Immunochemical detection of covalently modified protein adducts in livers of rats treated with methyleugenol.
Methyleugenol is an allylbenzene food flavoring which has been shown to form DNA and protein adducts, and to cause hepatotoxicity and carcinogenicity in rodents. In order to investigate the nature of the protein adducts, specific antisera were raised by immunizing rabbits with conjugates prepared by coupling 1'-acetoxymethyleugenol, or its acidic congener 3,4-dimethoxycinnamic acid, to rabbit serum albumin (RSA). These polyclonal antisera were shown by enzyme linked immunosorbent assay (ELISA) to contain antibodies which recognized the 3,4-dimethoxyphenyl ring portion of methyleugenol. Analysis of livers from rats given methyleugenol i.p. for 5 days, at doses between 10 and 300 mg/kg/day, revealed dose-dependent formation of novel protein adducts which were recognized by the antisera. The adducts were detected by ELISA and by immunoblotting and were concentrated in the microsomal fraction, and were shown in inhibition studies to be derived from methyleugenol. A 44 kDa adduct was the only protein adduct detected in livers of rats given low loses of methyleugenol (10 or 30 mg/kg/day) and was the major adduct detected in rats given high doses of the compound (100 and 300 mg/kg/day). This adduct was solubilized when microsomal fractions were extracted using 0.1 M sodium carbonate, implying that it is a peripheral membrane protein. A pattern of protein adducts which mirrored the in vivo situation was generated when rat hepatocytes were incubated with 1'-hydroxymethyleugenol in vitro, but could not be reproduced in experiments undertaken using liver microsomes or postmitochondrial supernatants. These findings imply that generation of protein adducts in livers of rats given methyleugenol in vivo proceeds via the 1'-hydroxy metabolite and requires crucial cofactors, and/or structural features, which are present in intact hepatocytes but not in broken cell preparations and which remain to be defined.
Gardner
I
I
Department of Pharmacology and Toxicology, Imperial College School of Medicine at St. Mary's, London, United Kingdom.
Bergin
P
P
Stening
P
P
Kenna
J G
JG
Caldwell
J
J
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005054:Eugenol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D005421:Flavoring Agents / Q000737:chemistry* / Q000633:toxicity; D005503:Food Additives / Q000737:chemistry* / Q000633:toxicity; D006241:Haptens / Q000737:chemistry / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008297:Male; D008862:Microsomes, Liver / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D011506:Proteins / Q000032:analysis; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D011933:Reagent Kits, Diagnostic; D012709:Serum Albumin / Q000737:chemistry* / Q000276:immunology; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D005421:Flavoring Agents; D005503:Food Additives; D006241:Haptens; D007106:Immune Sera; D011506:Proteins; D011933:Reagent Kits, Diagnostic; D012709:Serum Albumin; C005223:methyleugenol; D005054:Eugenol