Abstract
l-Methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with d-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.
Footnotes
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This research was supported by National Institutes of Health Grants R01 DK44295 and T32-ES-007015.
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A preliminary report of this work was previously presented at the following conference: Dever JT and Elfarra AA (2008) Sex-related differences in methionine toxicity in freshly isolated mouse hepatocytes and evidence for transamination, but not transmethylation playing a key mechanistic role. 2008 Society of Toxicology Annual Meeting; 2008 16–20 Mar; Seattle, WA. Society of Toxicology, Reston, VA.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.141044.
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ABBREVIATIONS: Met, l-methionine; TPN, total parenteral nutrition; TM, transmethylation; SAM, S-adenosyl-l-methionine; SAH, S-adenosyl-l-homocysteine; Hcy, homocysteine; TA, transamination; 3-MTP, 3-methylthiopropionic acid; AOAA, amino-oxyacetic acid; 3-DA, 3-deazaadenosine; LDH, lactate dehydrogenase; GSH, glutathione; GSSG, glutathione disulfide; d-Met, d-methionine; SSA, 5-sulfosalicylic acid; DMEM, Dulbecco's modified Eagle's medium; TB, trypan blue.
- Received May 9, 2008.
- Accepted June 12, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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