The long-term objectives of the proposed research are to gain a greater understanding of the enzymological details of sulfur and selenium methylation and demethylation and of the role of these reactions in the biochemical detoxification and anticarcinogenic effects of sulfur and selenium compounds.
The specific aims (bold) and experimental design to achieve them are as follows. 1) To purify thiol methyltransferase from mouse liver and to characterize its molecular properties. The purification will be carried out using conventional chromatographic methods of gel filtration, chromatofocusing, and affinity chromatography. The characterization will include molecular weight by SDS-polyacrylamide gel electrophoresis and kinetic constants for substrates. 2) To use purified thiol methyltransferase and thioether methyltransferases in combination to study the sequential methylation of sulfur and selenium compounds in vitro. The conditions and cofactors necessary for the conversion of 75Se-selenite to dimethyl 75Se-selenide and trimethyl 75Se-selenonium will be characterized. These products will be determined by HPLC with detection by a radioactivity flow monitor. The same system will be used to study the conversion of various thiols to methyl thioethers and dimethyl sulfonium ions. 3) To study the sequential methylation of sulfur compounds in vivo in mice. Conversion of various thiols and thioethers to methyl sulfonium ions will be tested by HPLC analysis of tissue and urine extracts from mice treated with the appropriate radioactive precursor. 4) To use this in vitro system to study the potential interferences between methylation of sulfur and selenium compounds. These two methylation pathways are proposed to share the above methyltransferases. Various thiols and thioethers will be used to determine if they interfere with the methylation of 75Se- selenite. 5) To study the interference between sulfur and selenium methylation in vivo. The interference by various thiols and thioethers with conversion of 75Se-selenite to dimethyl 75Se-selenide and trimethyl 75Se-selenonium ion will be tested by HPLC analysis of tissue and urine extracts from treated mice. 6) To purify S-adenosylmethionine:homocysteine methyltransferase from mouse liver for study as a candidate enzyme in the demethylation of anticarcinogenic selenonium compounds, and to test if this enzyme has this function in vivo. Chromatography by ion-exchange, gel filtration, chromatofocusing, and affinity methods will be used. The purified enzyme will be tested for its ability to transfer methyl groups from various sulfonium and selenonium ions to homocysteine. For in vivo studies, the effects of varying homocysteine levels on demethylation of trimethyl selenonium will be studied. 7) To study the influence of sex, development, lactation, and dietary selenium on the levels of thioether methyltransferase and thiol methyltransferase. Enzyme activities, Western blots of proteins, and Northern blots of mRNAs will be used to study the influence of these variables. 8) To study the ability of diallyl methyl sulfonium to form sulfonium ylides and react with ultimate carcinogens both in vitro and in vivo in mice. The in vitro system will contain mouse liver microsomes as a source of cytochrome P450 to activate 14C- dimethylnitrosamine and DNA. The ability of diallyl methyl sulfonium ion to block the formation of specific methylated bases in the DNA will be examine by HPLC base analysis of DNA hydrolysates. Similar analyses will be done on DNA from mice treated with 14C-dimethylnitrosamine and diallyl methyl sulfonium ion.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Toxicology Subcommittee 2 (TOX)
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University of Louisville
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