N-acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens. NAT1 alleles *10 and *11 have been proposed to alter protein level or enzyme activity compared to wild-type NAT1 *4 and to confer cancer risk, via uncertain pathways. This study characterizes regulatory polymorphisms and underlying mechanisms of NAT1 expression.
We measured allelic NAT1 mRNA expression and translation, as a function of multiple transcription start sites, alternative splicing, and three 3′-polyadenylation sites in human livers (one of which discovered in this study), B lymphocytes, and transfected cells. In a clinical study of 469 HIV/AIDS patients treated with the NAT1/NAT2 substrate sulfamethoxazole (SMX), associations were tested between SMX induced hypersensitivity and NAT1 *10 and *11 genotypes, together with known NAT2 polymorphisms.
NAT1*10 and *11 were determined to act as common regulatory alleles accounting for most NAT1 expression variability, both leading to increased translation into active protein. NAT1*11 (2.4% minor allele frequency) affected 3′polyadenylation site usage, thereby increasing formation of NAT1 mRNA with intermediate length 3′UTR (major isoform) at the expense of the short isoform, resulting in more efficient protein translation. NAT1 *10 (19% minor allele frequency) increased translation efficiency without affecting 3′-UTR polyadenylation site usage. Livers and B-lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 immunoreactivity and NAT1 enzyme activity than the reference genotype *4/*4. Patients who carry *10/*10 and *11/*4 (‘fast NAT1 acetylators’) were less likely to develop hypersensitivity to SMX, but this was observed only in subjects also carrying a slow NAT2 acetylator genotype.
NAT1 *10 and *11 significantly increase NAT1 protein level/enzyme activity, enabling the classification of carriers into reference and rapid acetylators. Rapid NAT1 acetylator status appears to protect against SMX toxicity by compensating for slow NAT2 acetylator status.
N-acetyltransferase; NAT1; polyadenylation; allelic expression imbalance; sulfamethoxazole; cotrimoxazole; protein translation; acetylator phenotype; idiosyncratic drug reactions
Antibody- and cell-mediated responses to sulfamethoxazole (SMX) were analyzed in AIDS patients with or without a history of hypersensitivity and in negative controls. In 20 of 20 (P < 0.01) human immunodeficiency virus (HIV)-seropositive patients with skin reactions to cotrimoxazole, we found SMX-specific antibodies, while only 9 of 20 and 17 of 20 HIV-seropositive patients without a history of hypersensitivity to cotrimoxazole had SMX-specific immunoglobulin M (IgM) and IgG, respectively. The levels of specific IgM and IgG were higher in patients with skin reactions than in patients without reactions (IgM, 1.0 +/- 0.19 versus 0.47 +/- 0.23 [P < 0.001]; IgG, 0.68 +/- 0.15 versus 0.47 +/- 0.14 [P < 0.001] [mean optical density values +/- standard deviations]). Seronegative controls with no history of exposure to sulfa compounds did not have SMX-specific IgG or IgM antibodies, and controls with a history of intake of SMX with or without reactions had low levels of IgG and IgM. The SMX-specific IgG subclasses were exclusively IgG1 and IgG3. None of the patients had detectable SMX-specific IgE or IgA antibodies nor did they exhibit a cell-mediated response as measured by a lymphocyte proliferation assay. Antibodies to SMX recognized N-acetyl-sulfonamide, N-(2-thiazolyl)-sulfanilamide, sulfadiazine, and sulfisoxazole but did not recognize sulfanilamide or 3-amino-5-methyl isoxazole in an inhibition assay. It is not known whether the SMX-specific antibodies associated with hypersensitivity reactions to SMX in HIV-seropositive patients have a pathogenic role in these reactions. Sulfanilamide or 3-amino-5-methyl isoxazole, on the other hand, could be potential alternative therapies in HIV-seropositive patients with a history of skin reactions to SMX.
Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-κB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-κB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-κB family members.
The incidence of hepatotoxicity related to trimethoprim/sulfamethoxazole (TMP/SMX) administered at a therapeutic dose may vary among study populations of different ethnicities and hepatotoxic metabolites of TMP/SMX may be decreased by drug-drug interaction with fluconazole. We aimed to investigate the incidence of hepatotoxicity and the role of concomitant use of fluconazole in HIV-infected patients receiving TMP/SMX for Pneumocystis jirovecii pneumonia. We reviewed medical records to collect clinical characteristics and laboratory data of HIV-infected patients who received TMP/SMX for treatment of P. jirovecii pneumonia at 6 hospitals around Taiwan between September 2009 and February 2013. Hepatotoxicity was defined as 2-fold or greater increase of aminotransferase or total bilirubin level from baselines. Roussel UCLAF Causality Assessment Method (RUCAM) was used to analyze the causality of drug-induced liver injuries. NAT1 and NAT2 acetylator types were determined with the use of polymerase-chain-reaction (PCR) restriction fragment length polymorphism to differentiate common single-nucleotide polymorphisms (SNPs) predictive of the acetylator phenotypes in a subgroup of patients. During the study period, 286 courses of TMP/SMX treatment administered to 284 patients were analyzed. One hundred and fifty-two patients (53.1%) developed hepatotoxicity, and TMP/SMX was considered causative in 47 (16.4%) who had a RUCAM score of 6 or greater. In multivariate analysis, concomitant use of fluconazole for candidiasis was the only factor associated with reduced risk for hepatotoxicity (adjusted odds ratio, 0.372; 95% confidence interval, 0.145–0.957), while serostatus of hepatitis B or C virus, NAT1 and NAT2 acetylator types, or receipt of combination antiretroviral therapy was not. The incidence of hepatotoxicity decreased with an increasing daily dose of fluconazole up to 4.0 mg/kg. We conclude that the incidence of TMP/SMX-related hepatotoxicity was 16.4% in HIV-infected Taiwanese patients who received TMP/SMX for pneumocystosis. Concomitant use of fluconazole was associated with decreased risk for TMP/SMX-related hepatotoxicity.
Background: Chronic progressive lung disease is the most serious complication of cystic fibrosis (CF). Glutathione plays an important role in the protection of the CF lung against oxidant-induced lung injury.
Objectives: We hypothesized that a polymorphism in a novel candidate gene that regulates glutathione synthesis might influence CF lung disease.
Methods: In a cross-sectional study, subjects were recruited from CF clinics in Seattle and multiple centers in Canada. We tested for an association between CF lung disease and a functional polymorphism in the glutamate-cysteine ligase catalytic subunit (GCLC) gene. Multiple linear regression was used to test for association between polymorphisms of GCLC and severity of CF lung disease while adjusting for age, Pseudomonas aeruginosa infection, and cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Analysis was repeated for patients with CF stratified by CFTR genotype.
Measurements and Main Results: A total of 440 subjects with CF participated in the study (51% male; mean [± SD] age, 26 ± 11 yr; mean FEV1, 62 ± 28% predicted). In the total population, there was a trend toward an association between GCLC genotypes and CF lung disease (linear regression coefficient [SEM], 1.68 [1.0]; p = 0.097). In the stratified analysis, there was a highly significant association between GCLC genotype and CF lung function in subjects with a milder CFTR genotype (linear regression coefficient [SEM], 5.5 (1.7); p = 0.001).
Conclusions: In patients with CF with a milder CFTR genotype, there is a strong association between functional polymorphisms of the GCLC gene and CF lung disease severity.
CFTR genotype; glutathione; modifier genes
Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate–cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothesized that overexpression of Gclc or Gclm to increase GSH synthesis would protect granulosa cells against oxidant- and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of Gclc, Gclm, both Gclc and Gclm or empty vector. GCL protein and enzymatic activity and total GSH levels were significantly increased in the GCL subunit-transfected cells. GCL-transfected cells were resistant to cell killing by treatment with hydrogen peroxide compared to control cells. Cell viability declined less in all the GCL subunit-transfected cell lines 1–8 h after 0.5 mM hydrogen peroxide treatment than in control cells. We next examined the effects of GCL overexpression on responses to ionizing radiation. ROS were measured using a redox-sensitive fluorogenic dye in cells irradiated with 0, 1 or 5 Gy of γ-rays. There was a dose-dependent increase in ROS within 30 min in all cell lines, an effect that was significantly attenuated in Gcl-transfected cells. Apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and activated caspase-3 immunoblotting, was significantly decreased in irradiated Gclc-transfected cells compared to irradiated control cells. Suppression of GSH synthesis in Gclc-transfected cells reversed resistance to radiation. These findings show that overexpression of GCL in granulosa cells can augment GSH synthesis and ameliorate various sequelae associated with exposure to oxidative stress and irradiation.
The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H2O2-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.
glutathione; glutamate cysteine ligase; GCLC; GCLM; TAT; protein transduction
Trimethoprim (TMP)/sulfamethoxazole (SMX) has consistently demonstrated great interindividual variability. Therapeutic drug monitoring may be used to optimize dosing. Optimal peak SMX concentration has been proposed as 100 to 150 μg/mL. The objective of our work was to determine the success rate of a TMP/SMX dosing guideline in achieving a targeted serum peak SMX concentration range.
Our retrospective cohort study enrolled 305 adult hospitalized patients who received treatment with TMP/SMX and underwent serum peak SMX concentration monitoring from January 2003 to November 2011. Patients receiving low-dose TMP/SMX therapy (TMP <15 mg/kg/d) were compared with those receiving high-dose therapy (TMP >15 mg/kg/d).
Patients were classified into peak and modified peak SMX concentration cohorts based on time between TMP/SMX dose and SMX quantification. The association between dosing group and the outcome of the SMX level within the goal range was measured using logistic regression models. The primary outcome measured was serum peak SMX concentration 100 to 150 μg/mL. Serum peak SMX concentrations were attained within range for the peak and modified peak cohort 29% and 26% of the time, respectively. The median peak SMX concentration was 144 μg/mL (range 25–471 μg/mL). The low daily dose cohort demonstrated a trend toward improvement in the odds of target peak concentration range attainment. The results were similar regardless of the method used to adjust for baseline characteristics. The pure peak and modified peak cohorts had 44% and 46% of patients with above-target SMX peak concentrations, respectively.
Attainment of the intended target concentration range was low with no difference in attainment between the low-dose and high-dose cohorts. Higher proportions of patients had an above-target SMX peak, which may indicate that the dosing algorithm is overly aggressive in obtaining the therapeutic goal.
Serum peak concentration; Sulfamethoxazole; Therapeutic drug monitoring; Trimethoprim
Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b5 (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity towards SMX-NO, and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue drug-adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b5, as well as significant decreases in SMX-HA reduction in SMX-NO treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in AIDS patients with antioxidant depletion.
Guinea pig; antioxidants; drug hypersensitivity; NADH hydroxylamine reductase
To test the role of acetylator status, and to investigate the reported discrepancy between acetylator phenotype and genotype in HIV-positive patients with sulphamethoxazole (SMX) hypersensitivity.
Forty HIV-positive patients (32 of whom were SMX-hypersensitive), and 26 healthy volunteers, were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis, and phenotyped using dapsone (50 mg) as a probe, for acetylator status. Sequencing of the NAT2 exon was performed where discrepancy between phenotyping and genotyping was detected. Our results were also pooled with published studies addressing slow acetylator status in HIV-positive SMX-hypersensitive patients.
Slow acetylator genotype and phenotype frequencies did not differ between HIV-positive SMX-hypersensitive and nonhypersensitive patients, and healthy controls, which was further confirmed in a meta-analysis of published studies (pooled odds ratio 2.25, 95% confidence interval 0.45, 11.17). Discordance between phenotype and genotype was resolved in four of the subjects by sequencing of the whole NAT2 exon, which revealed rare mutations, leaving three (9%) HIV-positive SMX-hypersensitive patients and one (4%) healthy volunteer who continued to demonstrate the discordance.
Slow acetylator phenotype or genotype is unlikely to predispose to SMX hypersensitivity in HIV-positive patients, although a minor role cannot be excluded. Phenotype–genotype discrepancies are partly due to nondetection of all rare alleles by PCR methodology, and can be circumvented by sequencing of the gene in patients showing a discrepancy.
adverse drug reactions; HIV; hypersensitivity; NAT2; polymorphism; sulphamethoxazole
Despite it being a quintessential Phase II detoxification gene, the transcriptional regulation of the rat γ-glutamate cysteine ligase catalytic subunit (GCLC) is controversial. Computer-based sequence analysis identified three putative antioxidant response elements (AREs) at positions −889 to −865 (ARE1), −3170 to −3146 (ARE2) and −3901 to −3877 (ARE3) in the 5’-flanking region of the transcriptional start site. Transfections of individual ARE-luciferase reporter gene constructs into H4IIE cells, a rat hepatoma cell line, identified ARE3 as the functional promoter. Chromatin immunoprecipitation assays using primary rat hepatocytes showed that the transcription factor Nrf2, which is known to regulate ARE-mediated genes, is associated with ARE3. Co-transfection of H4IIE cells with luciferase reporter plasmids containing Gclc ARE3 and an Nrf2 expression plasmid resulted in a 3-fold activation of ARE3-mediated transcription relative to controls. “Loss-of-function” analysis for Nrf2 by small interfering RNA (siRNA) revealed that ARE3-mediated expression was significantly impaired while site-directed mutagenesis of the ARE3-luciferase reporter abolished Nrf2-mediated induction. Treatment with two known Nrf2 inducers, R-(α)-lipoic acid and anetholedithiolethione, showed that the inducible expression of the GCLC gene was also regulated by the ARE3 element. Taken together, these results show that Nrf2 regulates the constitutive expression of rat Gclc through a distal ARE present in its 5’-flanking region. This is the first report showing that rat Gclc is under the transcriptional control of the Nrf2-ARE pathway on a constitutive basis.
NF-E2-related factor 2; glutathione, Chromatin immunoprecipitation; detoxification; anetholedithiolethione; R-(α)-lipoic acid; Phase II detoxification
Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser51 phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent.
glutamate-cysteine ligase catalytic subunit; glutamate-cysteine ligase modifier subunit; mRNA stability; sulfur amino acids; cysteine
Trivalent arsenite (As3+) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and cytotoxic effects of As3+. Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As3+ coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation and activity. As3+ increased the rate of transcription of both the Gclm and Gclc genes and induced the post-transcriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As3+-induced Gclc expression and attenuated induction of Gclm. As3+ induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and post-transcriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As3+ and highlight the potential importance of the GSH antioxidant defense system in regulating As3+-induced responses in hepatocytes.
arsenite; arsenic; glutamate cysteine ligase; GCL; GCLC; GCLM; glutathione; GSH; hepatocyte; Nrf2; gene transcription
Bioactivation of sulphamethoxazole (SMX) to chemically-reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro.Flow cytometry revealed binding of N-hydroxy (SMX-NHOH) and nitroso (SMX-NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX-NO was reduced by exogenous glutathione (GSH).SMX-NHOH and SMX-NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism.Fluorescence HPLC showed that SMX-NHOH and SMX-NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma.Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX-NHOH and SMX-NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX-NO than neutrophils.Partitioning of SMX-NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone.Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.
Sulphamethoxazole; hypersensitivity; cellular disposition; haptenation; reduction
Our recent finding that insulin increased the expression of glutamate cysteine ligase catalytic subunit (GCLc) and coincident increases in GCL activity and cellular glutathione (GSH) in human brain microvascular endothelial cells (IHECs) suggests a role for insulin in vascular GSH maintenance. Here, using IHECs stably transfected with promoter-luciferase reporter vectors, we found that insulin increased GCLc promoter activity, which required a prerequisite increase or decrease in media glucose. An intact antioxidant response element-4 (ARE4) was essential for promoter activation that was attenuated by inhibitors of PI3kinase/Akt/mTOR signaling. Interestingly, only at low glucose conditions did promoter activation correlated with increased GCLc expression and GSH synthesis. Low tert-butyl hydroperoxide (tBH) concentrations similarly mediated promoter activation, but the maximal activation dose was decreased 10-fold by insulin. Insulin-tBH co-administration abrogated the low or high glucose requirement for promoter activation, suggesting possible ROS involvement. ROS production was elevated at low glucose without or with insulin; however GSH increases were not inhibited by tempol, suggesting that ROS did not or achieve the threshold for driving GCLc promoter activation and de novo GSH synthesis. The minor effect of pyruvate also ruled out a major role for hypoglycemia (± insulin)-induced metabolic stress on GSH induction under these conditions.
The transcription factor NFE2-related factor 2 (Nrf2) mediates detoxification and antioxidant gene transcription following electrophile exposure and oxidative stress. Mice deficient in Nrf2 (Nrf2-null) are highly susceptible to acetaminophen (APAP) hepatotoxicity, and exhibit lower basal and inducible expression of cytoprotective genes, including NADPH quinone oxidoreductase 1 (Nqo1) and glutamate cysteine ligase (catalytic subunit, or Gclc). Administration of toxic APAP doses to C57BL/6J mice generates electrophilic stress and subsequently increases levels of hepatic Nqo1, Gclc and the efflux multidrug resistance-associated protein transporters 1–4 (Mrp1-4). It was hypothesized that induction of hepatic Mrp1-4 expression following APAP is Nrf2-dependent. Plasma and livers from wild-type (WT) and Nrf2-null mice were collected 4, 24 and 48 hrs after APAP. As expected, hepatotoxicity was greater in Nrf2-null compared to WT mice. Gene and protein expression of Mrp1-4 and the Nrf2 targets, Nqo1 and Gclc, was measured. Induction of Nqo1 and Gclc mRNA and protein after APAP was dependent on Nrf2 expression. Similarly, APAP treatment increased hepatic Mrp3 and Mrp4 mRNA and protein in WT, but not Nrf2-null mice. Mrp1 was induced in both genotypes after APAP, suggesting that elevated expression of this transporter was independent of Nrf2. Mrp2 was not induced in either genotype at the mRNA or protein levels. These results show that Nrf2 mediates induction of Mrp3 and Mrp4 after APAP, but does not affect Mrp1 or Mrp2. Thus coordinated regulation of detoxification enzymes and transporters by Nrf2 during APAP hepatotoxicity is a mechanism by which hepatocytes may limit intracellular accumulation of potentially toxic chemicals.
Nuclear factor-E2-related factor 2; Nrf2; acetaminophen; APAP; hepatotoxicity; multidrug resistance-associated proteins; Mrp3; Mrp4
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
antioxidant defense; diabetic retinopathy; Nrf2
Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insights into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to wild-type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using an S. cerevisiae model system. Mutant GCLC alone could not complement a glutathione-deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.
Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Uptake of homocysteine induces oxidative stress in macrophages. Antioxidant response elements (AREs) are regulatory elements within promoters of genes, which protect cells against oxidative stress. The current study investigated whether homocysteine induces transcription of glutamate-cysteine ligase (Gcl), via ARE driven gene expression in mouse macrophages. Gcl is the rate-limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Gcl is heterodimeric and the genes encoding the subunits of Gcl contain several AREs within their 5′-promoter regions. Treatment of mouse macrophages with d-/l-homocysteine (50 µM) induced depletion of intracellular glutathione and a compensatory increase in Gcl activity. Electro mobiliy shift assays demonstrated increased binding of nuclear proteins to ARE-containing oligonucleotides. Real-time RT-PCR revealed increased mRNA-expression of the catalytic subunit of Gcl (Gclc) after treatment with homocysteine, and this occurred via increased transcription as demonstrated with luciferase promoter reporter constructs for Gclc. Additional site directed mutagenesis demonstrated that ARE4 plays a direct role in mediating induction of Gclc by homocysteine. Supershift analysis and Western blotting revealed that Nrf2 signalling is critical in homocysteine-induced activation of ARE4. Inhibition of MAP kinase activity reduced binding of nuclear proteins to the AREs, nuclear expression of Nrf2 and mRNA expression of Gclc. Western blotting demonstrated phosporylation of ERK1/2 in homocysteine treated macrophages. These data suggest that ARE-driven gene expression of Gclc via a MEK/Nrf2 pathway could help to protect macrophages from oxidative stress due to hyperhomocysteinemia.
Homocysteine; Atherosclerosis; Oxidative stress; Glutamate-cysteine ligase; Antioxidant response element
Fucoxanthin, a natural carotenoid, is abundant in seaweed with antioxidant properties. This study investigated the role of fucoxanthin in the induction of antioxidant enzymes involved in the synthesis of reduced glutathione (GSH), synthesized by glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS), via Akt/nuclear factor-erythroid 2-related (Nrf2) pathway in human keratinocytes (HaCaT) and elucidated the underlying mechanism. Fucoxanthin treatment increased the mRNA and protein levels of GCLC and GSS in HaCaT cells. In addition, fucoxanthin treatment promoted the nuclear translocation and phosphorylation of Nrf2, a transcription factor for the genes encoding GCLC and GSS. Chromatin immune-precipitation and luciferase reporter gene assays revealed that fucoxanthin treatment increased the binding of Nrf2 to the antioxidant response element (ARE) sequence and transcriptional activity of Nrf2. Fucoxanthin treatment increased phosphorylation of Akt (active form), an up-regulator of Nrf2 and exposure to LY294002, a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, suppressed the fucoxanthin-induced activation of Akt, Nrf2, resulting in decreased GCLC and GSS expression. In accordance with the effects on GCLC and GSS expression, fucoxanthin induced the level of GSH. In addition, fucoxanthin treatment recovered the level of GSH reduced by ultraviolet B irradiation. Taken together, these findings suggest that fucoxanthin treatment augments cellular antioxidant defense by inducing Nrf2-driven expression of enzymes involved in GSH synthesis via PI3K/Akt signaling.
fucoxanthin; NF-E2-related factor 2; oxidative stress; cytoprotection; PI3K/Akt; GCLC; GSS; GSH
Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.
► Nrf1 knockout increased GCLC and GCLM expression in human bronchial epithelial cells. ► Overexpressed Nrf1 forms p120 or p65 increased GCLC- and GLCM-EpRE luciferase activity. ► Overexpressed Nrf2 competes with Nrf1 forms. ► Nrf1 forms are unchanged in the lungs of mice by age. ► Nrf1 forms are unchanged in exposure to nanoparticulate air pollution.
EpRE, electrophile response element; ER, endoplasmic reticulum; GCL, glutamate cysteine ligase; GCLC, catalytic subunit of GCL; GCLM, modifier subunit of GCL; HBE1, human bronchial epithelial cells; HO-1, heme oxygenase; Nrf1, nuclear factor-erythroid 2 p45 subunit-related factor 1; Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 2; nPM, nanoparticulate air pollution; Nrf1; Nrf2; Glutamate cysteine ligase; Electrophile response element; Air pollution; Phase II genes
Jeju ground water, containing vanadium compounds, was shown to increase glutathione (GSH) levels as determined by a colorimetric assay and confocal microscopy. To investigate whether the effects of Jeju ground water on GSH were specifically mediated by vanadium compounds, human Chang liver cells were incubated for 10 passages in media containing deionized distilled water (DDW), Jeju ground water (S1 and S3), and vanadyl sulfate (VOSO4). Vanadyl sulfate scavenged superoxide anion, hydroxyl radical and intracellular reactive oxygen species. Vanadyl sulfate effectively increased cellular GSH level and up-regulated mRNA and protein expression of a catalytic subunit of glutamate cysteine ligase (GCLC), which is involved in GSH synthesis. The induction of GCLC expression by vanadyl sulfate was found to be mediated by transcription factor erythroid transcription factor NF-E2 (Nrf2), which critically regulates GCLC by binding to the antioxidant response elements (AREs). Vanadyl sulfate treatment increased the nuclear translocation of Nrf2 and the accumulation of phosphorylated Nrf2. Extracellular regulated kinase (ERK) contributed to ARE-driven GCLC expression via Nrf2 activation. Vanadyl sulfate induced the expression of the active phospho form of ERK. Taken together, these results suggest that the increase in GSH level by Jeju ground water is, at least in part, due to the effects of vanadyl sulfate via the Nrf2-mediated induction of GCLC.
Jeju ground water; vanadyl sulfate; glutamate cysteine ligase; human Chang liver cells; erythroid transcription factor NF-E2
Conotruncal heart defects (CTDs) are among the most severe birth defects worldwide. Studies of CTDs indicate both lifestyle behaviors and genetic variation contribute to the risk of CTDs. Based on a hybrid design using data from 616 case-parental and 1645 control-parental triads recruited for the National Birth Defects Prevention Study between 1997 and 2008, we investigated whether the occurrence of CTDs is associated with interactions between 921 maternal and/or fetal single nucleotide polymorphisms (SNPs) and maternal obesity and tobacco use. The maternal genotypes of the variants in the glutamate-cysteine ligase, catalytic subunit (GCLC) gene and the fetal genotypes of the variants in the glutathione S-transferase alpha 3 (GSTA3) gene were associated with an elevated risk of CTDs among obese mothers. The risk of delivering infants with CTDs among obese mothers carrying AC genotype for a variant in the GCLC gene (rs6458939) was 2.00 times the risk among those carrying CC genotype (95% confidence interval: 1.41, 2.38). The maternal genotypes of several variants in the glutathione-S-transferase (GST) family of genes and the fetal genotypes of the variants in the GCLC gene interacted with tobacco exposures to increase the risk of CTDs. Our study suggests that the genetic basis underlying susceptibility of the developing heart to the adverse effects of maternal obesity and tobacco use involve both maternal and embryonic genetic variants. These results may provide insights into the underlying pathophysiology of CTDs, and ultimately lead to novel prevention strategies.
Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as γ-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique γ-carboxyl linkage from glutamate to cysteine and requires ATP and Mg++ as cofactors in this reaction. GCLM increases the Vmax and Kcat of GCLC, decreases the Km for glutamate and ATP, and increases the Ki for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.
Glutamate cysteine ligase; GCL; GCLC; GCLM; Glutathione; GSH; Post-translational
The synthetic organoselenium agent 1,4- phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG), are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamate cysteine ligase (GCL), through activation of antioxidant response elements (ARE) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular related kinase (ERK) (220%) and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), while the naturally-occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE reporter cells up to 3-fold for p-XSC and ≥5-fold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2 (siNrf2). The dependence of GCL induction on Nrf2 was confirmed in Nrf2 deficient mouse embryonic fibroblasts (MEF) where p-XSeSG induced GCL subunits in wildtype, but not Nrf2 deficient cells (p<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo, and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious inducer of GSH.
Organoselenium; p-XSC; p-XSeSG; glutathione; γ-GCL; Nrf2; MEF; antioxidant response element (ARE); HepG2-ARE; Lung cancer; Fisher 344 rat