Islam G A, Greibrokk T, Harvey R G, Ovrebø S
Department of Toxicology, National Institute of Occupational Health, Oslo, Norway.
Chem Biol Interact. 1999 Nov 30;123(2):133-48. doi: 10.1016/s0009-2797(99)00129-5.
Quantitation of protein-benzo[a]pyrene adducts represent a more sensitive analysis method than quantitation of benzo[a]pyrene-DNA adducts. By accurate analysis of benzo[a]pyrene-protein adducts several different molecular adduct forms can be studied. Male Wistar rats were injected i.p. with benzo[a]pyrene, and serum albumin was isolated and subjected to acid hydrolysis at 90 degrees C for 3 h. The hydrolysate was analyzed by HPLC with fluorescence detection. The HPLC profiles obtained after albumin hydrolysis from benzo[a]pyrene exposed animals were compared to similar HPLC profiles from in vitro adducted bovine serum albumin (BSA) and direct hydrolysis of both r-10,t-9-dihydrodiol-c-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE-III) and r-10,t-9-t-dihydrodiol-t-7,8-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-III). After acid hydrolysis of albumin from benzo[a]pyrene exposed rats, 6 fluorescent peaks were separated. Four of the peaks were isomers of benzo[a]pyrene-tetrahydrotetrols, (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol, (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol. In addition we found two fluorescent peaks, named X1 and X2 with retention times similar to the benzo[a]pyrene-tetrols. The unknown fluorescent peaks reacted similar to the four known tetrols in both dose response experiments and time course experiments. Fluorescent material with retention times equal to X1 and X2 were found after acid hydrolysis of syn-BPDE-III and anti-BPDE-III in acid and in hydrolysates from BSA treated in vitro with syn-BPDE-III and anti-BPDE-III. The ratio X1/X2 was relatively constant indicating epimerization equilibrium between these to species. Synchronous fluorescence analysis of fractions containing X1 or X2 from both in vivo and in vitro experiments showed fluorescence spectra characteristic of benzo[a]pyrene tetrols using a wavelength difference of 34 nm.
蛋白质 - 苯并[a]芘加合物的定量分析是一种比苯并[a]芘 - DNA加合物定量分析更灵敏的分析方法。通过对苯并[a]芘 - 蛋白质加合物的精确分析，可以研究几种不同的分子加合物形式。给雄性Wistar大鼠腹腔注射苯并[a]芘，分离血清白蛋白，并在90℃下进行3小时的酸水解。水解产物通过带荧光检测的高效液相色谱法进行分析。将苯并[a]芘暴露动物白蛋白水解后得到的高效液相色谱图谱与体外加合牛血清白蛋白（BSA）以及r - 10,t - 9 - 二氢二醇 - c - 7,8 - 氧基 - 7,8,9,10 - 四氢苯并[a]芘（顺式 - BPDE - III）和r - 10,t - 9 - t - 二氢二醇 - t - 7,8 - 氧基 - 7,8,9,10 - 四氢苯并[a]芘（反式 - BPDE - III）直接水解后的类似高效液相色谱图谱进行比较。对苯并[a]芘暴露大鼠的白蛋白进行酸水解后，分离出6个荧光峰。其中4个峰是苯并[a]芘 - 四氢四醇的异构体，即（±） - 苯并[a]芘 - r - 7,t - 8,9,10 - 四氢四醇、（±） - 苯并[a]芘 - r - 7,t - 8,9,c - 10 - 四氢四醇、（±） - 苯并[a]芘 - r - 7,t - 8,c - 9,t - 10 - 四氢四醇和（±） - 苯并[a]芘 - r - 7,t - 8,c - 9,10 - 四氢四醇。此外，我们还发现了两个荧光峰，命名为X1和X2，其保留时间与苯并[a]芘 - 四醇相似。在剂量反应实验和时间进程实验中，未知荧光峰的反应与四种已知四醇相似。在顺式 - BPDE - III和反式 - BPDE - III的酸水解产物以及体外经顺式 - BPDE - III和反式 - BPDE - III处理的BSA水解产物中，发现了保留时间与X1和X2相等的荧光物质。X1/X2的比值相对恒定，表明这两种物质之间存在差向异构化平衡。对体内和体外实验中含有X1或X2的馏分进行同步荧光分析，使用34 nm的波长差显示出苯并[a]芘四醇的荧光光谱特征。
