The location of binding sites for 3′-methyl-p-dimethylaminoazobenzene (3′-Me-DAB) or metabolites on components of rat liver cells and hepatoma cells in tumors induced by this carcinogen was determined at 2 stages during the induction of tumors in rats: (a) in normal liver immediately following the application of a massive dose of the azocarcinogen by intragastric feeding, and (b) in liver and tumor after hepatomas had developed following repeated exposures to the carcinogen by s.c. injections. Bound 3′-Me-DAB or metabolites were detected by the use of rabbit antisera directed against either p-azoazobenzene or p′-azo-p-dimethylaminoazobenzene in an indirect fluorescent antibody technique.

Soon after massive intragastric doses of 3′-Me-DAB, the staining observed when the anti-p-azoazobenzene antiserum was used was principally on cytoplasmic components of liver cells with some staining of the intranuclear components. When the second antiserum, anti-p′-azo-p-dimethylaminoazobenzene antiserum, was used, the most intense fluorescent staining was on the nuclear membranes, although there was some cytoplasmic and intranuclear staining as well.

The tumors induced by repeated s.c. injections of azocarcinogen were largely hepatocellular carcinomas and cholangiocarcinomas. Staining was observed in areas of liver around developing or spreading tumors using either of the antisera. The staining patterns for the two antisera were the same as was observed for livers of rats shortly after intragastric feeding. Nuclear membranes and intranuclear material of carcinoma cells showed good staining when anti-p′-azo-p-dimethylaminoazobenzene antiserum was used; however, little or no staining was observed in the carcinoma cells when anti-p-azoazobenzene antiserum was used although this antiserum does stain liver cells. In areas of bile duct proliferation, strong fluorescence was seen in the bile ducts while, again, there was little or none in the carcinoma cells surrounding the bile ducts.

The differences in staining observed between the two antisera indicate that at least two different compounds, either the carcinogen itself and a metabolite or two metabolites, are bound with different distributions to different cell constituents. Each antiserum appears to stain preferentially a particular compound, either the original bound carcinogen or a bound metabolite. The staining results also indicate that some cellular components capable of binding 3′-Me-DAB or its metabolites are retained in tumor cells, whereas other components also able to bind these substances are lost when normal cells become tumor cells.


This investigation was supported by Grant CA 13664 from the National Cancer Institute and General Research Support Grant RR 5648 from NIH, Department of Health, Education, and Welfare.

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