Islam G A, Greibrokk T, Rise F, Ovrebø S
Department of Toxicology, National Institute of Occupational Health, P.O. Box 8149 Dep., N-0033, Oslo, Norway.
Mutat Res. 1999 Jan 25;423(1-2):47-54. doi: 10.1016/s0027-5107(98)00224-3.
Determination of benzo[a]pyrene-DNA or protein adducts with high performance liquid chromatography (HPLC) after acid hydrolysis at high temperature (90 degrees C) enables four isomers of benzo[a]pyrene tetrahydrotetrol to be identified and quantitated. We have investigated the effect of acid treatment of benzo[a]pyrene-tetrahydrotetrol isomers using HPLC and nuclear magnetic resonance spectroscopy (NMR) analysis. By HPLC, we found reversible epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and of (+/-)-benzo[a]pyrene-r-7,t-8,c-9, t-10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,c-9, 10-tetrahydrotetrol, but no interconversion between the two isomer groups. After acid hydrolysis, we found an equilibrium of 87% (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol and 9% (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and 68% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol and 20% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. Minor amounts of two unknown compounds with similar chromatographic characteristics were also found. We have established a NMR method for determination of underivatized (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol confirming the epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10- tetrahydrotetrol. (+/-)-Benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol was treated with aqueous hydrochloric acid in tetrahydro- furan-d8 to give (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol at 57 degrees C while observing the 1H NMR resonances at 500 MHz. Gradient-selected correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed to confirm the assignments of the aliphatic hydrogens in the product (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-terahydrotetrol. Thus, when analyzing benzo[a]pyrene-DNA or protein adducts by cleaving the adducts with acid hydrolysis, the only ratio of biological significance is between (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol, due to interconversion (epimerization) at C-10.
在高温（90摄氏度）下进行酸水解后，采用高效液相色谱（HPLC）测定苯并[a]芘 - DNA或蛋白质加合物，能够鉴定和定量苯并[a]芘四氢四醇的四种异构体。我们利用HPLC和核磁共振光谱（NMR）分析研究了酸处理对苯并[a]芘 - 四氢四醇异构体的影响。通过HPLC，我们发现（±） - 苯并[a]芘 - r - 7，t - 8，9，10 - 四氢四醇可逆差向异构化为（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇，以及（±） - 苯并[a]芘 - r - 7，t - 8，c - 9，t - 10 - 四氢四醇可逆差向异构化为（±） - 苯并[a]芘 - r - 7，t - 8，c - 9，10 - 四氢四醇，但两个异构体组之间没有相互转化。酸水解后，我们发现平衡状态为87%的（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇、9%的（±） - 苯并[a]芘 - r - 7，t - 8，9，10 - 四氢四醇、68%的（±） - 苯并[a]芘 - r - 7，t - 8，c - 9，10 - 四氢四醇和20%的（±） - 苯并[a]芘 - r - 7，t - 8，c - 9，t - 10 - 四氢四醇。还发现了少量具有相似色谱特征的未知化合物。我们建立了一种NMR方法来测定未衍生化的（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇和（±） - 苯并[a]芘 - r - 7，t - 8，9，10 - 四氢四醇，证实了（±） - 苯并[a]芘 - r - 7，t - 8，9，10 - 四氢四醇向（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇的差向异构化。在5７摄氏度下，将（±） - 苯并[a]芘 - r - 7，t - 8，9，10 - 四氢四醇与四氢呋喃 - d8中的盐酸水溶液反应，得到（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇，同时在500 MHz下观察1H NMR共振。进行了梯度选择相关光谱（COSY）、异核多量子相关（HMQC）和异核多键相关（HMBC）实验，以确认产物（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇中脂肪族氢的归属。因此，当通过酸水解裂解加合物来分析苯并[a]芘 - DNA或蛋白质加合物时，由于在C - 10处的相互转化（差向异构化），唯一具有生物学意义的比例是（±） - 苯并[a]芘 - r - 7，t - 8，9，c - 10 - 四氢四醇加（±） - 苯并[a]芘 - r - 7，t - 8，9，10 - 四氢四醇与（±） - 苯并[a]芘 - r - 7,t - 8,c - 9,10 - 四氢四醇加（±） - 苯并[a]芘 - r - 7,t - 8,c - 9,t - 10 - 四氢四醇之间的比例。
