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Localization of Minor Liver-bound Metabolites of 3'-Methyl-p-dimethylaminoazobenzene with Anti-Hapten Antibodies¹

Orchard Park Laboratories, Roswell Park Memorial Institute,² Buffalo, New York 14263

The localization of known and possible metabolites of azocarcinogens such as p-amino-N,N-dimethylaniline, p-amino-N-monomethylaniline, and aniline or aminobenzene bound to components of liver cells of rats fed single or multiple doses of 3'-methyl-p-dimethylaminoazobenzene (3'-Me-DAB) has been determined with the use of antibodies raised against p-azo-N,N-dimethylaniline (p-azo-DA), p-azo-N-monomethylaniline (p-azo-MA), and azo-aniline (azo-B) in the indirect fluorescent antibody procedure. Antisera raised against p-azo-N-acetyl-N-methylaniline (p-azo-AMA) were also used since p-amino-N-acetyl-N-methylaniline (p-amino-AMA) is a possible metabolite of 3'-Me-DAB and is structurally related to the aniline derivatives examined.

Antisera raised against p-azo-AMA not only reacted in agar with p-azo-AMA-ovalbumin conjugate, but also cross-reacted with p-azo-MA-ovalbumin conjugate. Anti-azo-B and anti-p-azo-MA antisera reacted in agar only with azo-B-ovalbumin and p-azo-MA-ovalbumin conjugates, respectively. The p-azo-DA-ovalbumin conjugate did not react in agar with anti-p-azo-MA, anti-azo-B, or anti-p-azo-AMA antisera. Antisera raised against p-azo-AMA, azo-B, and p-azo-MA reacted in agar with the cytosol prepared from the livers of rats treated with 3'-Me-DAB or p-amino-AMA.

When antisera raised against p-azo-DA, p-azo-MA, and azo-B were absorbed with cytosol from normal rat liver, these absorbed antisera reacted with liver cells from rats fed 3'-Me-DAB. The antibodies responsible for this reaction were removed from the three antisera by absorption with cytosol from livers of rats fed 3'-Me-DAB, and the antibodies were also removed from the anti-p-azo-MA antisera by absorption with cytosol from the livers of rats fed p-amino-AMA. When anti-p-azo-DA, anti-p-azo-MA, and anti-azo-B antisera were absorbed with the cytosol from normal rat liver, only anti-p-azo-MA antisera reacted with liver cells from rats fed p-amino-AMA, and the antibodies responsible for this reaction were removed from the antisera by absorption with cytosol from rats fed p-amino-AMA but not by absorption with cytosol from rats fed 3'-Me-DAB. The antisera raised against p-azo-DA, p-azo-MA, or azo-B did not react with carcinoma cells induced in rat liver by 3'-Me-DAB, but they did react with liver cells adjacent to the carcinomas still capable of binding 3'-Me-DAB.

¹ This investigation was supported by a General Research Support Grant from NIH, Department of Health, Education and Welfare.

² A unit of the New York State Department of Health.

Received 9/23/76. Accepted 4/13/77.