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N-Phenylguanine, a base adduct possibly formed after arylation of DNA by benzene oxide, the first reaction metabolite during benzene metabolism, was synthesized in our laboratory and used as reference for the production and characterization of monoclonal antibodies. 2-Hydroxymethyl-7-phenylhypoxanthine, a molecule structurally similar to N-phenylguanine, was coupled by a linker molecule to different carrier proteins. The resulting conjugate was used to immunize BALB/c mice, the spleen cells of which were fused with mouse P3X63-Ag8.653 myeloma cells to obtain monoclonal antibodies. Several hybridoma lines were cultivated in defined media and characterized as to sensitivity and specificity by an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA demonstrated that all antibodies showed a very high affinity for N-phenylguanine but had a lower affinity towards various other samples including N-chlorophenylguanines and C-, N- and O-phenylguanine. As little as about 20 pg N-phenylguanine could be detected with one of the most sensitive antibodies, CE6/G11, with a colorimetric end point while the detection limit could be lowered to about 10 pg N-phenylguanine when a fluorescent end point was used. The detection limit of other methods used to determine N-phenylguanine so far is 10 ng for gaschromatography/mass-spectrometry and 1 ng for high-pressure liquid chromatography. Thus the use of specific monoclonal antibodies seems to be the most sensitive method for the detection of N-phenylguanine. [ABSTRACT FROM AUTHOR]

Chlorobenzene (CB) was administered to male Wistar rats. Twenty-four-hour urine samples were collected over a period of 7 days. p-Chlorophenylmercapturic acid, chlorophenols and a guanine adduct were determined by chromatographic methods. The excretion pattern of p-chlorophenyl-mercapturic acid did not seem to be significantly affected by phenobarbital. To analyze for the chlorophenols and guanine adducts, diluted urine was subjected to cation exchange chromatography using UV-detection. Fractions were found containing chlorophenols and a compound showing chromatographic properties similar to those of the synthetic N7-phenylguanine. The results of the HPLC analyses suggested the presence of a guanine adduct excreted on days 1 and 2 and between days 4 and 6 post-administration. There were no compounds detected which were identical with p-, m- or o-isomers of N7-chlorophenylguanine. The excretion of p- and m-chlorophenols by phenobarbital-pretreated animals was twice as high as that in untreated rats. A 4-fold increase was detected for o-chlorophenol. It is assumed that it is mainly direct hydroxylation that can be induced by phenobarbital. Dehalogenated phenolic metabolites may be capable of covalent binding to DNA.