Date of Award

2003

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Monica A. Valentovic

Second Advisor

Patrick I. Brown

Third Advisor

Carl Gruetter

Fourth Advisor

Elsa Mangiarua

Fifth Advisor

Gary O. Rankin

Abstract

The acetaminophen metabolite, p-aminophenol (PAP), is a known nephrotoxicant. The current study is directed at understanding the mechanism of this nephrotoxicity. Renal cortical slices isolated from F344 rats were incubated with 0 – 0.5 mM PAP with or without pretreatments. Renal slices exposed to PAP showed toxicity as measured by increased lactate dehydrogenase leakage (LDH), decreased pyruvate-stimulated gluconeogenesis, and decreased total adenine nucleotides. There was, however, an increase in ATP as percent of total, implying maintained mitochondrial function. These data show that PAP is directly toxic to renal cortical slices. Pretreatment with EDTA or EGTA provided protection from PAP- induced LDH leakage, while EGTA-AM did not. These data show that PAP toxicity is dependent on extracellular calcium and independent of small increases in intracellular calcium. PAP exposure decreased total glutathione within 30 min, and marked depletion was evident at 120 min. At 60 min, PAP increased glutathione disulfide (GSSG) as percent of total glutathione (%GSSG). PAP exposure also increased 4-hydroxynonenal- adducted proteins. Pretreatment with ascorbate prevented PAP mediated increases in LDH, while reduced glutathione (GSH) or N-acetylcysteine did not prevent PAP toxicity. These data support the hypothesis that PAP induces oxidative stress, which cannot be prevented by exogenous sulfhydryl containing compounds. Administration of pyruvate protected against PAP-induced time- and concentration-dependent effects on LDH and adenine nucleotides. Finally, pyruvate caused an increase in cellular total glutathione and reduced PAP effects on total glutathione and %GSSG. These data imply that pyruvate protects from PAP toxicity through increasing the concentrations of total glutathione and recycling GSSG back to GSH. Pretreatment with glucose provided slight protection from LDH and increases in %GSSG. Finally, pyruvate but not glucose induced a significant increase in NADPH, confirming the hypothesis that pyruvate protects through NADPH-dependent recycling of glutathione. The greater degree of protection provided by pyruvate implies that pyruvate may provide a more readily available source for the prevention of oxidative stress. Pyruvate protection from oxidative stress of PAP may be expanded to other toxins with oxidant mechanisms of toxicity. These results could also be extrapolated to acetaminophen nephrotoxicity, and may provide the basis for future treatments.

Subject(s)

Acetaminophen - Toxicology.

Nephrotoxicology.

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