Gopalakrishnan R, Morse M A, Lu J, Weghorst C M, Sabourin C L, Stoner G D, Murphy S E
Department of Pathology, College of Medicine and Public Health, The Ohio State University, Columbus 43210, USA.
Carcinogenesis. 1999 May;20(5):885-91. doi: 10.1093/carcin/20.5.885.
N-nitrosobenzylmethylamine (NBzMA) must be metabolically activated to exert its carcinogenic potential and is a potent inducer of tumors in the rat esophagus. The activation is believed to occur in the esophagus. Although the pathways of NBzMA metabolism are well studied, the principal cytochrome P450 enzyme(s) (P450) responsible for catalyzing its activation is unknown. Several preliminary studies have suggested that this enzyme may belong to the P450 2A family. We report here that P450 2A3 expressed in a baculovirus system metabolizes NBzMA, predominantly by methylene hydroxylation. To determine whether or not P450 2A3 is present in the rat esophagus, the relative level of P450 2A3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The mRNA levels of P450 2A3 were compared with the levels of P450 2A1 and 2A2 mRNA in the esophagus, liver, lung and nasal mucosa. P450 2A3 mRNA was detected in rat nasal mucosa, lung and esophagus, but not in liver, whereas P450 2A1 and 2A2 mRNAs were detected only in the liver. To determine the relative expression of P450 2A3 in each tissue, quantitative RT-PCR with PCR-MIMICS used as internal standards was performed. The expression level in the nasal mucosa was by far the greatest. The expression in the lung and esophagus was 60- and 1600-fold less, respectively. Using antibodies to P450 2A4/5 and P450 2A10/11 a 50 kDa immunoreactive protein was detected in all three tissues by western blot analysis. This is consistent with the expression of P450 2A3 in these tissues. However, the amount of protein detected in the nasal mucosa was much greater than that in the esophagus or lung. The expression of P450 2A protein was similar in the lung and esophagus. The rate of coumarin 7-hydroxylation in cultured rat esophagus was very low. This is a reaction efficiently catalyzed by P450 2A5, 2A6 and 2A10. In summary, our results clearly demonstrate the presence of P450 2A3 protein and mRNA in the esophagus, but the amounts are low and may not be sufficient to account for NBzMA activation in this tissue.
N-亚硝基苄基甲胺（NBzMA）必须经过代谢活化才能发挥其致癌潜力，它是大鼠食管肿瘤的强效诱导剂。这种活化作用被认为发生在食管中。尽管NBzMA的代谢途径已得到充分研究，但负责催化其活化的主要细胞色素P450酶尚不清楚。几项初步研究表明，这种酶可能属于P450 2A家族。我们在此报告，在杆状病毒系统中表达的P450 2A3可代谢NBzMA，主要通过亚甲基羟基化作用。为了确定大鼠食管中是否存在P450 2A3，通过逆转录聚合酶链反应（RT-PCR）测定了P450 2A3 mRNA的相对水平。将食管、肝脏、肺和鼻黏膜中P450 2A3 mRNA的水平与P450 2A1和2A2 mRNA的水平进行了比较。在大鼠鼻黏膜、肺和食管中检测到了P450 2A3 mRNA，但在肝脏中未检测到，而P450 2A1和2A2 mRNA仅在肝脏中被检测到。为了确定P450 2A3在每个组织中的相对表达，以PCR-MIMICS作为内标进行了定量RT-PCR。鼻黏膜中的表达水平迄今为止是最高的。肺和食管中的表达分别低60倍和1600倍。通过蛋白质印迹分析，使用针对P450 2A4/5和P450 2A10/11的抗体在所有三个组织中检测到了一种50 kDa的免疫反应性蛋白。这与P450 2A3在这些组织中的表达一致。然而，在鼻黏膜中检测到的蛋白量远大于食管或肺中的蛋白量。P450 2A蛋白在肺和食管中的表达相似。培养的大鼠食管中香豆素7-羟基化的速率非常低。这是一个由P450 2A5、2A6和2A10有效催化的反应。总之，我们的结果清楚地证明了食管中存在P450 2A3蛋白和mRNA，但含量较低，可能不足以解释该组织中NBzMA的活化作用。
