HYDROXYLAMINE AND PHENOL-INDUCED FORMATION OF METHEMOGLOBIN AND FREE-RADICAL INTERMEDIATES IN ERYTHROCYTES

As previously shown with isolated oxyhemoglobin, methemoglobin formati on can also be induced in intact erythrocytes by hydroxylamine compoun ds and substituted phenols such as butylated hydroxyanisole (BHA). Ele ctron spin resonance investigations revealed that, accordingly, free r adical intermediates were formed in erythrocytes from hydroxylamine, N ,N-dimethylhydroxylamine, and N-hydroxyurea. Due to the low stability of the dihydronitroxyl radicals, their detection required the use of a continuous flow system and relatively high amounts of the reactants. As has already been demonstrated with the solubilized hemoglobin syste m, hemoglobin of intact erythrocytes also reacts with the more hydroph ilic xenobiotics such as hydroxylamine. However, the reaction rate was slightly reduced, indicating the existence of an incomplete permeabil ity barrier for these compounds. The limited solubility of phenolic co mpounds in the aqueous buffer of suspended erythrocytes (in combinatio n with the strict requirement of osmolarity in order to prevent hemoly sis) impeded the direct detection of the respective phenoxyl radicals previously reported in hemoglobin solutions. However, in accordance wi th earlier findings in homogeneous reaction systems, chemiluminescence was observed as well, indicating the existence of a further reaction intermediate, which was also obtained in pure hemoglobin solutions whe n mixed with the respective reactants. As has recently been demonstrat ed, this light emission is indicative of the existence of highly proox idative compound I intermediates during methemoglobin formation. Proox idant formation in erythrocytes is reflected by a significant decrease in thiol levels even with those compounds where free radical formatio n was not directly detectable by ESR spectroscopy. The use of the spin -labeling technique revealed membrane effects as a result of oxidative stress. Oxidative metabolism of hemoglobin with hydroxylamine caused a release of low molecular weight iron. The marked hemolysis observed in the presence of BHA results from a direct membrane effect of this c ompound rather than a consequence of free radical induced oxidative st ress. A correlation of the different results is discussed in terms of possible toxicological consequences. Copyright (C) 1996 Elsevier Scien ce Inc.