THE EFFECTS OF XENOBIOTICS ON ERYTHROCYTES

1. Methemoglobin formation was observed when erythrocytes were incubat ed with xenobiotics such as hydroxylamines or phenols, other metabolit es resulting from the interaction of these compounds with erythrocytes being reactive free radicals derived from the respective xenobiotic, and a ferryl-heme ore-complex. 2. Steady-state levels of these reactio n products depended on the permeability of the erythrocyte membrane fo r the various methemoglobin (MetHb) generators and the presence of ant ioxidants that downregulate the radicals formed. 3. Electron spin reso nance (ESR) spectra of xenobiotic-derived free radicals could be obtai ned only from the readily water soluble hydroxylamines, whereas the po orly water soluble phenolic compounds did not allow the use of concent rations required for the generation of detectable amounts of ESR-sensi tive metabolites in erythrocytes. 4. Previous investigations with oxyh emoglobin solutions and with the MetHb/H2O2 model systems have shown t hat, apart from ESR-sensitive radical species, excited reaction interm ediates such as compound I ferryl hemoglobin can be detected as well b y using chemiluminescence measurements. 5. A strong correlation was fo und between the intensity of the emitted light and the MetHb formation rate, indicating that the production of compound 1 ferryl hemoglobin is closely related to the MetHb formation step. 6. The sensitivity of the photon-counting method allowed measurements of excited species in intact erythrocytes not only with the readily soluble hydroxylamines, but also with the less soluble phenolic compounds. 7. In addition, par ameters indicative of xenobiotic-induced oxidative alterations were fo und: a significant decrease in intraerythrocytic thiol levels was a re sult of all compounds that initiate MetHb formation, as also described for slowly reacting xenobiotics. 8. With the most reactive compound i nvestigated, unsubstituted hydroxylamine, a significant release of iro n from the oxidatively modified hemoglobin was detected, facilitated b y binding of this transition metal to hydroxylamine and its final oxid ation product, nitric oxide. 9. The use of the ESR spin-labeling techn ique revealed membrane alterations of erythrocytes exposed to the redu cing MetHb generators presented in this study. 10. A direct action of BHA and BHT on the integrity of the erythrocyte membrane was observed, leading to hemolysis independent of the formation of prooxidant speci es. 11. The presence of strong prooxidants (radicals) was indicated bo th by fluidity changes in the membrane and by an oxidative decrease in cytosolic thiol levels. GEN PHARMAC 31;3:343-347, 1998. (C) 1998 Else vier Science Inc.