DETERMINING N7-ALKYLGUANINE ADDUCTS BY IMMUNOCHEMICAL METHODS AND HPLC WITH ELECTROCHEMICAL DETECTION - APPLICATIONS IN ANIMAL STUDIES AND IN MONITORING HUMAN EXPOSURE TO ALKYLATING-AGENTS

Many xenobiotics exert their toxic effects through interaction with DN A in the cells of the exposed organism. This interaction may lead to t he formation DNA adducts. Some of these may give rise to mutations tha t initiate cell transformation and, ultimately, the formation of tumor s. Sensitive methods for determining DNA adducts are indispensable for the study of chemical mutagenesis and carcinogenesis and for biomonit oring human exposure to genotoxic agents. Alkylating agents form an im portant class of genotoxic compounds. They react preferentially at the N7-position of guanine. Under neutral or acidic conditions, the adduc ts can be readily released from the DNA backbone as the free base N7-a lkylguanine (N7-AlkGua). The imidazole ring of N7-alkyldeoxyguanosine (N7-AlkdGuo) can be opened under alkaline conditions, which results in formation of a more stable adduct in DNA. To develop immunochemical m ethods for the detection of N7-alkylations, we immunized mice with var ious alkylguanosines in the ring-opened form (RON7-AlkdGuo). Antibodie s were selected to detect adducts in isolated DNA by competitive ELISA and in single cells by immunofluorescence microscopy (IFM). Various m onoclonal antibodies were characterized in detail with respect to spec ificity and sensitivity toward methylated, ethylated, and hydroxyethyl ated DNAs. The antibodies showed extensive cross-reactivity toward N7- (m)ethyl- and N7-(2-hydroxyethyl)guanine modifications in the ring-ope ned form. The limits of detection in the direct and competitive ELISA were 5-10 and 1-2 adducts per 10(6) nucleotides, respectively. The det ection limit of the IFM method was about 20 adducts per 10(6) nucleoti des. To calibrate the immunochemical methods, we used an HPLC with ele ctrochemical detection (HPLC-EC) to quantify N7-methyl-, N7-ethyl-, an d N7-(2-hydroxyethyl)guanine released from DNA by neutral thermal hydr olysis. The base adducts were separated from nonmodified guanine and a denine on a reverse-phase HPLC column and quantified by use of an EC-d etector at an oxidation potential of 1.35 V. For each of the alkylated guanines mentioned, the detection limit was about 0.3 adducts per 10( 6) nucleotides. Results are presented of the application of the immuno chemical assays and HPLC-EC to investigate the induction of methylatio ns in liver DNA of rats treated in vivo with low dosages of hydrazine and the induction of DNA methylations in nucleated peripheral blood ce lls collected from a patient who received chemotherapy with the methyl ating cytostatic drug dacarbazine.