOH group and control group. It means that EtOH increased N-hydroxylation and N-acetylation in a similar degree. The N-acetylation ratio of PB group was relatively lower than control group because the PB increased N-hydroxylation induction. The N-acetylation ratios of all groups were higher than 1 during the entire experimental period. This result suggests that the effects of EtOH or PB need to be considered in the biochemical monitoring for the assessment of intermittent exposure of benzidine.