Rao D N, Cederbaum A I
Department of Biochemistry, Mount Sinai School of Medicine, City University of New York, NY 10029, USA.
Free Radic Biol Med. 1997;22(3):439-46. doi: 10.1016/s0891-5849(96)00335-8.
Rifamycin S and rifabutin are clinical drugs used to treat tuberculosis and leprosy. The formation of reactive oxygen species during the redox-cycling of rifamycin S (quinone) and rifabutin (quinonimine) was evaluated. The semiquinone (or semiquinonimine) and hydroquinone (or hydroquinonimine) formed during the reduction of the parent molecules by microsomal electron transfer in the presence of nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide-adenine dinucleotide, reduced (NADH) reoxidizes in air to generate superoxide radical and hydrogen peroxide. In the presence of added iron, hydroxyl radicals, formed by the Fenton reaction, were detected using 5,5'-dimethyl-1-pyroline-N-oxide as the spin-trap. Rifamycin S, a quinone, redox cycles more efficiently than rifabutin, a quinonimine, as approximately five times the concentration of hydroxyl radical adduct of 5,5'-dimethyl-1-pyroline-N-oxide (DMPO) was detected, when compared with rifabutin. The NADPH-dependent microsomal production of hydroxyl radical in the presence of rifamycin S was somewhat higher than the NADH-rifamycin S system with most iron chelators. However, with rifabutin, NADH-dependent microsomal production of hydroxyl radical was higher than that found with the NADPH-rifabutin system. An exception was the iron chelator, diethylene-triamine-pentacetic acid (DTPA), in which NADPH-dependent rates exceeded the rates with NADH with both antibiotics. Rat liver sub-mitochondrial particles also generated hydroxyl radical in the presence of NADH and either rifamycin S or rifabutin. The electron transport chain inhibitors such as rotenone and antimycin A enhanced the signal intensity of DMPO-OH, suggesting NADH dehydrogenase (complex I) as the major component involved in the reduction of rifamycin S. Rifamycin S was shown to be readily reduced to rifamycin SV, the corresponding hydroquinone by Fe(II); under similar conditions Fe(II) did not reduce rifabutin. Using optical spectroscopy, we determined that rifamycin S forms a complex with Fe(II). The stoichiometry of the complex was Fe(rifamycin S)3 in phosphate buffer at pH 7.4. Rifabutin did not form a detectable complex with Fe(II). The redox cycling of rifamycin S and rifabutin did not cause microsomal lipid peroxidation. In fact, the Fe:ATP induced lipid peroxidation was completely inhibited by these two molecules. These results indicate that rifamycin S and rifabutin can interact with rat liver microsomes to undergo redox-cycling, with the subsequent production of hydroxyl radicals when iron complexes are present. Compared to NADPH, NADH is almost as effective (rifamycin S) or even more effective (rifabutin) in promoting these interactions. These interactions may play a role in the hepatotoxicity associated with the use of these antibiotics.
利福霉素S和利福布汀是用于治疗结核病和麻风病的临床药物。评估了利福霉素S（醌）和利福布汀（醌亚胺）氧化还原循环过程中活性氧的形成。在烟酰胺腺嘌呤二核苷酸磷酸（还原型，NADPH）或烟酰胺腺嘌呤二核苷酸（还原型，NADH）存在下，通过微粒体电子转移使母体分子还原过程中形成的半醌（或半醌亚胺）和对苯二酚（或对苯二酚亚胺）在空气中再氧化，生成超氧自由基和过氧化氢。在添加铁的情况下，使用5,5'-二甲基-1-吡咯啉-N-氧化物作为自旋捕捉剂检测到由芬顿反应形成的羟基自由基。与利福布汀相比，醌类的利福霉素S氧化还原循环效率更高，因为检测到的5,5'-二甲基-1-吡咯啉-N-氧化物（DMPO）羟基自由基加合物浓度约为利福布汀的五倍。在大多数铁螯合剂存在下，利福霉素S存在时NADPH依赖的微粒体羟基自由基生成量略高于NADH-利福霉素S系统。然而，对于利福布汀，NADH依赖的微粒体羟基自由基生成量高于NADPH-利福布汀系统。铁螯合剂二乙烯三胺五乙酸（DTPA）是个例外，在这两种抗生素中，NADPH依赖的速率都超过了NADH依赖的速率。在NADH以及利福霉素S或利福布汀存在的情况下，大鼠肝脏亚线粒体颗粒也会产生羟基自由基。鱼藤酮和抗霉素A等电子传递链抑制剂增强了DMPO-OH的信号强度，表明NADH脱氢酶（复合体I）是参与利福霉素S还原的主要成分。已证明利福霉素S很容易被Fe(II)还原为相应的对苯二酚利福霉素SV；在类似条件下，Fe(II)不会还原利福布汀。通过光谱学方法，我们确定利福霉素S与Fe(II)形成复合物。在pH 7.4的磷酸盐缓冲液中，该复合物的化学计量比为Fe(利福霉素S)3。利福布汀未与Fe(II)形成可检测到的复合物。利福霉素S和利福布汀的氧化还原循环不会导致微粒体脂质过氧化。事实上，这两种分子完全抑制了Fe:ATP诱导的脂质过氧化。这些结果表明，利福霉素S和利福布汀可与大鼠肝脏微粒体相互作用进行氧化还原循环，当存在铁络合物时随后产生羟基自由基。与NADPH相比，NADH在促进这些相互作用方面几乎同样有效（利福霉素S）甚至更有效（利福布汀）。这些相互作用可能在与使用这些抗生素相关的肝毒性中起作用。
