Manno M, Tolando R, Ferrara R, Rezzadore M, Cazzaro S
Institute of Occupational Medicine, University of Padua Medical School, Italy.
Toxicology. 1995 Jun 26;100(1-3):175-83. doi: 10.1016/0300-483x(95)03083-r.
Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods, i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.
在1 mM连二亚硫酸钠作为还原剂存在的情况下，将人血红蛋白（Hb）、高铁血红蛋白（MHA）或大鼠肝脏微粒体细胞色素P-450（P-450）与1 mM四氯化碳（CCl4）、三氯溴甲烷（CCl3Br）、氯仿（CHCl3）或二氯甲烷（CH2Cl2）以微摩尔浓度进行厌氧孵育。孵育5分钟后，通过各种方法测量血红素，即与一氧化碳的结合光谱、吡啶-血红素原血红素测定法和卟啉荧光法，并对这四种类似物进行比较。观察到，对于CCl3Br、CCl4和CHCl3，在所有三种血红蛋白系统中均有统计学上显著的损失，但CH2Cl2没有。对于Hb，CCl3Br导致的损失（血红素测定法，63%；卟啉荧光法，48%；一氧化碳结合法，24%）大于CCl4（血红素测定法，31%）或CHCl3（血红素测定法，13%）。另一方面，对于MHA，CCl4导致了显著的损失（血红素测定法，88%；卟啉荧光法，83%；一氧化碳结合法，67%），这大于CCl3Br导致的损失（血红素测定法，49%；卟啉荧光法，38%；一氧化碳结合法，25%）。CHCl3未导致损失。最后，对于微粒体，CCl4导致的失活（一氧化碳结合法，58%；血红素测定法，50%；卟啉荧光法，33%）大于CCl3Br（一氧化碳结合法，33%；血红素测定法，10%）或CHCl3（血红素测定法，9%；一氧化碳结合法，6%）。在另一组类似实验中，离子对反相高效液相色谱法显示，在CCl3Br与Hb或微粒体孵育以及CCl4与Hb孵育过程中，形成了底物依赖性的血红素衍生产物。在所有用于Hb的方法中，观察到自由基形成潜力（CCl3Br > CCl4 > CHCl3 > CH2Cl2）与血红素失活程度之间存在相关性，但对于微粒体P-450或MHA则未观察到这种相关性。结果表明，这些卤代甲烷可能被不同的血红蛋白以不同方式激活，并表明它们进行还原代谢的潜在能力可能不是这些化学物质导致P-450血红素失活所涉及的唯一关键因素。
