An immunochemical approach for studying the role of protein alkylation in the hepatotoxicity of butylated hydroxytoluene (BHT) /
Several alkylphenols, such as butylated hydroxytoluene (BHT),
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1997.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739891451&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Several alkylphenols, such as butylated hydroxytoluene (BHT), form reactive quinone methide intermediates upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand White rabbits. Rabbit sera contained an antibody which was highly specific for BHT, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHTdependent protein alkylation over a wide molecular weight range. Although binding was somewhat dependent on the experimental system used, prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74 and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, omission of NADPH and deuterated BHT. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT quinone methide as in the enzyme-mediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h post dosing, and was dose-dependent. BHT specific antisera were used to aid in the purification, characterization and identification of the 34.5 and 74 kDa proteins. N-terminal amino acid sequencing revealed that the 34.5 kDa protein was the 32 kDa subunit of rat enoyl-CoA hydratase, a mitochondrial Poxidation enzyme. The 76 kDa protein was identified as a membrane/cytoskeletal linker protein, either radixin, ezrin or moesin. The possible role of these proteins in BHTmediated hepatotoxicity was discussed. Additionally, we investigated the possible role of alternative amino acid residues as binding sites for BHT-quinone methide by exposing a non-sulfhydryl containing protein, myoglobin, to chemically synthesized BHT-quinone methide. Western blotting experiments revealed that BHT can bind to these proteins, presumably via other basic amino acid side chains. Finally, we used BHT-specific antisera as an application to studies determining the relative rates of reactivity, rates of formation, protein binding propensity and toxicity of BHT and two related congeners, ethyl-BHT (E- BHT) and isopropyl-BHT (I-BHT). Binding, reactivity, and toxicity paralleled one another (BHT > E-BHT > I-BHT) in support of previous work and theory concerning carbon additions to the benzylic carbon atoms of simple alkylphenols. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". |
| Physical Description: | xiii, 120 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 104-119. |