Protein-Bound Dyes in the Serum and Liver of Rats Fed Aminoazo Dyes

Abstract

IT was previously reported (Dijkstra and Joubert, 1961) that the albumin fraction isolated from serum of rats fed a single dose of the carcinogen 3'-methyl-4-dimethylaminoazobenzene contained significant quantities of protein-bound dye, which was only released by prolonged hydrolysis of the protein. The present work reports observations on the relative importance of dye binding to liver and serum proteins after administration of various aminoazo dyes of different degrees of carcinogenicity. MATERIALS AND METHODSReagents. Treatment of animals.-Male albino rats (weight 180-220 g.) of the Wistar strain were fasted for 6 hours before the administration by stomach tube of 50 mg. of aminoazo dye in 2 ml. of olive oil. After dosing, the animals were fed ad libitum on stock diet (Dijkstra and Joubert, 1961) and tap water. At intervals the rats were anaesthetized with ether, the abdominal cavity was exposed and the blood was obtained from the abdominal aorta. The liver was immediately perfused in situ with cold 2 per cent sodium citrate (Miller and Miller, 1947).Estimation of dye.-Free (i.e. alcohol-extractable) dye and the protein-bound dye were determined (Dijkstra and Joubert, 1961) in the serum after precipitation of proteins with 50 ml. of absolute ethyl alcohol per 2 ml. of serum, and in the liver after homogenization of the left lateral lobe (2-3 g.) in 50 ml. of absolute ethyl alcohol. The optical absorption of the dye was measured at 520 m#t and it was ascertained that this involved no appreciable error in cases where the wavelength of maximum absorption differed from 520 m,u. The results of the bound dye determinations are expressed as the optical density (E) at 520 m, by the dye from 50 mg. of protein in a mixture of 2-0 ml. of ethyl alcohol and 2-5 ml. of 7 N hydrochloric acid using an absorption cell with a light path of 1 cm. (Miller and Miller, 1947). The results of the free dye estimations are similarly expressed, using the mean values of 7 0 and 20-0 per cent for the protein content in the serum and the liver respectively.The absorption spectra of the bound dyes from liver and serum were obtained as previously reported (Dijkstra and Joubert, 1961) at 38 hours after dye ad-

Studies on azo-dye carcinogenesis in rat liver. The effect of 4-dimethylamino-3′-methylazobenzene on the incorporation of [14C]leucine into rat-liver microsomal protein

“…1). The time-intervals chosen were based on the analysis of protein-bound 4-dimethylamino-3'-methylazobenzene in the microsome, pH 5-enzyme and pH 5-supernatant fractions (Table 1), which showed the same time-course of binding to proteins of the whole liver as reported by Dijkstra & Louw(1962),wheremaximunmbinding was found at about 40 hr. after dosing.…”

“…A stimulation of protein synthesis in vitro by rat-liver microsomal systems after the administration of methylcholanthrene in vivo has been observed (Decken & Hultin, 1960;Gelboin & Sokoloff, 1961). This has been considered to be associated with the increase in activities of certain microsomal enzymes after the administration of methylcholanthrene (Conney, Miller & Miller, 1956, 1957Decken & Hultin, 1960).…”

Early Stage in the Metabolism of Aminoazo Dyes in the Liver of Rats

“…of olive oil. In order to avoid any possible diurnal variation in the metabolic activity of the rats at the time of dosing, the dye was always administered between 9.00 and 10.00 p.m. After dosing, the animals were fed ad libitum on stock diet (Dijkstra and Joubert, 1961) and tap water. At intervals the livers were removed from the animals after decapitation and thorough bleeding.…”

“…QUANTITATIVE studies of the binding of various carcinogenic and non-carcinogenic aminoazo dyes to proteins (Dijkstra and Joubert, 1961;Dijkstra and Louw, 1962) led us to investigate the mechanism of the binding process after administration of a single dose of dye. As it has been suggested (Gelboin, Miller and Miller, 1958) that the binding to proteins takes place at the time of their synthesis in the liver, trichloroacetic acid (TCA) extracts of the liver were examined in the hope of finding the dye or its metabolites bound to soluble protein precursors.…”