Olsson G M, Brunmark A, Brunk U T
Department of Pathology II, University of Linköping, Sweden.
Virchows Arch B Cell Pathol Incl Mol Pathol. 1989;56(4):247-57. doi: 10.1007/BF02890023.
Irradiation of microsomes with visible light in the presence of externally-added acridine orange results in O2 uptake, malondialdehyde accumulation, and inactivation of the microsomal drug-metabolizing system. The latter effect is reflected by a decrease in NADPH-cytochrome P450- and NADH-cytochrome b5 reductase activities and cytochromes P450 and b5 content by 88-, 85-, 60-, and 34%, respectively, after 5-min irradiation. Anoxia prevented inactivation of both reductases by 70-90%, whereas it prevented completely cytochrome b5 destruction. The presence of reducing equivalents, at the expense of NADPH and NADH, exert a partial protection (40-54% residual activities) against photosensitization damage on both reductase activities, whereas it almost fully protected cytochrome b5. Photosensitization of lipid peroxidation, as well as inactivation of the microsomal drug-metabolizing system, appears to involve both a type I and type II process. Products of lipid peroxidation might also play a role in enzyme inactivation and cytochrome destruction, as suggested by kinetic and time course studies and the redox state of microsomes. The uptake of acridine orange by isolated lysosomes is linearly dependent on the concentration of added dye and the distribution between extra- and intralysosomal acridine orange is strongly dependent on the amount of lysosomes. Irradiation of acridine orange-loaded lysosomes (light intensity at the sample position approximately 320 mW/cm2) produces an impairment of the membrane which leads to a rapid release of enzyme (N-acetyl-beta-glucosaminidase activity) into the medium, accompanied by a loss of activity in the lysosome-containing pellet and a partial photodamage of the enzyme. Concomitantly, thiobarbituric acid-reactive material accumulation increases in the reaction mixture with increasing irradiation time. When light intensity at the position was reduced to approximately 3.6 mW/cm2, photodamage of lysosomes was of a lesser magnitude, allowing the demonstration of a lag phase, which decreased with irradiation time, probably reflecting the so-called first-stage activation of lysosomes, preceding the release of lysosomal enzymes.
在外部添加吖啶橙的情况下，用可见光照射微粒体可导致氧气摄取、丙二醛积累以及微粒体药物代谢系统失活。5分钟照射后，微粒体药物代谢系统的失活表现为NADPH-细胞色素P450还原酶和NADH-细胞色素b5还原酶活性以及细胞色素P450和b5含量分别下降88%、85%、60%和34%。缺氧可使两种还原酶的失活减少70 - 90%，而能完全防止细胞色素b5的破坏。以NADPH和NADH为代价存在还原当量时，对两种还原酶活性的光致敏损伤有部分保护作用（残余活性为40 - 54%），而对细胞色素b5几乎有完全保护作用。脂质过氧化的光致敏作用以及微粒体药物代谢系统的失活似乎涉及I型和II型过程。动力学和时间进程研究以及微粒体的氧化还原状态表明，脂质过氧化产物可能也在酶失活和细胞色素破坏中起作用。分离的溶酶体对吖啶橙的摄取与添加染料的浓度呈线性相关，且溶酶体内外吖啶橙的分布强烈依赖于溶酶体的数量。照射负载吖啶橙的溶酶体（样品位置的光强度约为320 mW/cm²）会导致膜损伤，从而使酶（N-乙酰-β-葡萄糖胺酶活性）迅速释放到培养基中，同时含溶酶体的沉淀中的活性丧失，且酶受到部分光损伤。与此同时，随着照射时间的增加，反应混合物中硫代巴比妥酸反应性物质的积累增加。当该位置的光强度降至约3.6 mW/cm²时，溶酶体的光损伤程度较小，从而能够显示出一个延迟期，该延迟期随照射时间而缩短，这可能反映了溶酶体酶释放之前溶酶体所谓的第一阶段激活。
