Microsomal Glutathione S-transferase Research Articles

Association of aspirin and NSAID use with risk of colorectal cancer according to genetic variants.

Use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with lower risk of colorectal cancer. To identify common genetic markers that may confer differential benefit from aspirin or NSAID chemoprevention, we tested gene × environment interactions between regular use of aspirin and/or NSAIDs and single-nucleotide polymorphisms (SNPs) in relation to risk of colorectal cancer. Case-control study using data from 5 case-control and 5 cohort studies initiated between 1976 and 2003 across the United States, Canada, Australia, and Germany and including colorectal cancer cases (n=8634) and matched controls (n=8553) ascertained between 1976 and 2011. Participants were all of European descent. Genome-wide SNP data and information on regular use of aspirin and/or NSAIDs and other risk factors. Colorectal cancer. Regular use of aspirin and/or NSAIDs was associated with lower risk of colorectal cancer (prevalence, 28% vs 38%; odds ratio [OR], 0.69 [95% CI, 0.64-0.74]; P = 6.2 × 10(-28)) compared with nonregular use. In the conventional logistic regression analysis, the SNP rs2965667 at chromosome 12p12.3 near the MGST1 gene showed a genome-wide significant interaction with aspirin and/or NSAID use (P = 4.6 × 10(-9) for interaction). Aspirin and/or NSAID use was associated with a lower risk of colorectal cancer among individuals with rs2965667-TT genotype (prevalence, 28% vs 38%; OR, 0.66 [95% CI, 0.61-0.70]; P = 7.7 × 10(-33)) but with a higher risk among those with rare (4%) TA or AA genotypes (prevalence, 35% vs 29%; OR, 1.89 [95% CI, 1.27-2.81]; P = .002). In case-only interaction analysis, the SNP rs16973225 at chromosome 15q25.2 near the IL16 gene showed a genome-wide significant interaction with use of aspirin and/or NSAIDs (P = 8.2 × 10(-9) for interaction). Regular use was associated with a lower risk of colorectal cancer among individuals with rs16973225-AA genotype (prevalence, 28% vs 38%; OR, 0.66 [95% CI, 0.62-0.71]; P = 1.9 × 10(-30)) but was not associated with risk of colorectal cancer among those with less common (9%) AC or CC genotypes (prevalence, 36% vs 39%; OR, 0.97 [95% CI, 0.78-1.20]; P = .76). In this genome-wide investigation of gene × environment interactions, use of aspirin and/or NSAIDs was associated with lower risk of colorectal cancer, and this association differed according to genetic variation at 2 SNPs at chromosomes 12 and 15. Validation of these findings in additional populations may facilitate targeted colorectal cancer prevention strategies.

High-resolution mass spectrometry-based background subtraction for identifying protein modifications in a complex biological system: detection of acetaminophen-bound microsomal proteins including argininosuccinate synthetase.

The detection and characterization of low-level protein modifications in a complex system without a methodology for modification enrichment is a very challenging task. This study describes a high-resolution LC/MS-based background subtraction methodology for the unbiased detection and identification of acetaminophen-bound proteins formed in incubations with mouse liver microsomes. The microsomal incubations were conducted using both acetaminophen and [(13)C2,(15)N]acetaminophen at a drug concentration of 200 μM. After tryptic digestion and high-resolution LC/MS analysis, data from the two drug treatment groups were each background-subtracted against the other. Thus, peptide signals that were identical in both groups were effectively canceled out, and drug-bound peptide peaks, differing in masses between the groups because of the isotopic mass shift, were retained after background subtraction and became highlighted in the resultant base peak ion chromatograms. Follow-up MS/MS experiments with these drug-bound peptides led to the identification of three acetaminophen-bound proteins: microsomal glutathione S-transferase, oligosaccharyltransferase subunit ribophorin I, and argininosuccinate synthetase. These initial findings demonstrate the utility of the methodology and may shed new light on the mechanism of acetaminophen-induced hepatotoxicity. The approach is potentially applicable to similar tasks of identification of protein modifications in other complex biological systems.

Relationship between liver and low rumen pH in goat.

The aim of this study was to analyze the response of dry goat liver to sub-acute ruminal acidosis induced by a highly concentrated diet. Non-pregnant, non-lactating female Poll-goats (N = 12) were randomly assigned to either a high-concentrate (HG) or a low-concentrate (LG) diet. Low rumen pH was successfully induced with HG (more than 3 h with rumen pH < 5.8). The plasma lipopolysaccharide concentration was significantly decreased in the HG compared with LG group (P < 0.05). Proteomic analysis showed that aldehyde dehydrogenases and microsomal glutathione S-transferase was downregulated in the HG group, whereas aldo-keto reductase was upregulated compared in the LG group. The abundance of mRNA for these proteins were also correspondingly increased (aldehyde dehydrogenases and microsomal glutathione-S-transferase) or decreased (aldo-keto reductase) in the HG group. Malondialdehyde content in the liver was decreased in the HG group compared to the LG group. These data indicate that the expression of hepatic proteins alters the regulation of endogenous lipopolysaccharide during low rumen pH in dry dairy goats. In particular, the protective effect of the liver may occur through inhibition of aldehyde and/or peroxide formation.

Glutathione S-transferase Activity in Diagnostic Pathology

Glutathione S-transferase (GST) is a generic term that describes a class of proteins involved in phase-II detoxification of endogenous compounds and xenobiotics. Regulation and function of GSTs have influences on cell growth, oxidative stress, as well as disease progression and prevention. Although not routinely assayed in clinical laboratories, the present review highlighted the application of GST activity in diagnostic pathology. Human GSTs are divided into three main families, namely, the cytosolic, mitochondrial and membranebound microsomal GSTs. The expression of GST isoforms in biologic systems may well be of relevance from a clinical and toxicity standpoints and applies in the renewed efforts in eradication and control of parasitic infections. A survey of frequency of polymorphism and measure of GST activity among human population groups are reliable and valuable diagnostic tools. The reliability of GST activity in diagnostic pathology is underscored in pathologic conditions/disorders whose etiologies are associated with overwhelming levels of oxidative stress and failure of GST-mediated detoxification mechanisms.

Molecular cloning, characterization and positively selected sites of the glutathione S-transferase family from Locusta migratoria.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily.

De novo analysis of the Tenebrio molitor (Coleoptera: Tenebrionidae) transcriptome and identification of putative glutathione S-transferase genes

We performed the Illumina sequencing of the yellow mealworm beetle, Tenebrio molitor L., a global stored grain pest and a model for insect physiological studies, and analyzed the transcriptome dataset. We obtained 82,001,576 clean reads that were assembled into 31,785 unigenes with a mean size of 1,022 bp. Among these unigenes, 16,025 (50.41 %) showed significant similarity (E value <10−5) to known proteins in the NCBI non-redundant database. Gene ontology, Cluster of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes analyses were used to classify functions of the unigenes. We identified 27 putative glutathione S-transferase (GST) genes, including 2 delta, 13 epsilon, 1 omega, 5 sigma, 1 theta and 1 zeta class GSTs, and 2 microsomal GSTs and 2 “unclassified” GSTs. Quantitative reverse transcription-PCR assay showed that 2 and 3 GST genes were mainly expressed in larval and pupal stages, respectively; while 10 genes were mainly expressed in the adult stage. Tissue-specific expression analysis found that, among the 27 GST genes, 8 were expressed predominately in the midgut, 13 were mainly detected in Malpighian tubules, and only one gene specifically distributed in the fat body. This work provides the first insights into the T. molitor GSTs at the molecular level: the results may assist in a functional study for the GSTs in this insect species.

Ovarian xenobiotic biotransformation enzymes are altered during phosphoramide mustard-induced ovotoxicity.

The anti-neoplastic prodrug, cyclophosphamide, requires biotransformation to phosphoramide mustard (PM), which partitions to volatile chloroethylaziridine (CEZ). PM and CEZ are ovotoxicants, however their ovarian biotransformation remains ill-defined. This study investigated PM and CEZ metabolism mechanisms through the utilization of cultured postnatal day 4 (PND4) Fisher 344 (F344) rat ovaries exposed to vehicle control (1% dimethyl sulfoxide (DMSO)) or PM (60μM) for 2 or 4 days. Quantification of mRNA levels via an RT(2) profiler PCR array and target-specific RT-PCR along with Western blotting found increased mRNA and protein levels of xenobiotic metabolism genes including microsomal epoxide hydrolase (Ephx1) and glutathione S-transferase isoform pi (Gstp). PND4 ovaries were treated with 1% DMSO, PM (60μM), cyclohexene oxide to inhibit EPHX1 (CHO; 2mM), or PM + CHO for 4 days. Lack of functional EPHX1 increased PM-induced ovotoxicity, suggesting a detoxification role for EPHX1. PND4 ovaries were also treated with 1% DMSO, PM (60μM), BSO (Glutathione (GSH) depletion; 100μM), GEE (GSH supplementation; 2.5mM), PM ± BSO, or PM ± GEE for 4 days. GSH supplementation prevented PM-induced follicle loss, whereas no impact of GSH depletion was observed. Lastly, the effect of ovarian GSH on CEZ liberation and ovotoxicity was evaluated. Both untreated and GEE-treated PND4 ovaries were plated adjacent to ovaries receiving PM + GEE or PM + BSO treatments. Less CEZ-induced ovotoxicity was observed with both GEE and BSO treatments indicating reduced CEZ liberation from PM. Collectively, this study supports ovarian biotransformation of PM, thereby influencing the ovotoxicity that ensues.

Limited protective properties of thymol and thyme oil on differentiated PC12 cells with downregulated Mgst1

In this study we analyzed molecular mechanisms of antioxidative protection of thymol and thyme oil using differentiated PC12 cells, a widely accepted neuronal model. Thymol due to multiple functions is commonly used for clinical applications. However, its action on nervous tissue remains poorly understood. We evaluated the effect of 24h incubation of the cells with thymol (100 and 400μM) and equivalent content of thyme oil. Microsomal glutathione transferase 1 (Mgst1) is an important player in anti-oxidative protection because of its transferase and peroxidase activities. Since its expression decreases during aging, we also used stable transfected cells with downregulated Mgst1 (PC12_M). We analyzed cell viability, lipid peroxidation level, glutathione content and expression of key enzymes responsible for cellular reduced glutathione – catalytic subunit of γ-glutamylcysteine ligase and glutathione reductase. Whereas thymol and thyme oil improved antioxidant capacity of control cells, diminished protection was observed in PC12_M line. Increasing interest in natural dietary components has focused attention on plants used as a rich source of bioactive phytochemicals. However, currently available information on the safe amount for thyme oil using appears to be insufficient. Our results indicate that the thyme oil should be used with caution, especially by elderly people.

Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (<2.5-fold) in the GST isoforms, ahr2 and AOE genes response. However, expression of cyp1a and cyp3a65 mRNA was markedly and consistently induced by high doses of atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes.

Short general anaesthesia induces prolonged changes in gene expression in the mouse hippocampus.

The long-term molecular changes in the central nervous system constitute an important aspect of general anaesthesia, but little is known about to what extent these molecular changes are affected by anaesthesia duration. The aim of the present study was to evaluate the effects of short duration (20 min) general anaesthesia with isoflurane or avertin on the expression of 20 selected genes in the mouse hippocampus at 1 and 4 days after anaesthesia. Nine to eleven-weeks-old male mice received one of the following treatments: 20 min of avertin-induced anaesthesia (n=11), 20 min of isoflurane-induced anaesthesia (n=10) and no anaesthesia (n=5). One and four days after anaesthesia, gene expression in the hippocampus was determined with reverse transcription quantitative real-time polymerase chain reaction. We found that anaesthesia led to the upregulation of six genes: Hspd1 (heat shock protein 1), Plat (tissue plasminogen activator) and Npr3 (natriuretic peptide receptor 3) were upregulated only 1 day after anaesthesia, whereas Thbs4 (thrombospondin 4) was upregulated only 4 days after anaesthesia. Syp (synaptophysin) and Mgst1 (microsomal glutathione S-transferase 1) were upregulated at both time points. Hspd1, Mgst1 and Syp expression was increased regardless of the anaesthetic used, Npr3 and Plat were increased only in mice exposed to avertin, and Thbs4 was upregulated only after isoflurane-induced anaesthesia. This study shows that some of the effects of short general anaesthesia on gene expression in the mouse hippocampus persist for at least 4 days.

PmMGST3, a novel microsomal glutathione S-transferase gene in the dinoflagellate Prorocentrum minimum, is a potential biomarker of oxidative stress.

In this study, we evaluated a novel microsomal glutathione S-transferase3 (MGST3) gene from the dinoflagellate Prorocentrum minimum, and examined its expression pattern in response to copper-and nickel-induced stresses. The full length of PmMGST3 was 732 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the poly (A) tail, covering a 441-bp ORF, 97-bp 5'UTR, and 194-bp 3'UTR. The PmMGST3 was up-regulated by metals, including copper and nickel. The highest up-regulation levels of the PmMGST3 were found under 0.1 mg/L copper and 0.5 mg/L nickel treatment, respectively. In addition, the PmMGST3 was gradually up-regulated by 0.1 mg/L copper with increasing exposure time. Furthermore, ROS production and reduced GSH was measured in the copper treated cells. A significant increased ROS production and reduced GSH were found in the copper treated cells. These results suggest that PmMGST3 may be related to defense mechanisms associated with oxidative stress in dinoflagellates.

The transcriptional network of WRKY53 in cereals links oxidative responses to biotic and abiotic stress inputs

The transcription factor WRKY53 is expressed during biotic and abiotic stress responses in cereals, but little is currently known about its regulation, structure and downstream targets. We sequenced the wheat ortholog TaWRKY53 and its promoter region, which revealed extensive similarity in gene architecture and cis-acting regulatory elements to the rice ortholog OsWRKY53, including the presence of stress-responsive abscisic acid-responsive elements (ABRE) motifs and GCC-boxes. Four proteins interacted with the WRKY53 promoter in yeast one-hybrid assays, suggesting that this gene can receive inputs from diverse stress-related pathways such as calcium signalling and senescence, and environmental cues such as drought and ultraviolet radiation. The Ser/Thr receptor kinase ORK10/LRK10 and the apoplastic peroxidase POC1 are two downstream targets for regulation by the WRKY53 transcription factor, predicted based on the presence of W-box motifs in their promoters and coregulation with WRKY53, and verified by electrophoretic mobility shift assay (EMSA). Both ORK10/LRK10 and POC1 are upregulated during cereal responses to pathogens and aphids and important components of the oxidative burst during the hypersensitive response. Taken with our yeast two-hybrid assay which identified a strong protein–protein interaction between microsomal glutathione S-transferase 3 and WRKY53, this implies that the WRKY53 transcriptional network regulates oxidative responses to a wide array of stresses.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-014-0374-3) contains supplementary material, which is available to authorized users.

Absence of MGST1 mRNA and protein expression in human neuroblastoma cell lines and primary tissue

A recent study identified a haplotype on a small region of chromosome 12, between markers D12S1725 and D12S1596, shared by all patients with familial neuroblastoma (NB). We previously localized the human MGST1 gene, whose gene product protects against oxidative stress, to this very same chromosomal region (12p112.1–p13.33). Owing to the chromosomal location of MGST1; its roles in tumorigenesis, drug resistance, and oxidative stress; and the known sensitivity of NB cell lines to oxidative stress, we considered a role for MGST1 in NB development. Surprisingly there was no detectable MGST1 mRNA or protein in either NB cell lines or NB primary tumor tissue, although all other human tissues, cell lines, and primary tumor tissue examined to date express MGST1 at high levels. The mechanism behind the failure of NB cells and tissue to express MGST1 mRNA is unknown and involves the failure of MGST1 pre-mRNA expression, but does not involve chromosomal rearrangement or nucleotide variation in the promoter, exons, or 3' untranslated region of MGST1. MGST1 provides significant protection against oxidative stress and constitutes 4 to 6% of all protein in the outer membrane of the mitochondria. As NB cells are extremely sensitive to oxidative stress, and often used as a model system to investigate mitochondrial response to endogenous and exogenous stress, these findings may be due to the lack of expression MGST1 protein in NB. The significance of this finding to the development of neuroblastoma (familial or otherwise), however, is unknown and may even be incidental. Although our studies provide a molecular basis for previous work on the sensitivity of NB cells to oxidative stress, and possibly marked variations in NB mitochondrial homeostasis, they also imply that the results of these earlier studies using NB cells are not transferable to other tumor and cell types that express MGST1 at high concentrations.

Prenatal expression of thioredoxin reductase 1 (TRXR1) and microsomal glutathione transferase 1 (MGST1) in humans

Thioredoxin reductase 1 (TRXR1) and microsomal glutathione transferase 1 (MGST1) are important redox and detoxifying enzymes in adult life. The aim of this study was to investigate the expression of these enzymes during fetal life. In addition, the role of gene methylation was studied since this might play an important role in the on-and-off switch of gene expression between fetal and adult life.To this end, the expression of the TRXR1-encoding gene TXNRD1 and the MGST1-encoding gene MGST1 was studied in fetal tissues. The mean mRNA expression of TXNRD1 in fetal livers were seven times higher compared to the mean expression in adult livers (p<0.001). Of the six studied splice variants of TXNRD1, four had a significantly higher expression in the fetal livers as compared to adult livers. The mean expression of MGST1 was twofold higher in adult compared to fetal liver tissue (p=0.01). For MGST1 the alternative first exon 1B was the predominant splice variant in both fetal and adult liver samples. The highest mRNA expression of both TXNRD1 and MGST1 was found in fetal adrenals, whereas expression was lower in fetal liver, lungs and kidneys. There was a significant correlation between the hepatic expression of TXNRD1 and MGST1. Treatment with the demethylating agent 5-AZA resulted in decreased levels of TXNRD1 in human liver HepG2 cells but did not affect the expression of MGST1.In conclusion, the expression of TXNRD1 is higher in fetuses than in adults and might be of importance during fetal life. Hepatic TXNRD1 and MGST1 are co-expressed in both fetuses and adults suggesting common regulatory mechanisms.

Nanoparticle-directed sub-cellular localization of doxorubicin and the sensitization breast cancer cells by circumventing GST-Mediated drug resistance

Resistance to single or multiple chemotherapeutic drugs is a major complication in clinical oncology and is one of the most common treatment limitations in patients with reoccurring cancers. Nanoparticle (NP)-based drug delivery systems (DDS's) have been shown to overcome drug resistance in cancer cells mainly by avoiding the activation of efflux pumps in these cells. We demonstrate in this work that polyester-based hyperbranched dendritic-linear (HBDL)-based NPs carrying doxorubicin (Dox) can effectively overcome microsomal glutathione transferase 1 (MGST1)-mediated drug resistance in breast cancer cells. Our DDS was much more effective at considerably lower intracellular Dox concentrations (IC50 6.3 μm vs. 36.3 μm) and achieved significantly greater reductions in viability and induced higher degrees of apoptosis (31% vs. 14%) compared to the free drug in the resistant cells. Dox-loaded HBDL NPs were found to translocate across the membranes of resistant cells via active endocytic pathways and to be transported to lysosomes, mitochondria, and the endoplasmic reticulum. A significantly lower amount of Dox accumulated in these cytoplasmic compartments in resistant cells treated with free Dox. Moreover, we found that Dox-HBDL significantly decreased the expression of MGST1 and enhanced mitochondria-mediated apoptotic cell death compared to free Dox. Dox-HBDL also markedly activated the JNK pathway that contributes to the apoptosis of drug-resistant cells. These results suggest that HBDL NPs can modulate subcellular drug distribution by specific endocytic and trafficking pathways and that this results in drug delivery that alters enzyme levels and cellular signaling pathways and, most importantly, increases the induction of apoptosis. Our findings suggest that by exploiting the cell transport machinery we can optimize the polymeric vehicles for controlled drug release to overcome drug resistance combat drug resistance with much higher efficacy.

The identification of 14 new genes for meat quality traits in chicken using a genome-wide association study

BackgroundMeat quality is an important economic trait in chickens. To identify loci and genes associated with meat quality traits, we conducted a genome-wide association study (GWAS) of F2 populations derived from a local Chinese breed (Beijing-You chickens) and a commercial fast-growing broiler line (Cobb-Vantress).ResultsIn the present study, 33 association signals were detected from the compressed mixed linear model (MLM) for 10 meat quality traits: dry matter in breast muscle (DMBr), dry matter in thigh muscle (DMTh), intramuscular fat content in breast muscle (IMFBr), meat color lightness (L*) and yellowness (b*) values, skin color L*, a* (redness) and b* values, abdominal fat weight (AbFW) and AbFW as a percentage of eviscerated weight (AbFP). Relative expressions of candidate genes identified near significant signals were compared using samples of chickens with High and Low phenotypic values. A total of 14 genes associated with IMFBr, meat color L*, AbFW, and AbFP, were differentially expressed between the High and Low phenotypic groups. These genes are, therefore, prospective candidate genes for meat quality traits: protein tyrosine kinase (TYRO3) and microsomal glutathione S-transferase 1 (MGST1) for IMFBr; collagen, type I, alpha 2 (COL1A2) for meat color L*; and RET proto-oncogene (RET), natriuretic peptide B (NPPB) and sterol regulatory element binding transcription factor 1 (SREBF1) for the abdominal fat (AbF) traits.ConclusionsBased on the association signals and differential expression of nearby genes, 14 candidate loci and genes for IMFBr, meat L* and b* values, and AbF are identified. The results provide new insight into the molecular mechanisms underlying meat quality traits in chickens.

Characterization of Microsomal GlutathioneS-Transferases MGST1, MGST2, and MGST3 in Cynomolgus Macaque

The glutathione S-transferase (GST) family comprises cytosolic, mitochondrial, and microsomal GSTs, all essential enzymes that metabolize a wide range of endogenous and exogenous substrates. Among the microsomal GSTs (MGSTs) in humans, MGST1, MGST2, and MGST3 are involved in detoxification; however, MGSTs have not been fully investigated in cynomolgus macaque, an important primate species widely used in drug metabolism and toxicity studies. In the present study, cynomolgus MGST2 and MGST3 cDNAs were isolated from liver tissue and characterized along with previously isolated cynomolgus MGST1. For comparison with the cynomolgus cDNAs, MGST2 and MGST3 cDNAs were also isolated from rhesus macaque (closely related to cynomolgus macaque) liver. Cynomolgus MGST2 and MGST3, respectively, were highly identical (99 and 98%) to human MGST2 and MGST3 and nearly identical to the amino acid sequences of the rhesus orthologs, and they were closely clustered with human MGST2 and MGST3 by phylogenetic analysis. The analysis of genome data indicated that MGST1, MGST2, and MGST3, respectively, had similar gene structures and genomic organization in macaque and human. Therefore, cynomolgus MGSTs have molecular similarities to the corresponding human MGSTs. Cynomolgus MGST2 and MGST3 were expressed in liver, jejunum, and kidney, but at lower levels than MGST1. GST activities were measured with 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane as substrates, using proteins heterologously expressed in Escherichia coli. Cynomolgus MGST1, MGST2, and MGST3 conjugated 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane, indicating that cynomolgus MGST1, MGST2, and MGST3 are functional enzymes. These results suggest that these functional cynomolgus MGST enzymes and the corresponding human MGSTs are molecularly similar.

The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers

Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10μgL−1) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver.

Catalytic Characterization of Human Microsomal Glutathione S-Transferase 2: Identification of Rate-Limiting Steps

Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S exhibit distinct catalytic and mechanistic properties, reflecting adaptation to broad and specific physiological functions, respectively.

Functional deficiency of aryl hydrocarbon receptor augments oxygen toxicity-induced alveolar simplification in newborn mice

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. New BPD is characterized as having alveolar simplification. We reported previously that aryl hydrocarbon receptor (AhR) deficiency increased susceptibility to hyperoxic lung injury in adult mice, and this was associated with decreased expression of cytochrome P450 1A enzymes and increased lung inflammation. Whether AhR protects newborn mice against hyperoxia-induced alveolar simplification is unknown. Thus, we tested the hypothesis that decreased activation of the pulmonary AhR augments hyperoxia-induced alveolar simplification and lung inflammation in newborn mice. Experimental groups included one-day old wild type (WT) and AhR dysfunctional (AhRd) mice exposed to 21% O2 (air) or 85% O2 (hyperoxia) for 14days. Exposure of newborn WT mice to hyperoxia resulted in increased protein, enzyme and mRNA expression of the AhR-regulated lung cytochrome P450 1A1, NAD(P)H quinone oxidoreductase-1, and microsomal glutathione S-transferase 1 enzymes, suggesting that hyperoxia increases activation of the pulmonary AhR. On the other hand, in the AhRd mice, hyperoxia induced the AhR-regulated enzymes to a lesser extent probably due to the dysfunctional AhR in these mice. Alveolar simplification and lung inflammation was increased in mice exposed to hyperoxia compared with those exposed to air, and AhRd mice were more susceptible to hyperoxia-induced alveolar simplification and lung inflammation compared with WT mice. These findings suggest that decreased activation of the pulmonary AhR in newborn AhRd mice augments hyperoxia-induced alveolar simplification and lung inflammation in these mice.