Susan H. Kenyon

The Effect of Ethanol and Its Metabolites Upon Methionine Synthase Activity In Vitro

The association of alcoholism with macrocytic anaemia has lead to investigation of the role of cobalamin-dependent methionine synthase in mediating alcohol toxicity. Several studies have found that long-term ingestion of large quantities of ethanol causes inhibition of liver methionine synthase activity in vivo: however, ethanol has not been found to inhibit the enzyme directly. The effect of ethanol and its breakdown products, acetate and acetaldehyde, on highly purified rat liver methionine synthase was tested in vitro. Enzyme activity was not inhibited by ethanol or acetate. Acetaldehyde was found to inhibit methionine synthase activity, with an apparent IC50 of 2 mM. The reported inhibition by acetaldehyde was found to become irreversible over time. Acetaldehyde-induced inhibition of liver methionine synthase activity is thus proposed as the most likely explanation of the reported in vivo effect of ethanol upon methionine synthase.

Taurine: protective properties against ethanol-induced hepatic steatosis and lipid peroxidation during chronic ethanol consumption in rats.

SummaryAlcohol was administered chronically to female Sprague Dawley rats in a nutritionally adequate totally liquid diet for 28 days. This resulted in hepatic steatosis and lipid peroxidation. Taurine, when co-administered with alcohol, reduced the hepatic steatosis and completely prevented lipid peroxidation. The protective properties of taurine in preventing fatty liver were also demonstrated histologically. Although alcohol was found not to affect the urinary excretion of taurine (a non-invasive marker of liver damage), levels of serum and liver taurine were markedly raised in animals receiving alcohol + taurine compared to animals given taurine alone. The ethanol-inducible form of cytochrome P-450 (CYP2E1) was significantly induced by alcohol; the activity was significantly lower than controls and barely detectable in animals fed the liquid alcohol diet containing taurine. In addition, alcohol significantly increased homocysteine excretion into urine throughout the 28 day period of ethanol administration; however, taurine did not prevent this increase. There was evidence of slight cholestasis in animals treated with alcohol and alcohol + taurine, as indicated by raised serum bile acids and alkaline phosphatase (ALP). The protective effects of taurine were attributed to the potential of bile acids, especially taurine conjugated bile acids (taurocholic acid) to inhibit the activity of some microsomal enzymes (CYP2E1). Thesein vivo findings demonstrate for the first time that hepatic steatosis and lipid peroxidation, occurring as a result of chronic alcohol consumption, can be ameliorated by administration of taurine to rats.

In vitro inactivation of mammalian methionine synthase by nitric oxide

The research described here provides one mechanism of uniting current effects of nitric oxide (NO) with the elevated levels of homocysteine detected in patients with cardiovascular and other disease. Time‐ and dose‐dependent studies of the inhibition of purified mammalian methionine synthase by NO were performed. The in vitro study gave an effective IC50 value of 3 μmol L‐1. Methionine synthase converts cellular homocysteine to methionine and is a major enzyme in the biosynthetic pathways for folates, S‐adenosylmethionine and biological methylations, sulphur amino acids and polyamines. Nitric oxide‐induced inactivation of methionine synthase alters the levels of these metabolites and could therefore provide a connection between the cardiovascular effects of NO, the plasma homo‐cysteine levels and cardiovascular diseases that is complementary to the more traditional NO‐induced stimulation of guanylate cyclase and the convertion of homocysteine to oxidized sulphur amino acids.

Methionine synthase activity and sulphur amino acid levels in the rat liver tumour cells HTC and Phi-1

Methionine dependence has been reported in tumour cells and suggested as a possible target for chemotherapeutic drugs. The underlying defect has not been extensively researched, nor have levels of sulphur amino acids been examined in these cells. This study compared two rat liver tumour cell lines. One was found to be methionine dependent (HTC) and the other found to be methionine independent (Phi-1). The methionine-dependent cell line (HTC) was discovered to contain markedly less methionine synthase activity, the enzyme activity being less responsive to methionine concentration than in the methionine-independent cells (Phi-1). HTC cells had lower cysteine requirements and contained larger concentrations of reduced glutathione (GSH) and taurine than the Phi-1 cells. Also, in contrast to Phi-1 cells, no glutathione was found in the media of the HTC cells, although large quantities of cysteinylglycine were detected. These results suggested that differences in methionine synthase activity might be partly responsible for methionine dependence and that methionine-dependent cells may have different metabolic requirements for other sulphur amino acids.

Effect of hydrazine upon vitamin B12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes

The effect of the industrial chemical, hydrazine (4-12 mM), on methionine synthase (EC 2.1.1.13) activity and levels of the sulphur amino acids homocysteine, cysteine, and taurine as well as GSH were investigated in vitro in isolated rat hepatocyte suspensions and monolayers in order to explain some of the adverse in vivo effects of hydrazine. None of the concentrations of hydrazine were overtly cytotoxic in hepatocyte suspensions (measured as lactate dehydrogenase [LDH] leakage) after 3 hr. However, after 24 hr in culture cells treated with 12 mM, hydrazine showed a significant increase in LDH leakage. Methionine synthase activity was reduced by hydrazine (8 and 12 mM) in suspensions (by 45 and 55%, after 3 hr) and monolayers (12 mM; 65-80% after 24 hr). This was not due to nitric oxide production and the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine, failed to protect against the hydrazine-induced loss of ATP and GSH and the reduction in urea synthesis at 24 hr. Homocysteine export was increased by 6 mM hydrazine, and total taurine content of treated cells was increased by 12 mM hydrazine. Thus, hydrazine was found to have several important and possibly deleterious effects on some parts of the sulphur amino acid pathway.