Ethanol-derived immunoreactive species formed by free radical mechanisms.
Recent studies have shown that the alpha-hydroxyethyl radical (CH3CHOH), a metabolite of ethanol, is produced in vitro and in vivo. We report studies that establish the immunogenicity of alpha-hydroxyethyl radical-derived protein adducts. Rat liver microsomes incubated in the presence of [14C]ethanol and NADPH (under aerobic conditions) incorporate 14C into acid-stable adducts. Incorporation was markedly inhibited by the free-radical scavenger alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. Rabbits immunized with rat liver microsomes that had been preincubated with ethanol and NADPH generated antibodies that recognized polylysine-acetaldehyde adducts and adducts formed by incubation of proteins with an alpha-hydroxyethyl radical-generating system (ethanol plus H2O2 plus Fe2+). Rabbits immunized with microsomes that had been preincubated with ethanol and NADPH plus alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone generated antibodies that recognized polylysine-acetaldehyde adducts. However, their reactivity against alpha-hydroxyethyl-derived protein epitopes was greatly reduced or was virtually abolished. Data indicate that microsomes metabolizing ethanol generate two types of adducts, acetaldehyde-derived adducts and alpha-hydroxyethyl radical-derived adducts, both of which are immunogenic. Immunization of rabbits with alpha-hydroxyethyl-bovine serum albumin adducts led to the production of antibodies that recognized alpha-hydroxyethyl-rabbit serum albumin adducts but did not recognize the native protein. Chronic alcohol feeding of rats led to the production of antibodies that recognized alpha-hydroxyethyl-rat serum albumin adducts but did not recognize rat serum albumin. The study (i) indicates that alpha-hydroxyethyl radical-derived protein adducts are immunogenic, (ii) supports earlier work that proposed that alpha-hydroxyethyl radicals generated in different systems bind covalently to proteins, and (iii) demonstrates the formation of antibodies to alpha-hydroxyethyl-derived protein adducts after chronic alcohol ingestion in vivo. The findings may have implications in the identification of chronic alcohol abuse and the pathogenesis of alcohol-induced organ damage.
Department of Pharmacology, University of Toronto, Canada.
C
C
Moncada
V
V
Torres
G
G
Varghese
E
E
Albano
Y
Y
Israel
D000079:Acetaldehyde / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D000431:Ethanol / Q000276:immunology*; D005609:Free Radicals; D008862:Microsomes, Liver / Q000378:metabolism; D011817:Rabbits; D051381:Rats
D002250:Carbon Radioisotopes; D005609:Free Radicals; C065424:1-hydroxyethyl radical; D000431:Ethanol; D000079:Acetaldehyde