The specific objective of the proposed work is to develop, validate and apply methods for the determination of excreted alkylated purines as markers of DNA alkylation in vivo in humans. The following approaches will be used: i) novel methods will be used for the preparation of antibodies to low molecular weight alkyl purines. The antibodies will be used to develop quantitative immunoassay methods for the determination of urinary alkyl purines (3-methyl-, 3-ethyl- and 3-carboxy- methyladenine, and 7-ethyl- and 7-carboxymethylguanine). ii) Methods for the determination of urinary alkylated purines will be validated in animal models. The level and pattern of urinary excretion of 3-methyladenine will be correlated with methylation in both lymphocyte DNA and target tissue DNA following administration of dimethylnitrosamine. Similar experiments will be carried out for the urinary 3-ethyladenine and and 7-ethylguanine following administration f diethylnitrosamine, and 3-carboxymethyladenine and 7-carboxymethylguanine following N-nitrosoglycocholic acid administration. iii) Urinary alkylpurine determination will be incorporated into epidemiological studies where either cancer incidence in defined populations or individual cases are the endpoints. Methods will be developed for the identification and determination of cyclophosphamide DNA adducts in blood and urine samples of cancer patients receiving chemotherapy. Immunoassay and mass spectrometric methods will be developed for case-control studies in which characteristic DNA adducts will be evaluated as markers of risk for second cancer. The overall goal of this project is to develop validated, practical methods for monitoring human exposure to alkylating carcinogens based on the determination of urinary alkyl purines. In the case of iatrogenic second cancers following cyclophosphamide therapy the methods have potential for identifying DNA adducts indicative of the risk of developing second cancer. This would enable key ideas in molecular epidemiology to be validated and also offer the specific benefit of identifying modifications in therapy required to reduce the risk of second cancers.
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