Our research has focused on studies of the effects and mechanisms of toxicity of hemolytic drugs (nitrofurantoin [NF]), related derivatives and aromatic hydrocarbons (benzenes, anilines, etc.) on red blood cells. We have demonstrated that NF stimulates the rate of HbO2 autoxidation resulting in the release of superoxide anion with concomitant Hb3+ formation. Furthermore, NF stimulated significant H2O2 production and metHb formation in red cells, while decreasing cellular levels of ATP and GSH. Inhibitors of heme and of electron transport, as well as free-radical scavengers protected against oxidative stress and ATP and/or GSH depletion and implicate Hb oxidase activity in toxicity. Derivatives of NF and various organic hydrocarbons were also effective in depleting cellular levels of ATP and GSH. An ATP-dependent proteolytic enzyme system has been shown to exist in the erythrocyte, and its activity was markedly stimulated by agents (phenylhydrazine, nitrite) which caused oxidative stress and toxicity to the red cell. In view of our previous work and interest in xenobiotic toxicity to the red cell, we conducted studies on the effects of several drugs and organic hydrocarbons on proteolytic activity in the erythrocyte. The results of these studies demonstrate a correspondence between xenobiotic-induced toxicity to the red cell and stimulation of proteolytic activity. Evidence obtained in our laboratory suggests that Hb may be the protein that is degraded via the ATP-dependent proteolytic system. Little information is available on the role of proteolysis in xenobiotic-mediated red cell toxicity, its correlation with Hb hemeoxygenase (monooxygenase/oxidase) activity and the clinical significance of proteolytic activity as an index of drug-induced toxicity to the red blood cell. Furthermore, no information is available on proteolysis or xenobiotic-induced proteolysis in fetal or enzyme-deficient red cells or in red cells from individuals with the sickle cell trait. Hence, we wish to characterize the correspondence between xenobiotic-induced toxicity to the red cell, Hb hemeoxygenase activity, and proteolysis in these cells. The time- and concentration-dependence of xenobiotic-stimulated proteolysis will be determined and proteolytic activity will be correlated with xenobiotic-induced metHb formation, reactive oxygen species production, free radical generation, and with alterations in cellular levels of ATP and GSH. Such work may provide a basis for the rapid clinical assessment of xenobiotic-induced toxicity to red cells in individuals suffering from various blood dyscrasias or hemoglobinopathies.

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National Institute of Environmental Health Sciences (NIEHS)
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Toxicology Study Section (TOX)
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Northwestern University at Chicago
School of Medicine & Dentistry
United States
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Starcevic, S L; Elferink, C; Novak, R F (2001) Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease. J Natl Cancer Inst 93:776-82
Shen, K; Novak, R F (1997) DDT stimulates c-erbB2, c-met, and STATS tyrosine phosphorylation, Grb2-Sos association, MAPK phosphorylation, and proliferation of human breast epithelial cells. Biochem Biophys Res Commun 231:17-21
Shen, K; Novak, R F (1997) Fas-signaling and effects on receptor tyrosine kinase signal transduction in human breast epithelial cells. Biochem Biophys Res Commun 230:89-93
Gruebele, A; Zawaski, K; Kaplan, D et al. (1996) Cytochrome P4502E1- and cytochrome P4502B1/2B2-catalyzed carbon tetrachloride metabolism: effects on signal transduction as demonstrated by altered immediate-early (c-Fos and c-Jun) gene expression and nuclear AP-1 and NF-kappa B transcription factor leve Drug Metab Dispos 24:15-22
Zangar, R C; Reiners Jr, J J; Novak, R F (1995) Gender-specific and developmental differences in protein kinase C isozyme expression in rat liver. Carcinogenesis 16:2593-7
Runge-Morris, M; Wu, N; Novak, R F (1994) Hydrazine-mediated DNA damage: role of hemoprotein, electron transport, and organic free radicals. Toxicol Appl Pharmacol 125:123-32
Zawaski, K; Gruebele, A; Kaplan, D et al. (1993) Evidence for enhanced expression of c-fos, c-jun, and the Ca(2+)-activated neutral protease in rat liver following carbon tetrachloride administration. Biochem Biophys Res Commun 197:585-90
Runge-Morris, M; Novak, R F (1993) Effects of phenelzine and hydralazine on hydrogen peroxide production and proteolysis in human red blood cells. J Pharmacol Exp Ther 267:1401-6
Mortensen, A M; Novak, R F (1992) Dynamic changes in the distribution of the calcium-activated neutral protease in human red blood cells following cellular insult and altered Ca2+ homeostasis. Toxicol Appl Pharmacol 117:180-8
Mortensen, A M; Novak, R F (1991) Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells. Toxicol Appl Pharmacol 110:435-49

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