Endogenous benzoic acid interferes with the signatures of amino acids and thiol compounds through perturbing N-methyltransferase, glutamate-cysteine ligase, and glutathione S-transferase activity in dairy products.

Endogenous benzoic acid causes adverse effects on individual health, but the potential mechanisms often remain elusive. The positive rate of benzoic acid in seventy-two goat milk samples in triplicate was 93.6 %, verifying the presence of endogenous benzoic acid. In this study, we investigated the differences in protein expression and metabolites among goat milk with different final concentrations of benzoic acid via combined proteomics and metabolomics (LOQ 3.25 to 56.63 µg L-1) analysis based on UHPLC-Q-Orbitrap HRMS. Integrated analysis showed that benzoic acid reduced the content of l-histidine (from 1.27 to 0.49 mg/L) and 1-methylhistidine (from 1.40 to 0.68 mg/L), due to the increase of benzoic acid (0-30 mg/L) concentration significantly reduced the level and activity of N-methyltransferase. Protein-metabolite interactions suggested that benzoic acid enhanced glutamate-cysteine ligase and glutathione S-transferase expression and affected l-glutamate (from 1.22 to 0.49 mg/L) and glutathione contents, eventually leading to the formation of off-flavors and oxidation of goat milk. Meanwhile, the level of l-phenylalanine (from 4.17 to 1.94 mg/L) and l-tyrosine (from 1.05 to 0.26 mg/L) progressively decreased with the increase of benzoic acid concentration, which had a deleterious effect on the nutritional value and flavor formation of goat milk. These findings clarified the mechanism by which low-dose benzoic acid negatively affects the nutritional quality and flavor formation of goat milk.

A Phase II Randomized Placebo-Controlled Trial of Oral N-acetylcysteine for Protection of Melanocytic Nevi against UV-Induced Oxidative Stress In Vivo.

Oxidative stress plays a role in UV-induced melanoma, which may arise from melanocytic nevi. We investigated whether oral administration of the antioxidant N-acetylcysteine (NAC) could protect nevi from oxidative stress in vivo in the setting of acute UV exposure. The minimal erythemal dose (MED) was determined for 100 patients at increased risk for melanoma. Patients were randomized to receive a single dose (1,200 mg) of NAC or placebo, in double-blind fashion, and then one nevus was irradiated (1-2 MED) using a solar simulator. One day later, the MED was redetermined and the irradiated nevus and a control unirradiated nevus were removed for histologic analysis and examination of biomarkers of NAC metabolism and UV-induced oxidative stress. Increased expression of 8-oxoguanine, thioredoxin reductase-1, and γ-glutamylcysteine synthase modifier subunit were consistently seen in UV-treated compared with unirradiated nevi. However, no significant differences were observed in these UV-induced changes or in the pre- and postintervention MED between those patients receiving NAC versus placebo. Similarly, no significant differences were observed in UV-induced changes between subjects with germline wild-type versus loss-of-function mutations in the melanocortin-1 receptor. Nevi showed similar changes of UV-induced oxidative stress in an open-label post-trial study in 10 patients who received NAC 3 hours before nevus irradiation. Thus, a single oral dose of NAC did not effectively protect nevi from UV-induced oxidative stress under the conditions examined. Cancer Prev Res; 10(1); 36-44. ©2016 AACR.

Carnosic acid protects against 6-hydroxydopamine-induced neurotoxicity in in vivo and in vitro model of Parkinson's disease: involvement of antioxidative enzymes induction.

The neuroprotective effects of carnosic acid (CA), a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), have been widely investigated in recent years, however, its protection in in vivo still unclear. In this study, we investigated the behavioral activity and neuroprotective effects of CA in a rat model of Parkinson's disease (PD) induced by 6-hydroxydopamine (6-OHDA). Rats were treated with 20mg/kg body weight of CA for 3 weeks before 6-OHDA exposure. Results indicated that CA improved the locomotor activity and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Significant protection against lipid peroxidation and GSH reduction was observed in the 6-OHDA rats pretreated with CA. Pretreatment with CA increased the protein expression of γ-glutamate-cysteine ligase catalytic subunit, γ-glutamate-cysteine ligase modifier subunit, superoxide dismutase, and glutathione reductase compared with 6-OHDA-stimulated rats and SH-SY5Y cells. Immunoblots showed that the reduction of the Bcl-2/Bax ratio, the induction of caspase 3 cleavage, and the induction of poly(ADP-ribose) polymerase (PARP) cleavage by 6-OHDA was reversed in the presence of SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) in SH-SY5Y cells. Rats treated with CA reversed the 6-OHDA-mediated the activation of c-Jun NH2-terminal kinase and p38, the down-regulation of the Bcl-2/Bax ratio, the up-regulation of cleaved caspase 3/caspase 3 and cleaved PARP/PARP ratio, and the down-regulation of tyrosine hydroxylase protein. However, BAM7, an activator of Bax, attenuated the effect of CA on apoptosis in SH-SY5Y cells. These results suggest that CA protected against 6-OHDA-induced neurotoxicity is attributable to its anti-apoptotic and anti-oxidative action. The present findings may help to clarify the possible mechanisms of rosemary in the neuroprotection of PD.

Deferoxamine promotes osteoblastic differentiation in human periodontal ligament cells via the nuclear factor erythroid 2-related factor-mediated antioxidant signaling pathway.

BACKGROUND AND OBJECTIVE: Recently it was reported that deferoxamine (DFO), an iron chelator, stimulates bone formation from MG63 and mesenchymal stem cells, but inhibits differentiation in rat calvarial cells; however, the effect of DFO on osteoblastic differentiation in human periodontal ligament cells (hPDLCs) has not been reported. The aim of this study was to investigate the effects and the possible underlying mechanism of DFO on osteoblastic differentiation of hPDLCs. MATERIAL AND METHODS: The effect of DFO on osteoblast differentiation was determined by the staining intensity of calcium deposits with Alizarin red and by RT-PCR analysis of the expression of osteoblastic markers. Signal transduction pathways were analyzed by western blotting. RESULTS: DFO increased osteogenic differentiation in a concentration-dependent manner by expression of the mRNA for differentiation markers and calcium nodule formation. Exposure of hPDLCs to DFO resulted in increases in the production of reactive oxygen species and in the levels of nuclear factor erythroid 2-related factor (Nrf2) protein in nuclear extractions, as well as a dose-dependent increase in the expression of Nrf2 target genes, including glutathione (GSH), glutathione S-transferase, γ-glutamylcysteine lygase, glutathione reductase and glutathione peroxidase. Pretreatment with Nrf2 small interfering RNA, GSH depletion by buthionine sulfoximine and diethyl maleate, and with antioxidants by N-acetylcysteine and vitamin E, blocked DFO-stimulated osteoblastic differentiation. Furthermore, pretreatment with GSH depletion and antioxidants blocked DFO-induced p38 MAPK, ERK, JNK and nuclear factor-kappaB pathways. CONCLUSION: These data indicate, for the first time, that nontoxic DFO promotes osteoblastic differentiation of hPDLCs via modulation of the Nrf2-mediated antioxidant pathway.

Rice protein improves oxidative stress by regulating glutathione metabolism and attenuating oxidative damage to lipids and proteins in rats.

AIMS: To evaluate the effects of rice protein (RP) on glutathione metabolism and oxidative damage. MAIN METHODS: Seven-week-old male Wistar rats were fed diets containing casein and RP without cholesterol for 3weeks. Plasma and liver lipid levels, hepatic accumulation of total glutathione (T-GSH), oxidized glutathione (GSSG), reduced glutathione (GSH), malondialdehyde (MDA) and protein carbonyl (PCO) were measured. In the liver, the total antioxidative capacity (T-AOC), mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modulatory subunit (GCLM), and the activities of hepatic catalase (CAT), total superoxide dismutase (T-SOD), γ-glutamylcysteine synthetase (γ-GCS), glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSHPx) were also measured. KEY FINDINGS: T-AOC, GCLC and GCLM mRNA levels, antioxidative enzyme activities (T-SOD and CAT) and glutathione metabolism related enzyme activities (γ-GCS, GST, GR and GSHPx) were effectively stimulated by RP feeding compared to casein, and RP significantly reduced the hepatic accumulation of MDA and PCO in rats. These results indicate that lipid-lowering activity was induced by RP feeding. SIGNIFICANCE: The present study demonstrates that RP improves oxidative stress primarily through enzymatic and non-enzymatic antioxidative defense mechanisms, reflected by enhancing the antioxidative status and attenuating the oxidative damage to lipids and proteins. These results suggest that RP can prevent hyperlipidemia in part through modifying glutathione metabolism, and sulfur amino acids may be the main modulator of this antioxidative mechanism.

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte-derived dendritic cells.

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides - which are the most frequent cause of adverse drug reactions - were co-incubated with THP-1, MUTZ-LC, or primary monocyte-derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1ß, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines.

Differential antiproliferation effect of 2'-benzoyloxycinnamaldehyde in K-ras-transformed cells via downregulation of thiol antioxidants.

2'-Benzoyloxycinnamaldehyde (BCA), one of the derivatives of 2'-hydroxycinnamaldehyde (HCA) isolated from the bark of Cinnamomum cassia, induces apoptosis in human cancer cells. We found that BCA induces stronger antiproliferative effects in K-ras-transformed cells (RK3E-ras) than in isogenic non-transformed cells (RK3E). Treatment of RK3E-ras with BCA resulted in increased ROS generation and depletion of intracellular glutathione, whereas BCA-treated RK3E showed no significant increase in the ROS level with concurrent increase in intracellular glutathione (GSH). Thiol antioxidants recovered cell proliferation inhibition caused by BCA in both cell lines, while non-thiol antioxidants failed to recover cell death. BCA decreased metallothionein (MT) expression in RK3E-ras, while inducing remarkable MT expression in RK3E. The increase of intracellular GSH in RK3E is partially caused by differential induction of γ-glutamylcysteine synthetase (γ-GCS) due to BCA treatment. To evaluate the upstream pathway for differential expression of γ-GCS and MT, we analyzed early DJ-1 (PARK7) and NF-E2 p45-related factor 2 (Nrf2) changes after BCA treatment. In RK3E, DJ-1 expression considerably increased for 3 h with concurrent induction of Nrf2, whereas in RK3E-ras cells BCA decreased these protein levels. Based on these findings, it seems that the therapeutic selectivity of BCA in RK3E-ras results from decreased thiol antioxidants via decreased DJ-1 and Nrf2 expression.

Diversity in antioxidant response enzymes in progressive stages of human nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD), which occurs in approximately 17 to 40% of Americans, encompasses progressive stages of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH). Inflammation and oxidative stress are known characteristics of NAFLD; however, the precise mechanisms occurring during disease progression remain unclear. The purpose of the current study was to determine whether the expression or function of enzymes involved in the antioxidant response, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione transferase (GST), and glutamate cysteine ligase, are altered in the progression of human NAFLD. Human livers staged as normal, steatotic, NASH (fatty), and NASH (not fatty) were obtained from the Liver Tissue Cell Distribution System. NQO1 mRNA, protein, and activity tended to increase with disease progression. mRNA levels of the GST isoforms A1, A2, A4, M3, and P1 increased with NAFLD progression. Likewise, GST A and P protein increased with progression; however, GST M protein levels tended to decrease. Of interest, total GST activity toward the substrate 1-chloro-2,4-dinitrobenzene decreased with NAFLD progression. GSH synthesis does not seem to be significantly dysregulated in NAFLD progression; however, the GSH/oxidized glutathione redox ratio seemed to be reduced with disease severity, indicating the presence of oxidative stress and depletion of GSH throughout progression of NAFLD. Malondialdehyde concentrations were significantly increased with disease progression, further indicating the presence of oxidative stress. Nuclear immunohistochemical staining of nuclear factor E2-related factor 2 (Nrf2), an indicator of activation of the transcription factor, was evident in all stages of NAFLD. The current data suggest that Nrf2 activation occurs in response to disease progression followed by induction of specific Nrf2 targets, whereas functionality of specific antioxidant defense enzymes seems to be impaired as NAFLD progresses.

Biomonitoring of antioxidant and oxidative stress responses in Perinereis gualpensis (Polychaeta: Nereididae) in Chilean estuarine regions under different anthropogenic pressure.

This study aimed to analyze oxidative stress parameters, including levels of the antioxidant glutathione (GSH), activity of glutamate-cysteine ligase (GCL) and glutathione-S-transferase (GST), total antioxidant capacity and protein oxidation, in the polychaete Perinereis gualpensis (Nereididae) collected from the Biobío, Itata, Valdivia and Lingue estuaries in Chile, which present different degrees of anthropogenic pressure. Sampling sites were characterized considering a geographic information system and the physicochemical characteristics of water and sediment. Significant differences (p<0.05) were observed between the sampling sites for most of the responses (GSH, GCL, GST and antioxidant capacity), mainly related to human activities such as agriculture, industry, among others. Multivariate correlation analysis indicates a certain relationship of antioxidant responses with human activities, salinity, and worm weight, this last employed to standardize GST and antioxidant capacity. These results clearly indicate biomarker responses in P. gualpensis in Biobío and Valdivia estuaries, the more affected by human activities.

Extract of Ginkgo biloba induces glutamate cysteine ligase catalytic subunit (GCLC).

The extract of Ginkgo biloba (EGb), containing 24% flavone glycosides and 6% terpenoids, is widely used to treat early-stage Alzheimer's disease, vascular dementia, peripheral claudication and vascular tinnitus. Its marked antioxidant activity has recently been demonstrated in both cell lines and animals. Glutathione (GSH) plays an important role in the antioxidant system by conjugating to xenobiotics to facilitate their export from cells. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme for GSH synthesis and its catalytic subunit (GCLC) determines this de novo synthesis. Thus, induction of GCLC is a strategy to enhance the antioxidant capability in cells. The present study aimed to investigate the induction effect of EGb on GCLC in HepG2 and Hep1c1c7 cell lines. Real-time PCR, Western blot and enzyme activity assay were used to detect induction and it was found that GCLC was induced by EGb in these two cell lines. It is suggested that the antioxidant activity of EGb is (or is partly) through the induction of GCLC.

Impaired glutathione synthesis in schizophrenia: convergent genetic and functional evidence.

Schizophrenia is a complex multifactorial brain disorder with a genetic component. Convergent evidence has implicated oxidative stress and glutathione (GSH) deficits in the pathogenesis of this disease. The aim of the present study was to test whether schizophrenia is associated with a deficit of GSH synthesis. Cultured skin fibroblasts from schizophrenia patients and control subjects were challenged with oxidative stress, and parameters of the rate-limiting enzyme for the GSH synthesis, the glutamate cysteine ligase (GCL), were measured. Stressed cells of patients had a 26% (P = 0.002) decreased GCL activity as compared with controls. This reduction correlated with a 29% (P < 0.001) decreased protein expression of the catalytic GCL subunit (GCLC). Genetic analysis of a trinucleotide repeat (TNR) polymorphism in the GCLC gene showed a significant association with schizophrenia in two independent case-control studies. The most common TNR genotype 7/7 was more frequent in controls [odds ratio (OR) = 0.6, P = 0.003], whereas the rarest TNR genotype 8/8 was three times more frequent in patients (OR = 3.0, P = 0.007). Moreover, subjects with disease-associated genotypes had lower GCLC protein expression (P = 0.017), GCL activity (P = 0.037), and GSH contents (P = 0.004) than subjects with genotypes that were more frequent in controls. Taken together, the study provides genetic and functional evidence that an impaired capacity to synthesize GSH under conditions of oxidative stress is a vulnerability factor for schizophrenia.

Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress.

Conjugated linoleic acid (CLA) exhibits anticancer and anti-inflammatory properties. Its ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (gammaGCL) protein expression was recently associated with the inhibition of typical pathological signs in MRL/MpJ-Fas(lpr) mice (MRL/lpr). In the present study the ability of CLA to modulate oxidative stress and phase 2 enzyme activity in the same animal model was investigated. Disease severity was associated with age-dependent production of anti-double-stranded DNA antibodies (anti-dsDNA IgGs) and with enhanced extent of oxidative stress markers: reduced total GSH, increased protein 3-nitrotyrosines (3-NT), and protein-bound carbonyl (PC) amounts. To examine the effect of CLA on antioxidant status, CLA or olive oil (as control) was administered to pregnant MRL/lpr mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. Finally, the antioxidant and chemopreventive properties of CLA were investigated in old MRL/lpr mice. Sera of CLA-treated mice contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, gammaGCL, glutathione S-transferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. In conclusion our data indicate that the activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in MRL/lpr mice.

Effect of Semecarpus anacardium Linn. nut milk extract on glutathione and its associated enzymes in experimentally induced mammary carcinoma.

Reduced glutathione (GSH) is a ubiquitous thiol-containing tripeptide that plays a key role in the etiology of many diseases and, in particular, cancer. GSH, the foremost internal protective system, participates directly in the destruction of free radical compounds and detoxification of carcinogens. The effect of Semecarpus anacardium nut milk extract was studied for gaining insight into the disease relationship to GSH and its metabolizing enzymes. Mammary carcinoma was induced by giving 7,12-dimethylbenz[a]anthracene (DMBA) (25 mg/mL of olive oil) perorally by gastric intubation, and nut milk extract of S. anacardium was administered orally (200 mg/kg of body weight/day) for 14 days to mammary carcinoma-bearing rats. The levels of GSH and its metabolizing enzyme activities were determined in liver and kidney homogenates. Significant decreases in GSH, glutathione peroxidase, glutathione S-transferase, glutathione reductase, and gamma-glutamylcysteine synthetase and a concomitant increase in oxidized glutathione, gamma-glutamyl transpeptidase, and glucose 6-phosphate dehydrogenase were observed in DMBA-induced mammary carcinoma in rats, while drug treatment reversed the conditions to near normal levels. There was a marked increase in GSH level and gamma-glutamylcysteine synthetase activity in drug control rats. These findings suggest that S. anacardium can exert its protective effect in maintaining the glutathione redox status by restoring the associated enzymes against oxidative stress in experimental mammary carcinoma.

Expression of antioxidant enzymes in rat retinal ischemia followed by reperfusion.

To evaluate the expression and protein levels of antioxidant enzymes in the rat retina exposed to oxidative stress induced by ischemia-reperfusion injury. Retinal ischemia was induced in female Wistar rats by ligation of the optic nerve and vessels behind the left eye bulb, and was followed by reperfusion for 0, 3, 6, or 24 hours. The right eye served as control. RNA and protein were extracted simultaneously from each retina. Expressions of the endogenous antioxidant enzymes glutathione peroxidase (GPx1), catalase (CAT), copper/zinc superoxide dismutase, manganese superoxide dismutase, and the catalytic subunit of glutamylcysteine ligase (GCLc) were analyzed with real-time reverse transcription polymerase chain reaction and related to the endogenous control cyclophilin B. Protein levels were measured with Western blot analysis. During the early phase (0 or 3 hours) of reperfusion, no changes were seen in enzyme expression. After 6 hours, GCLc expression increased by a factor of 1.14 (P = .034), followed by a decline of 0.80 after 24 hours (P = .00004), according to the comparative Ct method. After 24 hours of reperfusion, GPx1 expression increased by a factor of 1.14 (P = .028), and CAT had decreased by 0.82 (P = .022). Expressions of copper/zinc superoxide dismutase and manganese superoxide dismutase showed a tendency toward a decrease by factors of 0.86 (P = .055) and 0.88 (P = .053), respectively, after 24 hours. Protein levels did not differ for any of the antioxidants, regardless of reperfusion time. The slightly increased messenger RNA expression of GPx1 after 24 hours of reperfusion with a concomitant very modest decrease in CAT and GCLc expression and no change in protein levels indicate a very modest, if any, response to oxidative stress generated by ischemia followed by reperfusion in rat retina.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Antioxidant enzymes in renal cell carcinoma.

The aim of the study was to estimate the significance of oxidative/nitrosative damage and expression of antioxidant enzymes in renal cell carcinomas (RCC). For this we investigated immunohistochemically six antioxidant enzymes (AOEs) including MnSOD, ECSOD, thioredoxin, thioredoxin reductase, and gammaglutamyl cysteine synthetase heavy and light chain in 138 RCCs. As an indicator of oxidative/nitrosative damage, sections were stained with an antibody to nitrotyrosine. The extent of apoptosis was evaluated by TUNEL method and proliferation by immunohistochemistry to Ki67. Variable expression of all AOEs could be seen in RCC with expression of MnSOD being strongest. Nitrotyrosine was significantly associated with high grade tumors. MnSOD was associated with tumors of a lower stage. Cases showing ECSOD reactivity had higher and cases expressing thioredoxin lower apoptotic index than other tumors. No association with patient prognosis was observed. According to the results renal cell carcinomas show oxidative/nitrosative damage which, according to nitrotyrosine staining, was higher in high grade tumors. Of AOEs, MnSOD was more abundantly expressed in low stage tumors suggesting that its antioxidant function could play a main role to prevent development of oxidative damage leading to more aggressive tumors.

Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level.

Two hepatic enzymes, cysteine dioxygenase (CDO) and gamma-glutamylcysteine synthetase (GCS), play important regulatory roles in the response of cysteine metabolism to changes in dietary sulfur amino acid or protein levels. To examine the time-course of changes in CDO and GCS activities, CDO and GCS-catalytic or heavy subunit protein and mRNA levels, and cysteine and glutathione levels, we adapted rats to either a low protein (LP) or high protein (HP) diet, switched them to the opposite diet, and followed these parameters over 6 days. Hepatic CDO activity and amount, but not mRNA level, increased in response to higher protein intake; the t(1/2) of change for CDO activity or protein level was 22 h for rats switched from a LP to a HP diet and 8 h for rats switched from a HP to a LP diet, suggesting that the HP diet decreased turnover of CDO. Hepatic GCS activity, catalytic subunit amount and mRNA level decreased in response to a higher protein intake. GCS catalytic subunit level changed with a similar t(1/2) for both groups, but the change in GCS activity in rats switched from a LP diet to a HP diet was faster (approximately 16h) than for rats switched from a HP to a LP diet (approximately 74h). Hepatic cysteine and glutathione levels reached new steady states within 12 h (LP to HP) or 24 h (HP to LP). CDO activity appeared to be regulated at the level of protein, probably by diminished turnover of CDO in response to higher protein intake or cysteine level, whereas GCS activity appeared to be regulated both at the level of mRNA and activity state in response to the change in cysteine or protein availability. These findings support a role of cysteine concentration as a mediator of its own metabolism, favoring catabolism when cysteine is high and glutathione synthesis when cysteine is low.

Fluorescence-based microtiter plate assay for glutamate-cysteine ligase activity.

Glutamate-cysteine ligase (GCL; also known as gamma-glutamylcysteine synthetase) is the rate-limiting enzyme in glutathione (GSH) synthesis. Traditional assays for the activity of this enzyme are based either on coupled reactions with other enzymes or on high-performance liquid chromatography (HPLC) assessment of gamma-glutamylcysteine (gamma-GC) product formation. We took advantage of the reaction of naphthalene dicarboxaldehyde (NDA) with GSH or gamma-GC to form cyclized products that are highly fluorescent. Hepa-1 cells which were designed to overexpress mouse GCL and mouse liver homogenates were used to evaluate and compare the utility of the NDA method with an assay based on monobromobimane derivatization and HPLC analysis with fluorescence detection. Excellent agreement was found between GCL activities measured by HPLC and NDA-microtiter plate analyses. This assay should be useful for high-throughput GCL activity analyses.

Diminished immunoreactivity of gamma-glutamylcysteine synthetase in the airways of smokers' lung.

Glutathione (GSH) plays a major role in protecting the airways against oxidative stress. The rate-limiting enzyme in de novo GSH synthesis is gamma-glutamylcysteine synthetase (gamma-GCS), which is induced by acute exposure to GSH-depleting cytokines and oxidants, but downregulated by transforming growth factor beta and prolonged oxidant exposure, at least in vitro. Cell-specific expression or regulation of gamma-GCS may play an important role both in the defense against oxidants and in the pathogenesis of oxidant-associated airway diseases. In this study, the localizations of gamma-GCS heavy (gamma-GCS-HS) and light (gamma-GCS-LS) subunits were investigated by immunohistochemistry in 22 patients with chronic obstructive pulmonary disease (COPD), 20 smokers without COPD, and 13 lifelong nonsmokers. The ultrastructural distributions of both gamma-GCS subunits were assessed by immuno-electron microscopy. Both subunits were expressed most prominently in the large airways, and their ultrastructural localization was both cytoplasmic and along the plasma membrane. The expression of gamma-GCS-HS was stronger in the central bronchial epithelium than in the peripheral bronchioli (p = 0.020), or in alveolar macrophages (p = 0.008). The expression of gamma-GCS-HS in the central bronchial epithelium showed a tendency to be higher in nonsmokers compared with all smokers (p = 0.052). Alveolar macrophages of nonsmokers had higher levels of gamma-GCS-HS (p = 0.001) and gamma-GCS-LS (p = 0.001) than did smokers. The expression of gamma-GCS-HS in the central bronchial epithelium was more marked in nonsmokers than in patients with COPD (p = 0.015), the difference between smokers and patients with COPD was not significant. In conclusion, the heavy and light subunits of gamma-GCS are mainly expressed in the large airways. Their tendency to decrease in cigarette smokers may further predispose lung cells to ongoing oxidant stress, which contributes to the progression of lung injury.