Lynch A M, Murray S, Zhao K, Gooderham N J, Boobis A R, Davies D S
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Carcinogenesis. 1993 Feb;14(2):191-4. doi: 10.1093/carcin/14.2.191.
Incubation of mouse serum albumin with the food borne carcinogen [2-14C]-Amino-3,8,-dimethylimidazo[4,5-f]quinoxaline (14C-MeIQx) in the presence of mouse hepatic microsomes and an NADPH-regenerating system in vitro resulted in the formation of adducts of MeIQx with albumin, which increased proportionately with time for at least 120 min (approximately 1 pmol equivalents/mg of protein/min). We have previously shown in male Swiss Webster mice in vivo that 14C-MeIQx bound covalently to serum proteins and that the formation of adducts was dose dependent. 14C-MeIQx (100 mg/kg, i.p.) was administered to male (MF1) mice which were killed 24 h later. Serum albumin was purified by affinity chromatography and covalent binding of 14C-MeIQx was assessed. Total covalent binding of MeIQx to albumin was 14.0 +/- 5.2 pmol per mg albumin, which was 5-fold greater than to haemoglobin. Following mild acid hydrolysis, 1.25 pmol MeIQx per mg albumin was liberated as free amine, as determined by gas chromatography negative ion mass spectrometry (GC-MS). This represents 9% of total MeIQx adducted to albumin in vivo (cf 1.3% adducted to haemoglobin). These results suggested that adducts of MeIQx with serum albumin should provide a significantly more sensitive dosimeter than those with haemoglobin. We therefore investigated this approach with serum protein samples from three volunteers. Human serum albumin and non-serum albumin protein fractions were separated by affinity chromatography, before being subjected to GC-MS analysis for hydrolysable adducts of MeIQx. The levels of MeIQx in control samples, and from the release of the putative sulphinamide adducts in hydrolysed samples were below the limits of detection of the GC-MS assay (29 +/- 2.6 amol MeIQx/mg albumin). Despite an increase of 2 orders of magnitude in sensitivity, compared with haemoglobin, it is unlikely that the sulphinamide adduct of MeIQx with human serum albumin can be used as a dosimeter for human aminoimidazoazaarene exposure.
在体外，将小鼠血清白蛋白与食源性致癌物[2-¹⁴C]-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉（¹⁴C-MeIQx）在小鼠肝微粒体和NADPH再生系统存在的情况下进行温育，结果形成了MeIQx与白蛋白的加合物，其至少在120分钟内随时间成比例增加（约1 pmol当量/毫克蛋白质/分钟）。我们之前在雄性瑞士韦伯斯特小鼠体内已表明，¹⁴C-MeIQx与血清蛋白共价结合，且加合物的形成呈剂量依赖性。给雄性（MF1）小鼠腹腔注射¹⁴C-MeIQx（100毫克/千克），24小时后将其处死。通过亲和色谱法纯化血清白蛋白，并评估¹⁴C-MeIQx的共价结合情况。MeIQx与白蛋白的总共价结合量为每毫克白蛋白14.0±5.2 pmol，这比与血红蛋白的结合量高5倍。经过温和酸水解后，通过气相色谱负离子质谱法（GC-MS）测定，每毫克白蛋白释放出1.25 pmol的MeIQx作为游离胺。这占体内与白蛋白结合的总MeIQx的9%（相比之下，与血红蛋白结合的为1.3%）。这些结果表明，MeIQx与血清白蛋白的加合物应比与血红蛋白的加合物提供显著更灵敏的剂量计。因此，我们用三名志愿者的血清蛋白样本研究了这种方法。在通过GC-MS分析MeIQx的可水解加合物之前，通过亲和色谱法分离人血清白蛋白和非血清白蛋白蛋白组分。对照样本中以及水解样本中假定的亚磺酰胺加合物释放的MeIQx水平低于GC-MS测定的检测限（29±2.6 amol MeIQx/毫克白蛋白）。尽管与血红蛋白相比灵敏度提高了2个数量级，但MeIQx与人血清白蛋白的亚磺酰胺加合物不太可能用作人体氨基咪唑并氮杂芳烃暴露的剂量计。
