Nagai F, Ushiyama K, Satoh K, Kano I
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Japan.
Chem Biol Interact. 1990;76(2):163-79. doi: 10.1016/0009-2797(90)90086-3.
The interaction of o-phenylphenol (OPP) and its metabolites with DNA was examined. As a model system, the reactivities of OPP and its metabolites with DNA were studied by using pUC18 DNA. The major metabolite formed in vitro from OPP by mixed function oxidase was phenylhydroquinone (PHQ). This result corresponds to the findings that PHQ in the form of glucuronide conjugate was the main product detected in bladder of OPP fed rats in vivo. When supercoiled pUC18 DNA (form I) was incubated with PHQ at concentrations from 1 X 10(-6) M to 2 X 10(-1) M, DNA strand scission by PHQ was observed at a concentration as low as 1 X 10(-5) M and the amount of linear form (form III) increased with increasing PHQ concentration. PHQ causes DNA strand scission. The DNA cleavage by OPP and phenyl-p-benzoquinone (PBQ) was barely detectable. The DNA cleavage by PHQ was inhibited by superoxide dismutase (SOD), catalase and several oxygen radical scavengers such as polyethylene glycol, tert-butanol, dimethyl sulfoxide, sodium azide, sodium benzoate, bovine serum albumin and methionine. The production of superoxide radical from PHQ was confirmed by cytochrome c reduction assay. These results indicate that the oxygen radicals such as superoxide, hydroxyl radicals and some others generated in the process of oxidation of PHQ in aqueous solution are responsible for the DNA cleavage. In order to identify the sites of cleavage of DNA by PHQ, a 5'-end 32P-labeled 206 base-pair EcoRI-BglI fragment of pUC18 DNA was incubated with PHQ. The DNA was then analyzed by sequencing gel electrophoresis followed by autoradiography. When the DNA was incubated with PHQ and further treated with piperidine, cleavage was detected relatively more frequently at guanine residues. The attack seemed to occur at guanine residues in general, but was not restricted to guanines with specific residues in the vicinity.
研究了邻苯基苯酚（OPP）及其代谢产物与DNA的相互作用。作为一个模型系统，利用pUC18 DNA研究了OPP及其代谢产物与DNA的反应活性。由混合功能氧化酶在体外将OPP转化形成的主要代谢产物是苯氢醌（PHQ）。这一结果与以下发现相符：在体内给大鼠喂食OPP后，在其膀胱中检测到的主要产物是葡糖醛酸共轭形式的PHQ。当超螺旋pUC18 DNA（形式I）与浓度范围为1×10⁻⁶ M至2×10⁻¹ M的PHQ一起孵育时，在低至1×10⁻⁵ M的浓度下就观察到PHQ导致DNA链断裂，并且线性形式（形式III）的量随着PHQ浓度的增加而增加。PHQ会导致DNA链断裂。几乎检测不到OPP和对苯醌（PBQ）对DNA的切割。超氧化物歧化酶（SOD）、过氧化氢酶以及几种氧自由基清除剂如聚乙二醇、叔丁醇、二甲基亚砜、叠氮化钠、苯甲酸钠、牛血清白蛋白和蛋氨酸可抑制PHQ对DNA的切割。通过细胞色素c还原试验证实了PHQ会产生超氧自由基。这些结果表明，在水溶液中PHQ氧化过程中产生的超氧、羟自由基等氧自由基是导致DNA切割的原因。为了确定PHQ对DNA的切割位点，将一个5'端32P标记的pUC18 DNA的206个碱基对的EcoRI - BglI片段与PHQ一起孵育。然后通过测序凝胶电泳和放射自显影对DNA进行分析。当DNA与PHQ一起孵育并进一步用哌啶处理时，在鸟嘌呤残基处相对更频繁地检测到切割。这种攻击似乎总体上发生在鸟嘌呤残基处，但不限于附近具有特定残基的鸟嘌呤。
