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From preliminary experiments it was known that radiolabelled benzene and some of its metabolites during its metabolic activation process produce different in vitro DNA-phenyladducts in mitoplasts [5, 11]. As we reported previously [9] at least one of these adducts, N-7-phenylguanine, is excreted in the urine of rats in measurable amounts, probably through an excision-repair mechanism after an inhalation experiment. Now we found, after i.p. application of benzene in the urine of rats, a compound separated by cation-exchange chromatography that behaves like a synthezised N-7-phenylguanine reference substance with respect to its retention index and the UV-absorption. This finding could be confirmed by HPLC-measurements with reversed-phase carrier materials. Silylation and gaschromatographic/mass spectrometric (GC/MS) separation of the fraction, which contains the phenylguanine, revealed that these fractions contain further phenyl adducts. Furthermore we studied the time-dependent excretion of the DNA-base adduct. Surprisingly the excretion dropped to zero on the fourth day and showed a new increase thereafter. [ABSTRACT FROM AUTHOR]

Investigations with [C]benzene indicate the formation of base adducts . Experiments to separate adducts from urine of [C]benzene-exposed rats suggest the excretion of eight labeled compounds different from benzene metabolites. In order to obtain information about their structure we synthesized N7-, O-, C8- and N-phenylguanine. With regard to their chromatographic properties we compared these phenylguanines with products obtained by alkylation of guanine by metabolites of unlabeled and C-labeled benzene in with HPLC with UV detection and liquid scintillation counting. Furthermore GC/MS and ELISA techniques were used to detect N7-phenylguanine. Phenylguanines could not be identified in collected DNA fractions. The labeled compounds detected in urine of [C]benzene-exposed rats also showed deviations from the HPLC elution patterns of our reference substances. Even N7-phenylguanine, formerly suspected to be a urinary metabolite of benzene in the rat, could not be detected with these refined HPLC methods. With GC/MS a compound was found in trace amounts in concentrated rat urine samples, which had a similar fragmentation pattern to N7-phenylguanine. These data could not be confirmed by a sensitive immunological assay (ELISA). No. N7-phenylguanine was detected in purified rat urine samples. The results suggest the excretion of a hydroxylated phenylguanine which may be formed in liver or bone marrow DNA by highly reactive hydroxylated intermediates. The OH group might be lost because of the high temperatures during GC/MS measurements. A hydroxy group at the phenyl-ring of N7-phenylguanine will cause other elution properties in HPLC compared to N7-phenylguanine. [ABSTRACT FROM PUBLISHER]

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]