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Molecular Dosimetry in Rat Urine of Aflatoxin-N⁷-guanine and Other Aflatoxin Metabolites by Multiple Monoclonal Antibody Affinity Chromatography and Immunoaffinity/High Performance Liquid Chromatography¹

Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205 [J. D. G., A. P.]; Department of Biochemistry, University of Zimbabwe, Box MP 167, Harare, Zimbabwe [J. A. H.]; and Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 [L. J. T., P. R. D., G. N. W.]

The development of molecular dosimetry methods will simplify the identification of people at high risk for cancer. A combined monoclonal antibody immunoaffinity chromatography/high performance liquid chromatography method has been devised to isolate and quantify aflatoxin-DNA adducts and other metabolites in rat urine samples. We report the production of 11 different monoclonal antibodies recognizing aflatoxin B₁, aflatoxin Q₁, aflatoxin G₁, aflatoxicol, and aflatoxin M₁ and the application of these antibodies to a multiple monoclonal antibody affinity chromatography technique. Using the multiple monoclonal antibody affinity column with rat urines obtained from dosed animals, between 90 and 95% of total aflatoxin metabolites can be bound to the column and isolated. Analytical immunoaffinity chromatography/high performance liquid chromatography analysis of these isolated aflatoxins reveals that more than 55% of the aflatoxins in rat urine are aflatoxin-dihydrodiol, aflatoxin-N⁷-guanine, aflatoxin Q₁, aflatoxin M₁, aflatoxin P₁, and aflatoxin B₁, accounting for 1.5, 9.6, 1.8, 34.5, 8.0, and 1.0% of the total aflatoxins, respectively. Further, a perchloric acid digestion of the aflatoxin-N⁷-guanine peak was used to confirm its identity by its conversion to guanine. The measurement of aflatoxin-N⁷-guanine excretion in rat urine was examined to assess its utility as a marker of DNA adduct formation in the liver, and a dose-dependent excretion in urine was found with a correlation coefficient of 0.99. A comparison of the dose-dependent residual levels of aflatoxin binding to liver DNA with the amount of aflatoxin-N⁷-guanine excreted in urine showed a correlation coefficient of 0.98. Besides the nucleic acid adduct excretion data, aflatoxin M₁ and aflatoxin P₁ were evaluated as molecular dosimeters in the urine. Aflatoxin M₁ was found to be an excellent marker, whereas no linear relationship between dose and aflatoxin P₁ excretion in urine was found.

¹ This research was supported in part by USPHS Grants P01 ES00597 and RO1 CA54114. J. D. G. is a recipient of Research Career Development Award KO4 CA01517. J. A. H. is a recipient of a Senior African Fulbright Fellowship from the Council for the International Exchange of Scholars.

² To whom requests for reprints should be addressed.

Received 7/23/91. Accepted 10/31/91.

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