Date of Award

2009

Degree Name

Chemistry

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

John Wilkinson

Second Advisor

Leslie Frost

Third Advisor

William Price

Abstract

Cellular methylation and antioxidant metabolism are linked by the transsulfuration pathway, which converts the methionine cycle intermediate homocysteine to cysteine, a precursor for glutathione biosynthesis, principally in hepatic cells. In mammals, the transsulfuration pathway has been identified in liver, kidney, pancreas and brain. To determine whether the pathway exists in mammary cells human breast adenocarcinoma cells (MCF-7) and normal mammary epithelial cells (HMEC) were labeled with 35S-methionine for 24 hours following pre-treatment with a vehicle control, the cysteine biosynthesis inhibitor propargylglycine (PPG) or the gammaglutamyl cysteine synthesis inhibitor buthionine sulfoximine (BSO). Cell lysates were prepared and reacted with glutathione-S-transferase (GST) and the fluorescent labeling compound monochlorobimane (mCBi) to cause glutathione (GSH) to form a fluorescent GSHBimane conjugate with mCBi. Thin layer chromatography (TLC) was used to separate conjugated glutathione (GSH-Bi) from the free mCBi fluor. Fluorescent images and autoradiography of the TLC plate were compared; incorporation of 35S-methionine into glutathione-bimane spots identified through fluorescence (GSH-mCBi bands) indicated that functional transsulfuration occurs in mammary cells (there is no other known manner for the 35S of methionine to incorporate into cysteine and subsequently into GSH). Given the role that glutathione plays as a major cellular antioxidant, we were interested in determining the extent to which transsulfuration contributes to glutathione production under conditions of oxidant stress; enzymatic determination of the impact of PPG pre treatment and 2hr 300uM hydrogen peroxide treatment of HMEC’s on GSH levels indicated PPG reduced total GSH by one third, and blocked the ability of oxidatively stressed cells to upregulate GSH production. In summary, results of this study demonstrate the presence of the transsulfuration pathway in mammary epithelial cells and the importance of this pathway under oxidative stress conditions.

Subject(s)

Nervous system - Diseases - Genetic aspects.

Nervous system - Diseases - Molecular aspects.

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