Immunochemical Detection of Covalently Modified Protein Adducts in Livers of Rats Treated with Methyleugenol

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• 作者：Iain Gardner ; Pauline Bergin ; Peter Stening ; J. Gerald Kenna ; and John Caldwell
• 刊名：Chemical Research in Toxicology
• 出版年：1996
• 出版时间：June 1996
• 年：1996
• 卷：9
• 期：4
• 页码：713 - 721
• 全文大小：409K
• 年卷期：v.9,no.4(June 1996)
• ISSN：1520-5010

文摘

Methyleugenol is an allylbenzene food flavoring which has beenshown to form DNA andprotein adducts, and to cause hepatotoxicity and carcinogenicity inrodents. In order toinvestigate the nature of the protein adducts, specific antisera wereraised by immunizingrabbits with conjugates prepared by coupling 1'-acetoxymethyleugenol,or its acidic congener3,4-dimethoxycinnamic acid, to rabbit serum albumin (RSA). Thesepolyclonal antisera wereshown by enzyme linked immunosorbent assay (ELISA) to containantibodies which recognizedthe 3,4-dimethoxyphenyl ring portion of methyleugenol. Analysis oflivers from rats givenmethyleugenol ip for 5 days, at doses between 10 and 300 mg/kg/day,revealed dose-dependentformation of novel protein adducts which were recognized by theantisera. The adducts weredetected by ELISA and by immunoblotting and were concentrated in themicrosomal fraction,and were shown in inhibition studies to be derived from methyleugenol.A 44 kDa adductwas the only protein adduct detected in livers of rats given low dosesof methyleugenol (10 or30 mg/kg/day) and was the major adduct detected in rats given highdoses of the compound(100 and 300 mg/kg/day). This adduct was solubilized whenmicrosomal fractions wereextracted using 0.1 M sodium carbonate, implying that it is aperipheral membrane protein.A pattern of protein adducts which mirrored the in vivosituation was generated when rathepatocytes were incubated with 1'-hydroxymethyleugenol invitro, but could not be reproducedin experiments undertaken using liver microsomes or postmitochondrialsupernatants. Thesefindings imply that generation of protein adducts in livers of ratsgiven methyleugenol in vivoproceeds via the 1'-hydroxy metabolite and requires crucial cofactors,and/or structural features,which are present in intact hepatocytes but not in broken cellpreparations and which remainto be defined.