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

Abstract

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. MATERIALS AND METHODSReagents.-4'-Hydroxy-4-dimethylaminoazobenzene (4'-OH-DAB), m.p. -203 0°C., was prepared according to Miller and Miller (1948). The other aminoazo dyes used were described by Dijkstra and Louw (1962). They were dissolved in olive oil of B.P. quality.Treatment of animals.-Male albino rats (weight 220-260 g.) were fasted for 6 to 7 hours before administration by stomach tube of 50 mg. of aminoazo dye in 2 ml. 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.Preparation of TCA extracts.-The livers were minced with scissors and homogenized at 00 C. in 2 ml. of 10 per cent TCA per g. of liver, using a glass homogenizer with motor-driven perspex pestle. After standing for 10-15 minutes, the homogenate was centrifuged for 10 minutes at 1200 g at 30 C. The supernatant (ca. 1-8 ml.) was centrifuged a second time in order to remove all sedimentable matter. The residue of the TCA extraction was extracted repeatedly with 2-0 ml. of 7 per cent TCA per g. of liver, and the supernatants (ca. 2-0 ml.) were obtained in the same way. The TCA extracts were kept under an atmosphere of nitrogen.Estimation of dye.-The dye content of the TCA extract was determined spectrophotometrically at 520 m/,t after the addition of an equal volume of chilled concentrated hydrochloric acid. The results are expressed as the optical density (E'A) of these solutions using an absorption cell with a light path of 1 cm.Free, i.e. alcohol-extractable, dye was determined in the solution obtained after homogenization of 2 to 3 g. of minced liver tissue with 50 ml. of absolute ethyl alcohol, heating at 750 for 10 minutes, and centrifugation at 1200 g for 15 minutes.