Date of Award

12-3-2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Delon Barfuss - Chair

Second Advisor

Dr. Andrew Clancy

Third Advisor

Dr. Yuan Liu

Abstract

The bath-to-cell transport, cytosolic concentration, and tubular content of methylmercury (Me203Hg+) and the sulfhydryl-amino acids and sulfhydryl-amino acid derivatives conjugated to Me203Hg+ were studied in the non-perfused S2 segments of the proximal tubule of the rabbit kidney. Active transport of Me203Hg+ was established by a temperature dependent (greater than 100% reduction in bath-to-cell transport, 99% decrease in cytosolic concentration, 63% decline in the tubular contents at 12°C when compared to 37°C). Conjugates of Me203Hg+ showed mixed results, with L-cysteine and L-taurine demonstrating the most significant increase in uptake. Transport of Me203Hg+-L-cysteine was also temperature dependent with a 77% reduction in bath-to-cell transport, 76% decrease in cytosolic concentration, and 86% decline in tubular contents at 12°C when compared to 37°C. A significant decrease in transport was seen with the classic organic anion transport (OAT) inhibitors of PAH (71% and 67%) and probenicid (48% and 38%), as well as, the dicarboxylates, adepate (over 100%) and glutarate (69% and 52%) in both bath-to-cell and cytosolic concentration respectively. The addition of L-methionine to the Me203Hg+-L-cysteine conjugate significantly reduced the bath-to-cell transport by 64% and the cytosolic contents by 47%. The Me203Hg+-L-taurine conjugate also demonstrated temperature dependence (99% reduction and 91% decrease in bath-to-cell and cytosolic concentration respectively, at 12°C when compared to 37°C). Inhibition with PAH was also seen (77% reduction) in bath-to-cell transport and 67% decline in cytosolic concentration giving further evidence to the transport of the Me203Hg+-L-taurine conjugate via OAT. When Me203Hg+ was conjugated to L-methionine a 55% reduction in bath-to-cell transport was seen which was also temperature dependent (59% decrease at 12°C when compared to 37°), although no significant decrease in transport was noted with the addition of PAH. Analysis of the methylmercury conjugates via mass spectrometry demonstrated that L-cysteine, L-taurine, and L-methionine all are binding with methylmercury in the same unknown common configuration (MW=409). These results indicate the mercuric conjugate is gaining entry into the renal epithelial cells via the OAT and the amino acid transport system in the basolateral membrane of the proximal tubule.

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