DNA adduction by phenol, hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced 32P-postlabeling of adducts as labeled nucleoside bisphosphates, dinucleotides and nucleoside monophosphates.
DNA adduction by phenol, hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced 32P-postlabeling of adducts as labeled nucleoside bisphosphates, dinucleotides and nucleoside monophosphates.
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苯酚、氢醌或苯醌在体外而非体内进行 DNA 加合：核酸酶 P1 增强加合物的 32P 后标记，作为标记的核苷二磷酸、二核苷酸和核苷单磷酸。
DOI：
10.1093/carcin/11.8.1349
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发表时间：
1990
期刊：
影响因子：
4.7
通讯作者：
C. Mackerer
中科院分区：
文献类型：
--
作者：
M. Reddy;W. Bleicher;G. Blackburn;C. Mackerer
苯的致癌性被认为部分是由其化学反应性代谢产物苯醌（BQ）介导的，苯醌是由中间代谢物苯酚和氢醌（HQ）形成的。我们已经评估了这些化学品的DNA结合能力在体外和体内的postlabeling。用苯酚和HQ处理培养的大鼠Zymbal腺或BQ与DNA直接反应产生DNA加合物，其可通过核酸酶P1增强的32 P后标记试验检测为5 '-32 P标记的3'，5 '-二磷酸产物。该测定中灵敏度的增强是基于先前的发现，即核酸酶P1水解附着于正常核苷酸的3'侧的磷酸，但不水解大多数芳香族/大体积加合核苷酸的相应磷酸。同样基于核酸酶P1的这种水解性质，我们开发了一种额外的灵敏程序，该程序允许检测作为二核苷酸pXpN或核苷一磷酸pX的5 '-32 P标记产物的DNA损伤，其中X和N分别表示加合核苷和正常核苷。在后一种测定中，首先用核酸酶P1和酸性磷酸酶消化加合的DNA以产生XpN和N。然后将后者用32 P标记以产生[5 '-32 P] pXpN或用毒液磷酸二酯酶处理以获得[5'-32 P]pX。酶促条件的优化后，修改后的核酸酶P1测定产生的加合物回收率类似于那些通过双磷酸盐测定在体外苯酚，HQ-和BQ-DNA加合物。在雌性Sprague-Dawley大鼠的骨髓、Zymbal腺、肝脏和脾脏中，在4次单次每日p.o.剂量为75 mg/kg苯酚或150 mg/kg苯酚/HQ（1：1）。我们的研究结果表明，苯酚，HQ和BQ在体外产生加合物，但相应的加合物没有检测到在体内与苯酚和苯酚/HQ，即使当测量的标准和修改的核酸酶P1后标记方法能够检测1加合物在10（9-10）DNA碱基。
The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol/HQ, even when measured by the standard and modified nuclease P1 postlabeling methods capable of detecting 1 adduct in 10(9-10) DNA bases.