The binding of metabolites of two related azo dyes of different carcinogenic potency, the carcinogenic 4-dimethylaminoazobenzene ( D A B ) and the weakly carcinogenic 2-methyl-4-dimethylaminoazobenrene , to rat liver DNA and to subcellular fraction protein was studied following chronic oral administration for I to 3 weeks. Different techniques for measuring the amount of DAB metabolites bound to protein were first compared, then the whole study was performed with labelled DAB and 2-Me-DAB (aniline ringJ4C) of moderate specific activity. DAB metabolites were bound to liver DNA to a higher extent than those of 2-Me-DAB. In contrast, the binding of 2-Me-DAB metabolites was equal to or higher than that of DAB metabolites to protein. The amount of protein-bound metabolites was studied on the nucleo-mitochondrial fraction, microsomes, supernatant, nuclei, chromatin, nucleoplasm, nucleolar fraction and nuclear membrane. Following the administration of both dye diets, the supernatant protein bound the highest level of metabolites. The time-course of binding of DAB metabolites to DNA andprotein was different from that of 2-Me-DAB metabolites. These results show the possible involvement of carcinogen-D NA binding in the mechanism of carcinogenesis.

This binding required an enzymatic activation. We studied some characteristics of the enzymatic process, i.e. NADPH requirement, effect of pH, effect of various concentrations of microsomes and DNA, induction by 3-methylcholanthrene.This in vitro system is discussed in view of its validity for studying interactions between the carcinogenic azodyes and the cellular constituents.

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MODlFICATIONS OF CARBOHYDRATE METABOLISM OBSERVED DURING HEPATOCARCINOGENESIS INDUCED BY DAB. ' STUDIES O N GLYCOGEN METABOLISMI n an attempt 10 find an explanation for the hypoglycaeinia observed in rats with DAB-induced hepatomas, we studied glycogen metabolism in rat livers in which cancer invasion proceeded at different speeds.We measured the content of glycogen and ojglucose-6-phosphate in the liver and determined the activities of glycogen-syiithrtase, phosphorylase, and phosphoglucomutase.We demonstrated that the cause of the hypoglycaemia varies froin one case to another. I n some instances, glycogen accumulates in the liver but can no longer be broken down into glucose, wrhile in others glycogen is no longer synthesized.

The mouse is well known for its relative resistance to liver DAB carcinogenesis, when compared with the rat; the hamster is even more resistant (Miller and Miller, 1955; Homburger, 1968).Since the initial discovery of protein-bound azodyes in the liver of rats fed DAB (Miller and Miller, 1947), a large variety of azo compounds, either supplied in the diet or given by intraperitoneal injection, were shown to give rise to bound azo-dyes in rat liver (see review by Terayama, 1967;Lawson, 1968; Hughes, 1969/70;and Roberts, 1969). This binding was not specific to azo compounds and many other chemical carcinogens were found associated with proteins of target tissues (Hughes and Pilczyk, 1969;Sorof et al., 1969;Szafarz and Weisburger, 1969; Barry et al., 1969/70).In each case, soon after the beginning of carcinogen administration, the amount of bound dyes increased and reached a maximum; the decrease in binding which followed could possibly result in a gradual removal of the proteins able to combine with the dye.It is currently accepted that the molecule of DAB must be metabolized in order to express its carcinogenic effect and be bound to liver proteins (Terayama, 1958;Miller and Miller, 1969).