A Study on the Interaction between Hydroxylamine Analogues and Oxyhemoglobin in Intact Erythrocytes

The oxidative potency of hydroxylamine (HYAM) and its O-derivatives (O-methyl- and O-ethyl hydroxylamine) is generally larger than the effects of the N-derivatives (N-methyl-, N-dimethyl-, and N,O-dimethyl hydroxylamine). The effects of the two groups of hydroxylamines also differ in a qualitative sense. To elucidate this difference in toxicity profiles we investigated the hemoglobin dependence of the toxicity, the occurrence of cell-damaging products like superoxide and H2O2, and the cellular kinetics of the hydroxylamine analogues. All hydroxylamines were found to depend on the presence and accessibility of oxyhemoglobin to exert their toxicity. This did not provide an explanation for the different toxicity profiles. The interaction of some hydroxylamines with oxyhemoglobin is known to lead to the formation of radical intermediates. Differences in the stability of these radical products are known to occur, and in some cases secondary products are formed. This can contribute to the differences in toxicity. In this respect, production of superoxide radicals was demonstrated for all hydroxylamines in the reaction with oxyhemoglobin. Evidence for H2O2 generation during the reaction of HYAM, O-methyl, O-ethyl-, and N-dimethyl hydroxylamine with oxyhemoglobin was also found. Next to variations in the products formed, differences in cellular kinetics are likely to be among the most important factors that explain the different toxicity patterns seen for the hydroxylamines in erythrocytes. Indeed, differences were found to exist for the kinetics of methemoglobin formation in erythrocytes. Not only was the final level of methemoglobin formed much lower for the N-derivatives, but also the reaction rate with oxyhemoglobin was slower than with HYAM and its O-derivatives. Except for N,O-dimethyl hydroxylamine (NODMH), the same pattern was seen in hemolysates. NODMH tripled its effect on hemoglobin in hemolysate compared with incubations in erythrocytes. This implies that cellular uptake is a limiting factor for NODMH. Since formation of H2O2 is most likely a result of an interaction with hemoglobin, differences in kinetics of methemoglobin formation can be an explanation for the fact that NMH and NODMH did not produce H2O2 to a detectable level. These results indicate that (a) the toxicity of all hydroxylamines depends on an interaction with oxyhemoglobin; (b) the interaction with hemoglobin produces radical intermediates and concomitantly superoxide radicals and H2O2; and (c) differences in uptake, reaction rate with hemoglobin, and stability of the intermediates formed do exist for the different hydroxylamines and contribute to their differences in toxicity.