Krewet E, Verkoyen C, Müller G, Schell C, Popp W, Norpoth K
Institut für Hygiene und Arbeitsmedizin, Universitäts-Klinikum, Essen, Germany.
Carcinogenesis. 1993 Feb;14(2):245-50. doi: 10.1093/carcin/14.2.245.
Investigations with [14C]benzene indicate the formation of base adducts in vivo. Experiments to separate adducts from urine of [14C]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-, O6-, C8- and N2-phenylguanine. With regard to their chromatographic properties we compared these phenylguanines with products obtained by alkylation of guanine by metabolites of unlabeled and 14C-labeled benzene in vivo 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 [14C]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.
用[14C]苯进行的研究表明，体内会形成碱基加合物。从接触[14C]苯的大鼠尿液中分离加合物的实验表明，有8种标记化合物排出，这些化合物不同于苯的代谢产物。为了获取有关其结构的信息，我们合成了N7-、O6-、C8-和N2-苯基鸟嘌呤。关于它们的色谱性质，我们将这些苯基鸟嘌呤与未标记和14C标记苯的代谢产物在体内使鸟嘌呤烷基化所得到的产物进行了比较，采用带紫外检测的高效液相色谱法和液体闪烁计数法。此外，还使用气相色谱/质谱联用技术和酶联免疫吸附测定技术检测N7-苯基鸟嘌呤。在所收集的DNA组分中未鉴定出苯基鸟嘌呤。在接触[14C]苯的大鼠尿液中检测到的标记化合物在高效液相色谱洗脱模式上也与我们的参考物质有所偏差。即使是之前怀疑是大鼠体内苯的尿液代谢产物的N7-苯基鸟嘌呤，用这些改进的高效液相色谱方法也未能检测到。通过气相色谱/质谱联用技术在浓缩的大鼠尿液样本中发现了一种痕量化合物，其碎片模式与N7-苯基鸟嘌呤相似。这些数据无法通过灵敏的免疫测定法（酶联免疫吸附测定）得到证实。在纯化的大鼠尿液样本中未检测到N7-苯基鸟嘌呤。结果表明，可能是高反应性的羟基化中间体在肝脏或骨髓DNA中形成了一种羟基化苯基鸟嘌呤并排出。由于气相色谱/质谱联用测量过程中的高温，羟基可能会丢失。与N7-苯基鸟嘌呤相比，N7-苯基鸟嘌呤苯环上的羟基会导致其在高效液相色谱中具有不同的洗脱性质。
