Tayama S, Nakagawa Y
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Japan.
Mutat Res. 1994 Jul;324(3):121-31. doi: 10.1016/0165-7992(94)90056-6.
Phenylhydroquinone (PHQ), a metabolite of o-phenylphenol (OPP), is easily autoxidized to phenylbenzoquinone (PBQ) via the semiquinone (phenylsemiquinone, PSQ) with concomitant production of superoxide anion radicals (O2-.). We have used scavengers of active oxygen species to examine whether or not O2-. produced during oxidation of PHQ is related to cell damage in CHO-K1 cells. PHQ at 10 micrograms/ml (3-h treatment) induced sister-chromatid exchange (SCE), endoreduplication (ERD) and cell-cycle delay in CHO-K1 cells. These effects were inhibited by catalase (280 U/ml), a scavenger of hydrogen peroxide (H2O2), as well as by the reductants, ascorbate (3 mM) and GSH (1 mM). Mannitol (50 mM), a scavenger of hydroxyl radical (OH.), was ineffective and superoxide dismutase (SOD, 150 U/ml), a scavenger of O2-., or SOD plus catalase rather intensified the toxicity as did aminotriazole (20 mM), an inhibitor of catalase. Analyses of incubation solutions by HPLC showed that the extent of cell damage is correlated with PHQ loss; catalase suppressed PHQ loss, whereas SOD promoted it. The correlation was more clearly seen in the time courses of cell death and PHQ loss during incubation of PHQ with each of the scavengers of active oxygen species. These results show that neither O2-. nor OH. participates in the cell damage, but rather H2O2 generated via dismutation of O2-. may participate, probably by accelerating the autoxidation of PHQ and thus causing an increase in the production of toxic intermediates. In fact, conversion of PHQ to PBQ, a reactive product, was demonstrated during incubation with PHQ in phosphate-buffered saline by following the changes in UV-visible spectra of PHQ. Inclusion of H2O2 (0.2 or 1 mM) in the incubation mixture accelerated the PHQ loss. The present results can be explained in terms of the autoxidation mechanism of hydroquinone proposed by O'Brien (1991). Different from the results in the absence of S9 mix, the cell damage induced by 50 micrograms/ml OPP in the presence of S9 mix was not influenced by any of the scavengers of active oxygen species used. We conclude that PHQ causes cytotoxic and genotoxic effects through its autoxidation, both enzymatic and nonenzymatic, and that reactive intermediate(s) such as PSQ and/or PBQ may be ultimately responsible for the effects. H2O2 formed during the oxidation process participates in the damaging effects caused in the absence of S9 mix, probably by accelerating the autoxidation.
对苯二酚（PHQ）是邻苯基苯酚（OPP）的一种代谢产物，很容易通过半醌（苯半醌，PSQ）自动氧化为苯醌（PBQ），同时产生超氧阴离子自由基（O2-.）。我们使用活性氧清除剂来研究PHQ氧化过程中产生的O2-. 是否与CHO-K1细胞中的细胞损伤有关。10微克/毫升的PHQ（处理3小时）可诱导CHO-K1细胞发生姐妹染色单体交换（SCE）、核内复制（ERD）和细胞周期延迟。过氧化氢酶（280单位/毫升），一种过氧化氢（H2O2）清除剂，以及还原剂抗坏血酸（3毫摩尔）和谷胱甘肽（1毫摩尔）可抑制这些效应。甘露醇（50毫摩尔），一种羟基自由基（OH.）清除剂，无效，超氧化物歧化酶（SOD，150单位/毫升），一种O2-. 清除剂，或SOD加过氧化氢酶反而增强了毒性，氨基三唑（20毫摩尔），一种过氧化氢酶抑制剂也有同样的效果。通过高效液相色谱法对孵育溶液进行分析表明，细胞损伤程度与PHQ的损失相关；过氧化氢酶抑制了PHQ的损失，而SOD则促进了它的损失。在PHQ与每种活性氧清除剂孵育期间，细胞死亡和PHQ损失的时间进程中这种相关性更明显。这些结果表明，O2-. 和OH. 都不参与细胞损伤，而是O2-. 歧化产生的H2O2可能参与其中，可能是通过加速PHQ的自动氧化，从而导致有毒中间体的产生增加。事实上，在磷酸盐缓冲盐水中与PHQ孵育期间，通过跟踪PHQ紫外可见光谱的变化，证明了PHQ向PBQ（一种反应性产物）的转化。在孵育混合物中加入H2O2（0.2或1毫摩尔）加速了PHQ的损失。目前的结果可以根据奥布赖恩（1991年）提出的对苯二酚自动氧化机制来解释。与不存在S9混合物时的结果不同，在存在S9混合物的情况下，50微克/毫升OPP诱导的细胞损伤不受所用任何活性氧清除剂的影响。我们得出结论，PHQ通过其酶促和非酶促自动氧化引起细胞毒性和遗传毒性效应，并且反应性中间体如PSQ和/或PBQ可能最终对这些效应负责。氧化过程中形成的H2O2可能通过加速自动氧化参与在不存在S9混合物时引起的损伤效应。
