Lipid peroxidation stimulated by iron nitrilotriacetate in rat liver.

The complex of ferric iron with nitrilotriacetate (iron-NTA) given i.p. is an unusually potent stimulus for lipid peroxidation (LP) in vivo, as monitored by exhaled alkanes. Localization of 59Fe-labeled NTA radioactivity in mouse liver and accumulation of thiobarbituric acid (TBA)-reacting material in liver after i.p. injection suggested that the effect of i.p. iron-NTA could be primarily hepatic. It was found that 100 microM iron-NTA added to a hepatocyte suspension gassed with air stimulated ethane production (3 +/- 1 pmoles/10(6) cells/min) versus an undetectable control, and at a sensitivity of 0.083 pmole/10(6) cells/min. Under similar conditions, hepatocytes stimulated by iron-NTA generated low level chemiluminescence (CL) in parallel with formation of TBA-reactants; the generation of CL was concentration related. Liver was homogenized and fractionated by ultracentrifugation: iron-NTA stimulated CL in whole liver homogenate as in intact cells. The greater part of this activity localized to the microsomal and mitochondrial fractions where NADH or NADPH was required. Using rat liver microsomes, it was shown that iron-NTA in the presence of NADPH stimulated two phases of CL with an initial phase maximum in 1-2 min (phase 1) which decreased abruptly to be followed by a prolonged rise (phase 2); NADH could replace NADPH. Ferrous iron (as chloride) caused a burst of CL, whereas ferric iron was inactive. However, complex differences exist between CL stimulated by Fe(II) and by iron-NTA in the presence of reducing equivalents. Under conditions resulting in the production of CL, a microsomal system with iron-NTA and reducing equivalent accumulated TBA-reactants in parallel with the stimulated CL and rapid increase in oxygen consumption. Both desferrioxamine and butylated hydroxyanisole were able to strongly inhibit the CL stimulated by iron-NTA. When iron-NTA and iron-ADP were compared in the microsomal system, similar responses were obtained but major differences characterized the effects of these iron chelates on whole cells with the ADP complex being relatively inactive. We conclude that iron-NTA stimulated free radical reactions in liver by undergoing cyclic oxidation and reduction and that these reactions utilized oxygen, generated CL, and formed TBA-reactants and ethane. At a subcellular level, the reactions of iron-NTA resembled those reported for iron-ADP.