Kim P M, Wells P G
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Mol Pharmacol. 1996 Jan;49(1):172-81.
Bioactivation of phenytoin and related teratogens by peroxidases such as prostaglandin H synthase (PHS) may initiate hydroxyl radical (.OH) formation that is teratogenic. Salicylate is hydroxylated by .OH at the third and fifth carbon atoms, forming 2,3- and 2,5-dihydroxybenzoic acids (DHBA). In vivo salicylate metabolism produces only the 2,5-isomer, so 2,3-DHBA formation may reflect .OH production. In the present study, we validated the salicylate assay using the known .OH generator paraquat and evaluated .OH production by phenytoin. Female CD-1 mice were treated with paraquat (30 mg/kg, intraperitoneally) given 30 min after acetylsalicylic acid (ASA) (200 mg/kg, intraperitoneally). Blood was collected at 5, 15, and 30 min and 1 and 2 hr after paraquat, and plasma was analyzed for DHBA isomers and glucuronide conjugates by high performance liquid chromatography with electrochemical detection. Paraquat increased 2,3-DHBA formation 19.2-fold, with substantial inter-individual variability in the time of maximal formation (p = 0.0001). The 2,3-DHBA glucuronide conjugates in vivo and in hepatic microsomal studies amounted to approximately 11% and 0.43%, respectively, of total 2,3-DHBA equivalents. To investigate putative .OH production initiated via PHS-catalyzed phenytoin bioactivation, ASA was given 30 min before phenytoin (65 or 100 mg/kg, intraperitoneally), resulting in respective 7.6-fold (p = 0.02) and 14.2-fold (p = 0.003) increases in phenytoin-initiated maximal 2,3-DHBA formation. Maximal 2,3-DHBA formation was 2.1-fold higher when ASA was administered after rather than before the same dose (65 mg/kg) of phenytoin (p = 0.03), indicating ASA inhibition of PHS-catalyzed phenytoin bioactivation. Urinary analysis was much less sensitive, and the 2,5-isomer reflected enzymatic rather than .OH-mediated hydroxylation. The paraquat studies demonstrate the importance of timing in accurately quantifying 2,3-DHBA formation and suggest that glucuronidation does not interfere. The substantial, dose-dependent initiation of 2,3-DHBA formation by phenytoin, and its inhibition by ASA, provide the first in vivo evidence that PHS-dependent .OH formation could contribute to the molecular mechanism of phenytoin teratogenesis.
诸如前列腺素H合酶（PHS）等过氧化物酶可使苯妥英及相关致畸剂发生生物活化，这可能引发具有致畸性的羟基自由基（·OH）的形成。水杨酸可被·OH在其第三个和第五个碳原子处羟基化，形成2,3 - 二羟基苯甲酸（DHBA）和2,5 - 二羟基苯甲酸（DHBA）。体内水杨酸代谢仅产生2,5 - 异构体，因此2,3 - DHBA的形成可能反映·OH的产生。在本研究中，我们使用已知的·OH生成剂百草枯验证了水杨酸测定法，并评估了苯妥英引发的·OH产生。雌性CD - 1小鼠在腹腔注射乙酰水杨酸（ASA）（200 mg/kg）30分钟后，腹腔注射百草枯（30 mg/kg）。在注射百草枯后的5、15和30分钟以及1和2小时采集血液，通过高效液相色谱 - 电化学检测法分析血浆中的DHBA异构体和葡糖醛酸苷缀合物。百草枯使2,3 - DHBA的形成增加了19.2倍，最大形成时间存在显著的个体间差异（p = 0.0001）。体内和肝微粒体研究中2,3 - DHBA葡糖醛酸苷缀合物分别占2,3 - DHBA总当量的约11%和0.43%。为了研究通过PHS催化的苯妥英生物活化引发的假定·OH产生，在腹腔注射苯妥英（65或100 mg/kg）前30分钟给予ASA，导致苯妥英引发的最大2,3 - DHBA形成分别增加了7.6倍（p = 0.02）和14.2倍（p = 0.003）。当在相同剂量（65 mg/kg）的苯妥英之后而非之前给予ASA时，最大2,3 - DHBA形成高2.1倍（p = 0.03），表明ASA抑制了PHS催化的苯妥英生物活化。尿液分析的敏感性要低得多，并且2,5 - 异构体反映的是酶促羟基化而非·OH介导的羟基化。百草枯研究证明了准确量化2,3 - DHBA形成时时间的重要性，并表明葡糖醛酸化不会产生干扰。苯妥英大量的、剂量依赖性地引发2,3 - DHBA形成以及其被ASA抑制，首次提供了体内证据，表明PHS依赖性·OH形成可能参与苯妥英致畸作用的分子机制。
