Age-Related Changes in Sulfur Amino Acid Metabolism in Male C57BL/6 Mice.

Alterations in sulfur amino acid metabolism are associated with an increased risk of a number of common late-life diseases, which raises the possibility that metabolism of sulfur amino acids may change with age. The present study was conducted to understand the age-related changes in hepatic metabolism of sulfur amino acids in 2-, 6-, 18- and 30-month-old male C57BL/6 mice. For this purpose, metabolite profiling of sulfur amino acids from methionine to taurine or glutathione (GSH) was performed. The levels of sulfur amino acids and their metabolites were not significantly different among 2-, 6- and 18-month-old mice, except for plasma GSH and hepatic homocysteine. Plasma total GSH and hepatic total homocysteine levels were significantly higher in 2-month-old mice than those in the other age groups. In contrast, 30-month-old mice exhibited increased hepatic methionine and cysteine, compared with all other groups, but decreased hepatic S-adenosylmethionine (SAM), S-adenosylhomocysteine and homocysteine, relative to 2-month-old mice. No differences in hepatic reduced GSH, GSH disulfide, or taurine were observed. The hepatic changes in homocysteine and cysteine may be attributed to upregulation of cystathionine ß-synthase and down-regulation of γ-glutamylcysteine ligase in the aged mice. The elevation of hepatic cysteine levels may be involved in the maintenance of hepatic GSH levels. The opposite changes of methionine and SAM suggest that the regulatory role of SAM in hepatic sulfur amino acid metabolism may be impaired in 30-month-old mice.

Sulfur amino acid metabolism in Zucker diabetic fatty rats.

The present study was aimed to investigate the metabolomics of sulfur amino acids in Zucker diabetic fatty (ZDF) rats, an obese type 2 diabetic animal model. Plasma levels of total cysteine, homocysteine and methionine, but not glutathione (GSH) were markedly decreased in ZDF rats. Hepatic methionine, homocysteine, cysteine, betaine, taurine, spermidine and spermine were also decreased. There are no significant difference in hepatic S-adenosylmethionine, S-adenosylhomocysteine, GSH, GSH disulfide, hypotaurine and putrescine between control and ZDF rats. Hepatic SAH hydrolase, betaine-homocysteine methyltransferase and methylene tetrahydrofolate reductase were up-regulated while activities of gamma-glutamylcysteine ligase and methionine synthase were decreased. The area under the curve (AUC) of methionine and methionine-d4 was not significantly different in control and ZDF rats treated with a mixture of methionine (60mg/kg) and methionine-d4 (20mg/kg). Moreover, the AUC of the increase in plasma total homocysteine was comparable between two groups, although the homocysteine concentration curve was shifted leftward in ZDF rats, suggesting that the plasma total homocysteine after the methionine loading was rapidly increased and normalized in ZDF rats. These results show that the AUC of plasma homocysteine is not responsive to the up-regulation of hepatic BHMT in ZDF rats. The present study suggests that the decrease in hepatic methionine may be responsible for the decreases in its metabolites, such as homocysteine, cysteine, and taurine in liver and consequently decreased plasma homocysteine levels.

L-serine supplementation attenuates alcoholic fatty liver by enhancing homocysteine metabolism in mice and rats.

BACKGROUND: Hyperhomocysteinemia plays an important role in the development of hepatic steatosis, and studies indicate that homocysteine-lowering treatment inhibits the development of fatty liver. OBJECTIVE: We evaluated the effects of L-serine on alcoholic fatty liver and homocysteine metabolism. METHODS: In a binge ethanol study, male C57BL/6 mice were divided into 4 groups: control, ethanol + vehicle, and ethanol + 20 or 200 mg/kg L-serine. Mice were gavaged with ethanol (5 g/kg body weight) 3 times every 12 h with or without L-serine which was given twice 30 min before the last 2 ethanol doses. Control mice were fed isocaloric dextran-maltose. In a chronic ethanol study, male Wistar rats were divided into 3 groups: control, ethanol, and ethanol + L-serine. Rats were fed a standard Lieber-DeCarli ethanol diet (36% ethanol-derived calories) for 4 wk with or without dietary L-serine supplementation (1%; wt:vol) for the last 2 wk. In control rats, the ethanol-derived calories were replaced with dextran-maltose. The effects of L-serine were also tested in AML12 cells manipulated to have high homocysteine concentrations by silencing the genes involved in homocysteine metabolism. RESULTS: Binge ethanol treatment increased serum homocysteine and hepatic triglyceride (TG) concentrations by >5-fold vs. controls, which were attenuated in the 200-mg/kg L-serine treatment group by 60.0% and 47.5%, respectively, compared with the ethanol group. In the chronic ethanol study, L-serine also decreased hepatic neutral lipid accumulation by 63.3% compared with the ethanol group. L-serine increased glutathione and S-adenosylmethionine by 94.0% and 30.6%, respectively, compared with the ethanol group. Silencing betaine homocysteine methyltransferase, cystathionine ß-synthase, or methionine increased intracellular homocysteine and TG concentrations by >2-fold, which was reversed by L-serine when L-serine-independent betaine homocysteine methyltransferase was knocked down. CONCLUSION: These results demonstrate that L-serine ameliorates alcoholic fatty liver by accelerating L-serine-dependent homocysteine metabolism.

Effects of age increase on hepatic expression and activity of cytochrome P450 in male C57BL/6 mice.

Effects of aging on hepatic expression and activity of cytochrome P450 (CYP) isoforms were investigated in male mice aged 2, 6, 18, and 30 months. Microsomal protein, total CYP, cytochrome b5 and NADPH-dependent cytochrome P450 reductase contents in liver were fully expressed in young (2-month-old) mice. Neither Cyp1a1 nor Cyp2c was detected in any aged mice. And Cyp1a2 was maximally expressed at 2 months and decreased with age. Hepatic levels of Cyp2b10 and Cyp3a11 were decreased in 30-month-old mice. Hepatic Cyp2e1 levels were constantly maintained from 2-month to 30-month old mice. Hepatic activities of ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase were gradually decreased after 6 months. The 30-month-old mice exhibited the lowest activity of midazolam 1'-hydroxylase. Pentoxyresorufin-O-depenthylase activity was decreased in 30-month-old mice, but not statistically significant. There were no significant differences in hepatic activities of chlorzoxazone 6-hydroxylase and midazolam 4-hydroxylase. The present study shows that increasing age, especially 30-month-old mice, leads to decrease in expression and activity of hepatic CYP isoforms, suggesting that aging mice exhibit poor hepatic drug-metabolizing capacity.

HepG2 cells as an in vitro model for evaluation of cytochrome P450 induction by xenobiotics.

Although various in vitro assays have been developed to evaluate the cytochrome P450 (CYP)-inducing potential of drug candidates, there is a continuing need for the development of a reliable model in drug discovery. The objective of the present study was to compare CYP induction by chemicals in HepG2 cells with Huh7, NKNT-3, and reverted NKNT-3 cells. HepG2 cells showed more similarity to human liver than the other cell lines in comparisons of the expression of cellular proteins. In evaluation of basal CYP activity, Huh7 cells exhibited the highest CYP1A2 and CYP3A4 activity, and HepG2 cells showed the highest CYP2B6 activity. The inducibility of CYP1A2, CYP2B6, and CYP3A4 by prototypical inducers was determined using enzyme assay, immunoblot analysis, and real-time PCR. Among the cells tested, HepG2 cells were highly responsive to CYP inducers, such as 3-methylcholanthrene for CYP1A2 and phenobarbital for CYP2B6 and CYP3A4. Moreover, HepG2 cells were responsive to various CYP1A2, CYP2B6, and CYP3A4 inducers as determined using fluorogenic and LC-MS/MS substrates. Thus, HepG2 cells may be comparable to human hepatocytes for the evaluation of CYP induction or slightly less sensitive. These results suggest HepG2 cells as a cell-based model in screening for CYP inducers in drug discovery.

Expression of hepatic antioxidant enzymes in non-obese type-2 diabetic Goto-Kakizaki rats.

Diabetes mellitus and its complications have been attributed in part to oxidative stress, against which antioxidant enzymes constitute a major protective mechanism. The present study was performed to investigate the effects of early stage type 2 diabetes in the absence of obesity and liver damage on hepatic antioxidant enzyme expression and oxidative stress using 9-week-old Goto-Kakizaki (GK) rats. Hepatic total antioxidant capacity determined by total oxygen radical scavenging capacity and lipid peroxidation determined by malondialdehyde in plasma and liver were not significantly different between normal Wistar rats and GK rats. These results indicated that oxidative stress is not evident in these type 2 diabetic rats. Hepatic expression levels of antioxidant enzymes, including superoxide dismutase-1, catalase, glutathione peroxidase and reductase, thioredoxin-1, mu- and pi-class glutathione S-transferase (GST), and the gamma-glutamylcysteine ligase catalytic subunit, were not different between normal rats and GK rats. But, hepatic level and activity of alpha-class GST were decreased and peroxiredoxin-1 level was increased in GK rats, suggesting that upregulation of peroxiredoxin-1 compensates for downregulation of alpha-class GST. These results suggest that alpha-class GST and peroxiredoxin-1 in liver can be altered during the early stages of type 2 diabetes in the absence of obesity and severe oxidative stress.

Alterations in hepatic metabolism of sulfur amino acids in non-obese type-2 diabetic Goto-Kakizaki rats.

Elevated plasma homocysteine has been identified as a risk factor for cardiovascular disease and non-alcoholic liver disease, which are major complications of diabetes. Hence, hepatic homocysteine metabolism has become a major focus of diabetes research. However, little information is available regarding plasma homocysteine levels in non-obese diabetic animals. Therefore, we investigated the hepatic metabolism of sulfur-amino acids in non-obese type-2 diabetic Goto-Kakizaki rats. The experiments were performed using 9-week-old Goto-Kakizaki rats and age-matched Wistar rats. The major finding of this study is that homocysteine levels in the liver and plasma are maintained by a balance between the up-regulation of betaine homocysteine methyltransferase and the inhibition of cystathionine ß-synthase in non-obese type-2 diabetic rats. Hepatic levels of cysteine and its metabolites, such as hypotaurine, taurine, and glutathione, were increased despite inhibition of the transsulfuration of homocysteine to cysteine. The elevated hepatic taurine and glutathione levels may be attributed to the up-regulation of cysteine dioxygenase expression and increased cysteine availability for glutathione synthesis. Inhibition of hepatic methionine adenosyltransferase activity in Goto-Kakizaki rats was associated with a decrease in hepatic S-adenosylmethionine, which serves as an allosteric activator of cystathionine ß-synthase. The non-obese type-2 diabetic condition results in profound changes in hepatic sulfur-amino acid metabolism and raises the possibility that sulfur-amino acid metabolism may be regulated by obesity- as well as diabetes-associated factors. Further study to elucidate the pathological significance of sulfur-amino acid metabolism in chronic liver disease in type-2 diabetic animals is underway in this laboratory.

Elevation of cysteine consumption in tamoxifen-resistant MCF-7 cells.

Tamoxifen (TAM) resistance is a main cause of therapeutic failure in breast cancers. Although methionine dependency is a phenotypic characteristic of tumor cells, the role of sulfur amino acid metabolism in chemotherapy resistance remains to be elucidated. This study compared metabolite profiles of sulfur amino acid metabolism from methionine to taurine or glutathione (GSH) between normal MCF-7 and TAM-resistant MCF-7 (TAMR-MCF-7) cells. TAMR-MCF-7 cells showed elevated levels and activities of enzymes involved in both transsulfuration from methionine to cysteine and metabolism of cysteine to GSH and taurine. Cysteine concentrations in TAMR-MCF-7 cells and medium conditioned by cell culture for 42h were markedly decreased, while GSH, hypotaurine, and taurine concentrations in the medium were increased. These results show that TAMR-MCF-7 cells display enhanced cysteine utilization. The addition of propargylglycine, a specific cystathionine γ-lyase inhibitor, and buthionine sulfoximine, a specific γ-glutamylcysteine ligase inhibitor, to TAMR-MCF-7 cells, but not to MCF-7 cells, resulted in cytotoxicity after sulfur amino acid deprivation. These results suggest that cell viability of TAMR-MCF-7 cells is affected by inhibition of sulfur amino acid metabolism, particularly cysteine synthesis from homocysteine and GSH synthesis from cysteine. Additionally, the S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in TAMR-MCF-7 cells increased to ~3.6-fold relative to that in MCF-7 cells, a finding that may result from upregulation of methionine adenosyltransferase IIa and S-adenosylhomocysteine hydrolase. In conclusion, this study suggests that TAMR-MCF-7 cells display enhanced cysteine utilization for synthesis of GSH and taurine, and are sensitive to inhibition of cysteine metabolism.

The impact of intratracheally instilled carbon black on the cardiovascular system of rats: elevation of blood homocysteine and hyperactivity of platelets.

Carbon black (CB) is an industrial chemical with high potential for human exposure. Although the relationship between exposure to particulate matter (PM) and cardiovascular disease is well documented, the risk of adverse cardiovascular effects attributed to CB particles has not been clearly characterized. This study was performed to (1) investigate the effects of CB on cardiovascular system and (2) identify the target tissue or potential biomarkers. Carbon black with a distinct particle size, N330 (ultrafine particle) and N990 (fine particle), was intratracheally instilled into rats at a doses of 1, 3, or 10 mg/kg. Measurements of thrombotic activity and determination of plasma homocysteine levels, cardiac functionality, and inflammatory responses were conducted at 24-h and 1-wk time points. Exposure to N330 accelerated platelet-dependent blood clotting at 10 mg/kg, the highest exposure tested. Unexpectedly, both N330 and N990 led to prolongation of activated partial thromboplastin time (aPTT), whereas these CB particles failed to affect prothrombin time (PT). N990 produced a significant elevation in the level of plasma homocysteine, a well-established etiological factor in cardiovascular diseases. Both N330 and N990 induced apparent inflammation in the lungs; however, both particles failed to initiate systemic inflammation. Neither CB particle produced observable cardiac symptoms as detected by electrocardiography. Taken together, data show CB exposure enhanced the cardiovascular risk by inducing hyperhomocysteinemia and platelet hyperactivity, although these effects may be variable depending on particle size and exposure duration. Homocysteine may be a potential biomarker for cardiovascular toxicity following CB exposure.

Hepatic expression of cytochrome P450 in type 2 diabetic Goto-Kakizaki rats.

Although hepatic expression of cytochrome P450 (CYP) changes markedly in diabetes, the role of ketone bodies in the regulation of CYP in diabetes is controversial. The present study was performed to determine the expression and activity of CYP in non-obese type II diabetic Goto-Kakizaki (GK) rats with normal levels of ketone bodies. In the present study, basal serum glucose levels increased 1.95-fold in GK rats, but acetoacetate and ß-hydroxybutyrate levels were not significantly different. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats, and consequently, activities of CYP reductase and midazolam 4-hydroxylase, mainly catalyzed by CYP3A2, increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated and the activities of 7-ethoxyresorufin-O-deethylase and 7-methoxyresorufin-O-demethylase, mainly catalyzed by CYP1A, also decreased in GK rats. Hepatic levels of microsomal protein and total CYP and hepatic expression of cytochrome b(5), CYP1B1, CYP2B1 and CYP2C11 were not significantly different between the GK rats and normal Wistar rats. Moreover, the expression and activity of CYP2E1, reported to be up-regulated in diabetes with hyperketonemia, were not significantly different between GK rats and control rats, suggesting that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. Our results showed that the expression of hepatic CYP is regulated in an isoform-specific manner. The present results also show that the GK rat is a useful animal model for the pathophysiological study of non-obese type II diabetes with normal ketone body levels.

Sulfur amino acid metabolism in doxorubicin-resistant breast cancer cells.

Although methionine dependency is a phenotypic characteristic of tumor cells, it remains to be determined whether changes in sulfur amino acid metabolism occur in cancer cells resistant to chemotherapeutic medications. We compared expression/activity of sulfur amino acid metabolizing enzymes and cellular levels of sulfur amino acids and their metabolites between normal MCF-7 cells and doxorubicin-resistant MCF-7 (MCF-7/Adr) cells. The S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in MCF-7/Adr cells decreased to ~10% relative to that in MCF-7 cells, which may have resulted from down-regulation of S-adenosylhomocysteine hydrolase. Expression of homocysteine-clearing enzymes, such as cystathionine beta-synthase, methionine synthase/methylene tetrahydrofolate reductase, and betaine homocysteine methyltransferase, was up-regulated in MCF-7/Adr cells, suggesting that acquiring doxorubicin resistance attenuated methionine-dependence and activated transsulfuration from methionine to cysteine. Homocysteine was similar, which is associated with a balance between the increased expressions of homocysteine-clearing enzymes and decreased extracellular homocysteine. Despite an elevation in cysteine, cellular GSH decreased in MCF-7/Adr cells, which was attributed to over-efflux of GSH into the medium and down-regulation of the GSH synthesis enzyme. Consequently, MCF-7/Adr cells were more sensitive to the oxidative stress induced by bleomycin and menadione than MCF-7 cells. In conclusion, our results suggest that regulating sulfur amino acid metabolism may be a possible therapeutic target for chemoresistant cancer cells. These results warrant further investigations to determine the role of sulfur amino acid metabolism in acquiring anticancer drug resistance in cancer cells using chemical and biological regulators involved in sulfur amino acid metabolism.