Amphotericin B as an intracellular antioxidant: protection against 2,2'-azobis(2,4-dimethylvaleronitrile)-induced peroxidation of membrane phospholipids in rat aortic smooth muscle cells.

The antifungal activity of amphotericin B (AmB) and its side-effects (e.g. nephrotoxicity and hemolytic action) are suggested to be associated with its prooxidant effects in target cells. To test this hypothesis, we have undertaken studies to examine the role of AmB in oxidative stress in cultured rat aortic smooth muscle cells (SMC) incubated in the absence or in the presence of a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). No changes in the pattern of membrane phospholipids could be detected by two-dimensional high performance thin-layer chromatography (HPTLC) after oxidative stress induced by AMVN in which the cells remained viable, as judged by trypan blue exclusion. To improve the sensitivity of detection of oxidative stress in the cells, cis-parinaric acid (PnA) was incorporated biosynthetically into the membrane phospholipids [using PnA-human serum albumin (hSA) complex]. Incubation of the cells under aerobic conditions in the presence of up to 10 microM AmB showed no significant change in the pattern of PnA-labeled phospholipids, suggesting that AmB was not affecting the oxidative state of the cells. In contrast, treatment with AMVN (0.5 mM, incubation in the dark for 2 hr at 37 degrees--conditions in which the viability of the cells was maintained) caused a significant reduction of all fluorescently labeled phospholipid fractions separated by HPLC. When PnA-labeled cells were subjected to oxidative stress by incubation with 0.5 mM AMVN in the presence of AmB, the loss of fluorescent phospholipids was reduced in a concentration-dependent manner over a concentration range of 0.25 to 10 microM. Thus, AmB does not produce any prooxidant effect but rather acts as an intracellular antioxidant.