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SEVERAL aminoazo dyes are known to induce liver tumours when fed to rats (Miller and Miller, 1953, 1955). The dyes which appear in the liver after feeding have been classified as " free ", "polar bound " and " non-polar bound " by Miller and Miller (1947). The free dye is that fraction which can be extracted by ethyl alcohol from the dried tissue protein. The bound dye, in contrast, cannot be removed from the intact proteins by even the most rigorous extraction, but is only liberated after alkaline hydrolysis. The non-polar fraction of the bound dye can be extracted from the hydrolysis mixture by petroleum ether, whereas the polar fraction, which constitutes the major part, can be extracted only by an ethyl alcohol-ethyl ether mixture. The non-polar dyes have been identified as N-demethylation products, while the polar bound dyes are believed to owe their polar properties to their attachment to some amino acid or peptide fragment released from the liver protein by the alkaline hydrolysis In rats fed aminoazo dyes, either continuously at a low level, or in a single large dose, the only organ in which bound dye is found is the liver (Miller and Miller, 1947; Gelboin, Miller and Miller, 1958). Studies on the accumulation of protein-bound dye in the liver under various conditions indicate that the formation of this bound dye has a role in the carcinogenic process, possibly through deletion of the protein involved in the binding However, the observation that high levels of bound dyes are found in the livers of rats fed the weakly carcinogenic 2-methyl-4-dimethylaminoazo-benzene and 3-methyl-4-dimethylaminoazobenzene (Miller et al., 1949) and in the livers of refractory rats after feeding 3'-methyl-4-dimethylaminoazobenzene that the formation of bound dye in the liver may be a necessary, but not sufficient, requirement for the induction of liver tumours by aminoazo dyes (Miller and Miller, 1955). Besides its presence in the liver, a relatively small amount of bound dye has also been detected in the blood plasma of rats fed 4-dimethylaminoazobenzene for two months (Miller and Miller, 1947). No further attention has, however, been given to the occurrence or possible importance of bound dye in blood or serum. We have, therefore, undertaken a study of this aspect of the problem. The present work reports the determination of bound dye in the serum and observations on the protein fractions to which it is attached.

Protein-Bound Azo Dyes in the Serum of Rats Fed 3'-Methyl-4-Dimethylaminoazobenzene