The erythrocyte, by virtue of its presence in blood, interacts rapidly with a diverse array of xenobiotics to include therapeutic agents and environmental pollutants such as polycyclic aromatic hydrocarbons and volatile organic compounds. Hemoglobin constitutes 95% of all the cytosolic proteins in the red cell and it has been shown to catalyze the oxidative modification of a wide variety of xenobiotics in a manner analogous to that of the hepatic monooxygenase system. Moreover, recent studies have demonstrated the presence of xenobiotic-hemoglobin adducts in smokers and in populations exposed to volatile organic compounds and investigations have sought to exploit the presence of adducts for human dosimetry experiments. The presence of flavoproteins and a large concentration of the oxygen-binding pigment Hb in the red cell, also implies that the red cell experiences continual oxidative stress. Recent results suggest that certain proteolytic enzymes specifically target proteins modified by either xenobiotic or oxidative insult for degradation and elimination and that such a system represents a novel, relatively uncharacterized mechanism for cellular defense against xenobiotic insult. Thus, the objectives of the proposed research are: (1) To continue characterization of the effects of insult by xenobiotic and reactive oxygen species on protein degradation in human red cells and hemolysates and to establish the effects of endogenous protective systems on protein degradation following insult. (2) To examine whether xenobiotic insult or oxidant stress results in degradation of specific cytosolic or membrane proteins. (3) To purify the high molecular weight alkaline protease implicated in degradation of damaged proteins, prepare polyclonal or monoclonal antibodies against this protease, demonstrate the presence of this enzyme in human red cells by immunoblot techniques, and establish the role of this enzyme in xenobiotic- damaged protein degradation via immunotitration experiments. The proposed studies will serve to identify the mechanism(s) of protein damage (i.e. oxidative stress versus covalent modification), the proteins affected and to begin the arduous task of using immunochemical techniques to identify the protease(s) involved in degarding proteins rendered abnormal by xenobiotic modification of oxidative stress.

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Pharmacology A Study Section (PHRA)
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Wayne State University
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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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