• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.6 no.1
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• pp.28-37
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• 1996

Benzidine, an aromatic amine used primarily in the manufacture of azo dyes, is recognized as a urinary bladder carcinogen in humans. In rats, mice, and hamsters, chronic exposure to benzidine resulted in tumors of the liver. The present study was undertaken to suggest analyzing the metabolites of benzidine with the optimal condition, identify the metabolites of benzidine, and observe time variance of the metabolites in the isolated perfusated rat liver. N-acetylbenzidine was synthesized by acetylation of benzidine with acetic anhydride and separated by thin layer chromatography(TLC) and high performance liquid chromatography(HPLC). To analysis benzidine and the metabolites of benzidine, HPLC operating condition has been optimized by means of preliminary experiment. The mobile phase consisted of acetonitrile(37%) in phosphate buffer, flow rate maintained at 1.0 ml/min. Optimal detective conditions were electrochemicaldetector(ECD) at 0.75 V for benzidine and N-acetylbenzidine and ultravioletdetector(UVD) at 287 nm for N,N'-diacetylbenzidine. The separation system was composed of a guard column and a separation column(Polymer C18, $4.6{\times}250cm$) at a temparature of $40^{\circ}C$. The perfusion system was equilibrated for 30 minutes before addition of benzidine to the perfusate. Samples of the perfusate were collected at time intervals(0, 10, 20, 30, 60, 90, 120 min) during the 2 hour perfusion. Before analyzing samples by HPLC/ECD/UVD, samples had been treated with sep-pak. Samples of perfusate analyzed by HPLC/ECD/UVD and the metabolites of benzidine in the isolated perfused rat liver were N-acetylbenzidine and N,N'-diacetylbenzidine. Benzidine metabolized over 60% during the initial 30 minutes of perfusion, extensively by 1 hour, and was undetectable in the perfusate. N-acetylbenzidine increased by 30 minutes of perfusion, declined. N,N'-diacetylbenzidine increased the 0-90 minutes period, remained constant during the 90-120 minutes period.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.6 no.1
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• pp.156-164
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• 1996

Benzidine is recognized as a urinary bladder carcinogen in humans. The use of benzidine in industries was prohibited because of its carcinogenecity, but, production and usage of benzidine-based dye was still permitted in most countries. This study was performed to compare the excretory patterns of urinary metabolites between benzidine-based dye(Direct Black 38) and benzidine in rats Benzidine-based dye was administered orally at the doses of 0.3, 0.5, 0.7 mmol/kg and benzidine was administered orally at the doses of 0.2, 0.4, 0.6 mmol/kg into Sprague-Dawley rats. To analyze benzidine and its metabolites, the high performance liquid chromatography with an electric chemical and ultraviolet detector were used. N-acetylbenzidine, N,N'-diacetylbenzidine and 4-aminobiphenyl were identified in the urine of the rats receiving dye and benzidine. The excreted amount of the urinary benzidine from dye was almost 1/10 of that from benzidine. Excretion rates of metabolites were more prolonged in the dye receiving group than those of the benzidine group. Peak concentration time of urinary N,N'-diacetylbenzidine was more prolonged than other metabolites in both groups. The excreted amount of N-acetylbenzidine was more than the others in both group. These results suggested that N-acetylbenzidine may be an useful Biological exposure index for benzidine-based dye.