Booth E D, Aston J P, van den Berg P T, Baan R A, Riddick D A, Wade L T, Wright A S, Watson W P
Shell Research Ltd, Sittingbourne Research Centre, Kent, UK.
Carcinogenesis. 1994 Oct;15(10):2099-106. doi: 10.1093/carcin/15.10.2099.
An immunoenrichment procedure has been developed for applications in the detection and identification of a broad range of polycyclic aromatic hydrocarbon (PAH)-DNA adducts at very low abundance. The procedures are based on a monoclonal antibody raised to r-7,trans-8,trans-9-trihydroxy-cis-10-(N2-deoxyguanosyl-5'-phospha te)- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-N2-dG) which has been tested for cross-reactivity towards DNA and proteins (bovine serum albumin, chicken gamma globulin and human globin) covalently modified with a range of PAH diol-epoxides. The antibody recognised DNA adducted with the diol-epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene, dibenz[a,h]anthracene or picene. The antibody also cross-reacted with the 7,8,9,10-tetraol derived from benzo[a]pyrene and the 1,2,3,4-tetraols of benz[a]anthracene and chrysene. The degree of cross-reactivity was greatest for PAH adducts with structural features similar to anti-BPDE-N2-dG proximate to the base attachment. The antibody also recognised a range of PAHs adducted to human globin; these included adducts of benzo[a]pyrene, benz[a]anthracene and chrysene diol-epoxides. This wide range of recognition provides good evidence for the class-specific recognition of PAH adducts by the antibody. When immobilized on Sepharose 4B and used in the immunoadsorption purification of adducted nucleotides, the antibody selectively enriched adducts of benzo[a]pyrene, benz[a]anthracene and chrysene from normal nucleotides. Quantitative measurements with [14C]benzo[a]pyrene-DNA adducts showed that the immobilized antibody was able to enrich benzo[a]pyrene adducts from a DNA hydrolysate containing adducts at a level of 1 adduct/10(10) normal nucleotides. In addition, this immunoadsorption technique was effective in enriching a mixture of DNA adducts formed in the skin of CF1 mice treated cutaneously with a mixture of [3H]benzo[a]pyrene and [3H]chrysene. Class-specific immunoenrichment procedures for DNA adducts are important in assisting the identification of genotoxic components in complex mixtures. The performance characteristics of this immobilized antibody suggest that it may be suitable for application in the detection, identification and monitoring of human exposures to low levels of PAHs.
已开发出一种免疫富集程序，用于检测和鉴定极低丰度下的多种多环芳烃（PAH）-DNA加合物。该程序基于一种针对r-7,反式-8,反式-9-三羟基-顺式-10-（N2-脱氧鸟苷-5'-磷酸）-7,8,9,10-四氢苯并[a]芘（抗-BPDE-N2-dG）产生的单克隆抗体，该抗体已针对与一系列PAH二醇环氧化物共价修饰的DNA和蛋白质（牛血清白蛋白、鸡γ球蛋白和人球蛋白）的交叉反应性进行了测试。该抗体识别与苯并[a]芘、苯并[a]蒽、屈、二苯并[a,h]蒽或苊的二醇环氧化物加成的DNA。该抗体还与源自苯并[a]芘的7,8,9,10-四醇以及苯并[a]蒽和屈的1,2,3,4-四醇发生交叉反应。对于在碱基连接附近具有与抗-BPDE-N2-dG相似结构特征的PAH加合物，交叉反应程度最大。该抗体还识别一系列与人球蛋白加成的PAHs；这些包括苯并[a]芘、苯并[a]蒽和屈二醇环氧化物的加合物。这种广泛的识别为该抗体对PAH加合物的类别特异性识别提供了有力证据。当固定在琼脂糖4B上并用于加合核苷酸的免疫吸附纯化时，该抗体从正常核苷酸中选择性富集苯并[a]芘、苯并[a]蒽和屈的加合物。用[14C]苯并[a]芘-DNA加合物进行的定量测量表明，固定化抗体能够从含有1个加合物/10(10)个正常核苷酸水平加合物的DNA水解物中富集苯并[a]芘加合物。此外，这种免疫吸附技术有效地富集了用[3H]苯并[a]芘和[3H]屈的混合物经皮处理的CF1小鼠皮肤中形成的DNA加合物混合物。DNA加合物的类别特异性免疫富集程序对于协助鉴定复杂混合物中的遗传毒性成分很重要。这种固定化抗体的性能特征表明它可能适用于检测、鉴定和监测人类低水平接触PAHs的情况。
