BIOCHEMICAL INTERACTION OF TRYPAN BLUE AND P-DIMETHYLAMINOAZOBENZENE IN THE LIVER OF RAT (I)

The striking fact that the subcutaneous injections of trypan blue into rats inhibit liver tumor formation by p-dimethylaminoazobenzene (DAB), was studied particularly in regard to certain changes in the metabolic pattern and to the chemical nature of this inhibitory mechanism. Investigations were also made to determine which of the impurities present in Merck's trypan blue are responsible for the toxicity noted. Regarding the enzymes (succinic dehydrogenase, cytochrome oxidase, catalase) and riboflavin subjected to determination, the livers of rats that received 60mg of trypan blue and 600-700mg of DAB for over 225 days showed in general a metabolic pattern which was significantly different from that seen in liver cancer, resembling though not perfectly, that in normal liver. However, there were seen some increase in hepatic desoxyribonucleic acid content, and some decrease in the activities of xanthine oxidase and d-amino acid oxidase, indicating a pattern which is intermediate between normal and cancerous livers. Based on Schneider's and Dounce's methods, the trypan blue within the rat liver was found to be bound to cellular components other than the nucleus, probably to the protein or proteins. But with the method described by Miller et al., trypan blue produces no considerable effect on the levels of the total protein bound DAB contained in the livers of rats within the period studied. Some attempts at analysis were carried out from the view points of electrophoretic analyses and other similar methods, and the results were discussed in relation to the enzymedeletion hypothesis of cancer formation, but the ultimate cause of liver cancer inhibition still remains a matter for speculation. The Merck product of trypan blue used in our experiments was not pure, but apparently contains at least, two or three types of dye. Also the starting materials for the synthesis contained frequently in the various commercial products do not seem to have a connection with the toxicity of a 1per cent aqueous solution of trypan blue (Merck). There was no evidence as to which component of the commercial dye is responsible for the toxicity and inhibition of liver cancer induction.