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
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
NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA.
Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semi-quantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced.
Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06–1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (p < 0.0001, r = 0.42; p = 0.01, r = 0.23, respectively), and expression of both proteins correlated with one another (p < 0.0001; r = 0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency.
These studies indicate that while novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low frequency cSNPs only appear to minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.
sulfamethoxazole; sulfonamide hypersensitivity; cytochrome b5; NADH cytochromeb5 reductase; single nucleotide polymorphism
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.
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
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.
Cystic fibrosis (CF) is a monogenic disease caused by CFTR gene mutations, with clinical expression similar to complex disease, influenced by genetic and environmental factors. Among the possible modifier genes, those associated to metabolic pathways of glutathione (GSH) have been considered as potential modulators of CF clinical severity. In this way it is of pivotal importance investigate gene polymorphisms at Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC), Glutathione S-transferase Mu 1 (GSTM1), Glutathione S-transferase Theta 1 (GSTT1), and Glutathione S-transferase P1 (GSTP1), which have been associated to the GSH metabolic pathway and CF clinical severity.
A total of 180 CF’s patients were included in this study, which investigated polymorphisms in GCLC and GST genes (GCLC -129C>T and -3506A>G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G) by PCR and PCR-RFLP associating to clinical variables of CF severity, including variables of sex, clinical scores [Shwachman-Kulczycki, Kanga e Bhalla (BS)], body mass index, patient age, age for diagnosis, first clinical symptoms, first colonization by Pseudomonas aeruginosa, sputum’s microorganisms, hemoglobin oxygen saturation in the blood, spirometry and comorbidities. The CFTR genotype was investigated in all patients, and the genetic interaction was performed using MDR2.0 and MDRPT0.4.7 software.
The analysis of multiple genes in metabolic pathways in diseases with variable clinical expression, as CF disease, enables understanding of phenotypic diversity. Our data show evidence of interaction between the GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G polymorphism with CFTR gene mutation classes, and BS (Balance testing accuracy= 0.6824, p= 0.008), which measures the commitment of bronchopulmonary segments by tomography.
Polymorphisms in genes associated with metabolism of GSH act on the CF’s severity.
We present a 46-year-old African-American man with AIDS who was admitted on two different occasions within three weeks for signs and symptoms of meningitis after using trimethoprim/sulfamethoxazole (TMP/SMX). TMP/SMX is primarily used for the treatment of pneumocystis carinii pneumonia prophylaxis in AIDS patients. Drug-induced aseptic meningitis (DIAM) is commonly seen with nonsteroidal anti-inflammatory drugs (NSAIDS), antibiotics (with TMP/SMX being the most frequently implicated), intravenous immunoglobulins and OKT3 antibodies. However, the implication of TMP/SMX inducing aseptic meningitis has been underreported to FDA/MEDWATCH program. This might be due to the fact that it has also been used to treat bacterial meningitis from organisms like Listeria monocytogenes, which is a common pathogen in the elderly and in infants. We reviewed the literature in an attempt to characterize the pattern and predictors of TMP/SMX-induced aseptic meningitis.
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.
Infant laboratory abnormalities have been associated with exposure to antiretrovirals and to trimethoprim/sulfamethoxazole (TMP/SMX).
We analyzed data from International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) Protocol P1025, a prospective cohort study of human immunodeficiency virus type 1 (HIV)–infected women and their infants. Live-born, singleton, HIV-uninfected infants with at least 6 months of follow-up who represented the first pregnancy on study of HIV-infected mothers with at least 1 prenatal visit, CD4 count, and viral load during pregnancy and who used at least 1 antiretroviral during pregnancy were eligible for inclusion in this analysis.
The study population comprised 1524 infants. During the first 6 months of life, 7.4% of laboratory serious adverse events (SAEs) were related to glucose, 7.2% were related to hemoglobin, 8.7% were related to absolute neutrophil count, and 4.0% were related to total lymphocyte count. The likelihood of laboratory SAEs decreased with increasing age for hemoglobin, absolute neutrophil count, and glucose. Infant preterm birth and current receipt of antiretroviral(s) were the factors with the strongest associations with laboratory SAEs.
The overall frequency of laboratory SAEs was low and decreased with age. Preterm infants are at higher risk of hemoglobin- and total lymphocyte count–related SAEs.
Inhalation of ambient fine particulate matter (PM2.5) is associated with adverse respiratory and cardiovascular effects. A major fraction of PM2.5 in urban settings is diesel exhaust particulate (DEP), and DEP-induced lung inflammation is likely a critical event mediating many of its adverse health effects. Oxidative stress has been proposed to be an important factor in PM2.5-induced lung inflammation, and the balance between pro- and antioxidants is an important regulator of this inflammation. An important intracellular antioxidant is the tripeptide thiol glutathione (GSH). Glutamate cysteine ligase (GCL) carries out the first step in GSH synthesis. In humans, relatively common genetic polymorphisms in both the catalytic (Gclc) and modifier (Gclm) subunits of GCL have been associated with increased risk for lung and cardiovascular diseases.
This study was aimed to determine the effects of Gclm expression on lung inflammation following DEP exposure in mice.
Materials and methods
We exposed Gclm wild type, heterozygous, and null mice to DEP via intranasal instillation and assessed lung inflammation as determined by neutrophils and inflammatory cytokines in lung lavage, inflammatory cytokine mRNA levels in lung tissue, as well as total lung GSH, Gclc, and Gclm protein levels.
The Gclm heterozygosity was associated with a significant increase in DEP-induced lung inflammation when compared to that of wild type mice.
Discussion and conclusion
This finding indicates that GSH synthesis can mediate DEP-induced lung inflammation and suggests that polymorphisms in Gclm may be an important factor in determining adverse health outcomes in humans following inhalation of PM2.5.
Diesel exhaust particulate; lung inflammation; glutathione; oxidative stress; glutamate cysteine ligase
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
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
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 challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistant Mycobacterium tuberculosis strains. While TMP alone was not effective against M. tuberculosis, the combination of TMP and SMX was bacteriostatic against M. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDR M. tuberculosis strains. Treatment of M. tuberculosis with TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistance in vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMX in vivo to prevent the emergence of drug-resistant M. tuberculosis.
The sulfamethoxazole (SMX)-trimethoprim drug combination is routinely used as prophylaxis against Pneumocystis pneumonia during the first 3 to 6 months after renal transplantation. The objective of this study was to examine the impact of N-acetyltransferase 2 (NAT2) and CYP2C9 polymorphisms on the pharmacokinetics of SMX in 118 renal transplant recipients. Starting on day 14 after renal transplantation, patients were administered 400 mg/day-80 mg/day of SMX-trimethoprim orally once daily. On day 14 after the beginning of SMX therapy, plasma SMX concentrations were determined by a high-performance liquid chromatography method. The SMX area under the concentration-time curve from 0 to 24 h (AUC0-24) for 15 recipients with the NAT2 slow acetylator genotype (NAT2*5/*6, -*6/*6, -*6/*7, and -*7/*7) was significantly greater than that for 56 recipients with the NAT2 rapid acetylator genotype (homozygous for NAT2*4) (766.4 ± 432.3 versus 537.2 ± 257.5 μg-h/ml, respectively; P = 0.0430), whereas there were no significant differences in the SMX AUC0-24 between the CYP2C9*1/*1 and -*1/*3 groups. In a multiple regression analysis, the SMX AUC0-24 was associated with NAT2 slow acetylator polymorphisms (P = 0.0095) and with creatinine clearance (P = 0.0499). Hepatic dysfunction in NAT2 slow acetylator recipient patients during the 6-month period after SMX administration was not observed. SMX plasma concentrations were affected by NAT2 polymorphisms and renal dysfunction. Although standard SMX administration to patients with NAT2 slow acetylator polymorphisms should be accompanied by monitoring for side effects and drug interaction effects from the inhibition of CYP2C9, SMX administration at a low dose (400 mg) as prophylaxis may not provide drug concentrations that reach the level necessary for the expression of side effects. Further studies with a larger sample size should be able to clarify the relationship between SMX plasma concentration and side effects.
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.
Trimethoprim-sulfamethoxazole (TMP/SMX) is a widely prescribed antimicrobial for the management of several uncomplicated infections. It is commonly used for the treatment and prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in the HIV-infected population. The adverse reaction to TMP/SMX is more frequent and severe in HIV-infected patients as compared to the general population. Here, we report a case of Stevens-Johnson syndrome (SJS) secondary to TMP/SMX. The patient had a generalized cutaneous reaction with involvement of the eyes, oral cavity, and genitals. He had elevated hepatic alanine aminotransferase and aspartate aminotransferase enzyme. TMP/SMX therapy was stopped and supportive treatment was started. His condition improved after eight days of stopping TMP/SMX therapy.
Desensitization; HIV infection; glutathione enzyme; P. jirovecii pneumonia; Steven Johnson syndrome; trimethoprim-sulfamethoxazole
Using two strains of Staphylococcus aureus, one susceptible and one heterogeneously resistant to methicillin, for which MICs and MBCs of trimethoprim-sulfamethoxazole (TMP-SMX) were 0.06 and 0.06 micrograms/ml and 0.06 and 0.25 microgram/ml, respectively (concentrations are those of TMP), we studied the efficacies of TMP-SMX and cloxacillin, teicoplanin, and vancomycin for treatment of experimental staphylococcal endocarditis. Rabbits were treated with dosages of TMP-SMX selected to achieve concentrations in serum equivalent to that obtained in humans treated for Pneumocystis carinii pneumonia. The overall mortality rate of rabbits treated with TMP-SMX was 84% at day 3, not different from that of the control groups (P > 0.1). No sterile vegetations were observed to be present in control groups or in animals treated with TMP-SMX. However, 26, 60, and 75% of rabbits treated with teicoplanin, cloxacillin, and vancomycin, respectively, showed sterile vegetations. For methicillin-susceptible S. aureus (MSSA), the mean vegetation counts were not significantly different between the control group and the group treated with TMP-SMX (P > 0.1). For methicillin-resistant S. aureus (MRSA), treatment with TMP-SMX was more effective than no therapy, decreasing the number of organisms in vegetations (P < 0.01). For both strains, therapy with cloxacillin and therapy with teicoplanin or vancomycin were significantly more effective than therapy with TMP-SMX. Despite high concentrations of teicoplanin in serum which exceeded MBCs for staphylococci more than 50 times at the peak and 10 times at the trough, therapy with cloxacillin or vancomycin was superior to therapy with teicoplanin against both MSSA and MRSA. These data do not support the use of TMP-SMX in treatment of endocarditis and other severe staphylococcal infections with high bacterial counts.
This study investigated the mechanisms responsible for the disrupted homeostasis of reduced glutathione (GSH) in aging muscles with stress (14 days of hind-limb unloading [HU]). Adult and old rats were randomized into four groups: weight bearing and 3, 7, and 14 days of HU. Soleus muscles were harvested to investigate the activity or content of enzymes involved in GSH metabolism (utilization and synthesis). The activities of glutathione S transferase, glutathione reductase, γ-glutamyl transpeptidase, and glutamate cysteine ligase (GCL) were determined. The protein content of the two subunits of GCL, catalytic subunit (GCLC) and modifier subunit (GCLM), were evaluated. The major results, failure to maintain the accelerated GCLC production and GCL activity, are associated with the GSH depletion in aging muscles with 14 days of HU. The results suggest that the regulation of GCL, especially the catalytic subunit, with stress may be compromised in aging muscles.
GCL; GCLC; Hind-limb unloading
Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a hetero-dimer composed of a catalytic subunit GCLC and a modifier subunit GCLM.
We generated apo E−/− mice deficient in GCLM (apoE−/−/Gclm−/−) and transgenic mice that over-express GCLC specifically in macrophages (apoE−/−/Gclc-Tg) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis.
Methods and Results
Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40 and 50 weeks of age and in the aortic sinus at 40 or 50 weeks of age. The apoE−/−/Gclm−/− mice more rapidly developed complex lesions while the apoE−/−/Gclc-Tg mice had reduced lesion development as compared to the littermate apo E−/− control mice. Transplant of bone marrow from the apoE−/−/Gclm−/− and apoE−/−/Gclc-Tg mice into apo E−/− mice with established lesions also stimulated or inhibited further lesion development at 30 weeks post-transplant.
Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apo E−/− mice.
Atherosclerosis; macrophages; glutathione; apo E−/−
Among 5,043 invasive pneumococcal disease (IPD) isolates identified through South African national surveillance from 2003 to 2007, we estimated the effect of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis on antimicrobial resistance. Patients on TMP-SMX prophylaxis were more likely to have a pneumococcal isolate nonsusceptible to TMP-SMX, penicillin, and rifampin. TMP-SMX nonsusceptibility was associated with nonsusceptibility to penicillin, erythromycin, and rifampin and multidrug resistance. This study informs empirical treatment of suspected IPD in patients with a history of TMP-SMX use.
Glutathione plays a crucial role in free radical scavenging, oxidative injury, and cellular homeostasis. Previously, we identified a non-synonymous polymorphism (P462S) in the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC), the rate-limiting enzyme in glutathione biosynthesis. This polymorphism is present only in individuals of African descent. Presently, we report that this ethnic-specific polymorphism (462S) encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. In addition, overexpression of the 462P wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the 462S isoform. We also demonstrate that apoptotically stimulated mammalian cells overexpressing the 462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type 462P enzyme. Finally, we genotyped several African and African-descent populations and demonstrate that the 462S polymorphism is in Hardy-Weinberg dysequilibrium, with no individuals homozygous for the 462S polymorphism identified. These findings describe a glutathione production pathway polymorphism present in individuals of African descent with significantly decreased in vitro activity.
Ninety patients with urinary tract infections were treated in a randomized double-blind study with either a combination of trimethoprim and sulfamethoxazole (TMP-SMX) or sulfamethoxazole alone (SMX). Thirty of 42 patients treated with TMP-SMX were cured by the time of follow-up compared with 26 of 48 treated with SMX alone. Of the 29 patients infected with SMX-resistent organisms, the combination TMP-SMX cured 12 of 17, whereas SMX alone cured 2 of 12. Of the 61 patients infected with SMX-sensitive organisms, TMP-SMX cured 18 of 25; SMX alone cured 24 of 36. In 50 women the infection was found localized to The upper urinary tract by the use of the Fairley bladder washout technique. TMPsmx cured 16 or 24 of these patients with proved upper tract infections and SMX alone cured 11 of 26. Although none of these differences were significant, TMP-SMX appears to be an effective drug combination for the therapy of proved upper tract infection and is also effective in eradicating sulfonamide-resistant organisms.
4-Hydroxynonenal (HNE), a major electrophilic product of lipid peroxidation, is regarded as both a marker of oxidative stress and a mediator of oxidative damage. At subtoxic concentrations, however, this compound has been shown to be a signalling molecule that can induce the expression of various antioxidant/detoxification enzymes, including glutamate-cysteine ligase (GCL), the rate-limiting enzyme in the de novo synthesis of glutathione. GCL consists of a catalytic (GCLC) and modulatory (GCLM) subunit, which are encoded by separate genes. Here, we investigated the effect of submicromolar concentrations of HNE on the expression of the GCL genes and the transcription factors involved. We demonstrated that submicromolar concentrations of HNE (as little as 0.3 µM) could increase the expression of both GCLC and GCLM. We also found that the induction of GCL expression was abrogated by siRNA for Nrf2. Our data suggest that a submicromolar concentration of HNE, as found in human plasma under physiological conditions, can induce GCL transcription in cultured cells implying that ‘basal’ expression of GCL is under regulation by lipid peroxidation that occurs under physiological conditions. Moreover, this induction is mediated through the EpRE-Nrf2 signalling pathway thought to be predominantly active only during stress.
4-Hydroxynonenal; glutamate-cysteine ligase; EpRE-Nrf2 pathway