Effects of hyperhomocysteinemia and betaine-homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome.

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1mM and 2mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine-homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.

Quantification of homocysteine-related metabolites and the role of betaine-homocysteine S-methyltransferase in HepG2 cells.

We optimized and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of six metabolites of homocysteine metabolism: homocysteine, methionine, cysteine, S-adenosylmethionine, S-adenosylhomocysteine and betaine. The detection limits for these metabolites were in the nanomolar range, and the intra- and inter-day precisions were lower than 20% of the relative standard deviations. The method was specifically designed for the determination of the intracellular concentrations of the metabolites in cultured cells. To study the role of betaine-homocysteine S-methyltransferase (BHMT), HepG2 cells and HepG2 cells that were stably transfected with BHMT ((BHMT) HepG2) were treated with homocysteine or with a specific inhibitor of BHMT, and metabolite levels were subsequently measured. Severely compromised methyl group metabolism in the HepG2 cells, which is typical of cancer-derived cells, prevented clear evaluation of the changes caused by the external manipulations of homocysteine metabolism. However, the ease of handling these cells and the almost unlimited source of experimental material supplied by cells in permanent culture allowed us to develop a reliable methodology. The precautions concerning intracellular metabolite determinations using LC-MS/MS in cultured cells that are expressed in this work will have global validity for future metabolomics studies.

Excretion of urinary N7 guanine and N3 adenine DNA adducts in mice after inhalation of styrene.

New urinary adenine adducts, 3-(2-hydroxy-1-phenylethyl)adenine (N3alphaA), 3-(2-hydroxy-2-phenylethyl)adenine (N3betaA), were found in the urine of mice exposed to styrene vapour. These styrene 7,8-oxide derived adenine adducts as well as previously identified guanine adducts, 7-(2-hydroxy-1-phenylethyl)guanine (N7alphaG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7betaG) were quantified by HPLC-ESI-MS(2) and the excretion profile during and after a repeated exposure to 600mg/m(3) or 1200mg/m(3) of styrene for 10 consecutive days (6h/day) was determined. The excretion was dose dependent. Total N3 adenine adducts (N3alphaA+N3betaA) excreted amounted to nearly 0.8x10(-5)% of the absorbed dose while urinary N7 guanine adducts (N7alphaG+N7betaG) amounted to nearly 1.4x10(-5)% of the dose. No accumulation of the adducts was observed. Due to rapid depurination from the DNA, the excretion of both N3 adenine and N7 guanine adducts ceased shortly after finishing the exposure. Both N3 adenine and N7 guanine adducts may be used as non-invasive biomarkers of effective dose reflecting only a short time exposure to styrene.

Targeted Metabolomics for Homocysteine-Related Metabolites in Primary Hepatocytes.

Liquid chromatography-tandem mass spectrometry has become the most convenient method to identify and quantify low molecular weight metabolites from various sources. Metabolomics studies of hepatocytes hold promise for the identification of the mechanisms of toxicant-related disease processes. In this chapter, we present a rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of intracellular concentrations of nine homocysteine-based metabolites, namely homocysteine, methionine, cysteine, dimethylglycine, cystathionine, S-adenosylmethionine, S-adenosylhomocysteine, choline, and betaine. The method is specifically designed for the analysis of cultured primary hepatocytes.

Vinylphenylmercapturic acids in human urine as biomarkers of styrene ring oxidation.

New metabolites of styrene, three isomeric vinylphenylmercapturic acids (2-, 3-, and 4-VPMA), were recently identified by LC-ESI-MS in the urine of mice. In this study, 4-VPMA together with traces of 2- and 3-VPMA were found also in the urine of hand-lamination workers, which were exposed to styrene vapours at concentrations ranging from 23 to 244mg/m(3). Concentrations of 4-VPMA in these end-of-shift samples were 4.59±3.64ng/mL (mean±S.D.; n=10), those found next morning after the work-shift were 2.14±2.07ng/mL (mean±S.D.; n=10). Strong correlation (R=0.959) was found in the next-morning samples between concentrations of 4-VPMA and phenylglyoxylic acid, whereas correlations found between 4-VPMA and mandelic acid in both end-of-shift and next-morning samples were much weaker. The excretion of 4-VPMA accounted for only about 3.5×10(-4)% of the absorbed dose of styrene. Despite very low metabolic yield, formation of VPMAs clearly indicates occurrence and extent of styrene ring oxidation considered to be a toxicologically relevant metabolic pathway.

Urinary N3 adenine DNA adducts in humans occupationally exposed to styrene.

Urine samples from humans occupationally exposed to styrene, with mandelic acid levels ranging from 400 to 1145 mg/g creatinine and from 68 to 400mg/g creatinine for high and low exposure group, respectively, were analysed for N3 adenine DNA adducts, namely, 3-(2-hydroxy-1-phenylethyl)adenine (N3 alpha A) and 3-(2-hydroxy-2-phenylethyl)adenine (N3 beta A). A sensitive LC-ESI-MSMS method was developed with the limit of quantification of 1 pg/mL for both analytes. Peaks corresponding to N3 alpha A and/or N3 beta A were found in seven of nine end-of-shift samples of the high exposure group and in six of 19 end-of-shift samples of the low exposure group. Concentration of N3 alpha A+N3 beta A amounted to 2.8+/-1.6 pg/mL (mean+/-S.D.; n=9) and 1.8+/-1.3 pg/mL (mean+/-S.D.; n=19) in the high and low exposure group, respectively. Of other 10 samples taken the next morning after exposure, two contained low but quantifiable concentrations of N3 alpha A and none contained N3 beta A. However, interfering peaks were detected also in some control urine samples. Out of 22 controls, six and two samples contained peaks co-eluting with N3 alpha A and N3 beta A, respectively. Therefore, the method used was found insufficiently specific to be applicable for biological monitoring. Comparing the excretion of N3 alpha A+N3 beta A to that reported previously in mice it can be estimated that at the same absorbed dose, humans excreted not more than 1/30 of the amount of adenine adducts excreted by mice. As a consequence, the damage to DNA caused by styrene 7,8-oxide (SO), a reactive metabolite of styrene, appears to be much lower in humans than in mice.

Design of a partial PPARdelta agonist.

Structure based ligand design was used in order to design a partial agonist for the PPARdelta receptor. The maximum activation in the transactivation assay was reduced from 87% to 39%. The crystal structure of the ligand binding domain of the PPARdelta receptor in complex with compound 2 was determined in order to understand the structural changes which gave rise to the decrease in maximum activation.