Characterization of hydrazine-stimulated proteolysis in human erythrocytes.
The ability of hydrazine, acetylphenylhydrazine, methylhydrazine, and phenylhydrazine to stimulate proteolysis in red cells has been characterized. All four hydrazines effectively stimulated proteolysis in red cells and in hemolysate as evidenced by a two- to threefold increase in the rate of tyrosine release. The rate of tyrosine release varied linearly with time, increased with increasing concentration of hydrazine, and also increased as a function of hematocrit. The rank order for stimulation of proteolysis in red cells was phenylhydrazine greater than methylhydrazine greater than hydrazine approximately equal to acetylphenylhydrazine. Inhibitors of glycolysis in red cells only minimally (13-27%) decreased the rate of tyrosine release stimulated by the different hydrazines. Agents which diminished electron transport decreased the rate of tyrosine release. NADP inhibited the rate of tyrosine release stimulated by hydrazine, methylhydrazine, and acetylphenylhydrazine by approximately 36 to 41%; 2'-AMP was less effective. The rate of tyrosine release resulting from insult by the hydrazines was increased slightly by methylene blue, moderately inhibited (approximately 10 to 27%) by the chelator o-phenanthroline and inhibited approximately 30 to 40% by N-ethylmaleimide. Use of an oxygen-depleted atmosphere (N2) increased slightly the rate of tyrosine release stimulated by the hydrazines; in contrast, carbon monoxide decreased proteolysis stimulated by hydrazine, methylhydrazine, and acetylphenylhydrazine by approximately 50%. Although the antioxidants dimethylfuran, dimethylthiourea, and methylsulfoxide failed to diminish proteolysis stimulated by the hydrazines, N-acetylcysteine exerted a protective effect, decreasing hydrazine-stimulated tyrosine release in red cells approximately 30 to 50%. Inclusion of 3-amino-1,2,4-triazole in the incubation failed to increase further the rate of hydrazine-stimulated proteolysis. These data suggest that more reactive free radicals generated from the hydrazine are responsible for protein damage, that damaged protein (hemoglobin) is degraded via proteolysis, and that an ATP-independent process primarily participates in the degradation of abnormal proteins in the red cell. Thus, proteolytic enzymes present in the erythrocyte appear to exert a protective effect against cellular damage through the removal of abnormal proteins generated as a consequence of xenobiotic insult. The ability of proteolytic enzymes to recognize and degrade abnormal proteins may be of importance in using protein (hemoglobin)-xenobiotic adducts to assess exposure to toxic agents (risk assessment).
Runge-Morris
M A
MA
Department of Pharmacology, Northwestern University, Chicago, Illinois 60611.
Iacob
S
S
Novak
R F
RF
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000328:Adult; D000640:Amitrole / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001798:Blood Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D004579:Electron Transport; D004792:Enzyme Precursors / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008706:Methemoglobin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D000975:Antioxidants; D001798:Blood Proteins; D004792:Enzyme Precursors; D006834:Hydrazines; D014443:Tyrosine; D000255:Adenosine Triphosphate; D008706:Methemoglobin; D006861:Hydrogen Peroxide; D002154:Calpain; C047018:procalpain; D000640:Amitrole