Human mitochondrial glutathione transferases: Kinetic parameters and accommodation of a mitochondria-targeting group in substrates.

Glutathione-S-transferases are key to the cellular detoxification of xenobiotics and products of oxidative damage. GSTs catalyse the reaction of glutathione (GSH) with electrophiles to form stable thioether adducts. GSTK1-1 is the main GST isoform in the mitochondrial matrix, but the GSTA1-1 and GSTA4-4 isoforms are also thought to be in the mitochondria with their distribution altering in transformed cells, thus potentially providing a cancer specific target. A mitochondria-targeted version of the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), MitoCDNB, has been used to manipulate the mitochondrial GSH pool. To finesse this approach to target particular GST isoforms in the context of cancer, here we have determined the kcat/Km for the human isoforms of GSTK1-1, GSTA1-1 and GSTA4-4 with respect to GSH and CDNB. We show how the rate of the GST-catalysed reaction between GSH and CDNB analogues can be modified by both the electron withdrawing substituents, and by the position of the mitochondria-targeting triphenylphosphonium on the chlorobenzene ring to tune the activity of mitochondria-targeted substrates. These findings can now be exploited to selectively disrupt the mitochondrial GSH pools of cancer cells expressing particular GST isoforms.

Absolute protein quantification based on calibrated particle counting using electrospray-differential mobility analysis.

The traceability of in vitro diagnostics or drug products is based on the accurate quantification of proteins. In this study, we developed an absolute quantification approach for proteins. This method is based on calibrated particle counting using electrospray-differential mobility analysis (ES-DMA) coupled with a condensation particle counter (CPC). The absolute concentration of proteins was quantified with the observed protein particle number measured with ES-DMA-CPC, and the detection efficiency was determined by calibrators. The measurement performance and quantitative level were verified using two certificated reference materials, BSA and NIMCmAb. The linear regression fit for the detection efficiency values of three reference materials and one highly purified protein (myoglobin, BSA, NIMCmAb and fibrinogen) indicated that the detection efficiency and the particle size distribution of these proteins exhibited a linear relationship. Moreover, to explore the suitability of the detection efficiency-particle size curve for protein quantification, the concentrations of three typical proteinaceous particles, including two high molecular weight proteins (NIST reference material 8671 and D-dimer) and one protein complex (glutathione S-transferase dimer), were determined. This work suggests that this calibrated particle counting method is an efficient approach for nondestructive, rapid and accurate quantification of proteins, especially for measuring proteinaceous particles with tremendous size and without reference standards.

Construction of a dual-signal readout platform for effective glutathione S-transferase sensing based on polyethyleneimine-capped silver nanoclusters and cobalt-manganese oxide nanosheets with oxidase-mimicking activity.

A novel dual-mode fluorometric and colorimetric sensing platform is reported for determining glutathione S-transferase (GST) by utilizing polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) and cobalt-manganese oxide nanosheets (CoMn-ONSs) with oxidase-like activity. Abundant active oxygen species (O2•-) can be produced through the CoMn-ONSs interacting with dissolved oxygen. Afterward, the pink oxDPD was generated through the oxidation of colorless N,N-diethyl-p-phenylenediamine (DPD) by O2•-, and two absorption peaks at 510 and 551 nm could be observed. Simultaneously, oxDPD could quench the fluorescence of PEI-AgNCs at 504 nm via the inner filter effect (IFE). However, in the presence of glutathione (GSH), GSH prevents the oxidation of DPD due to the reducibility of GSH, leading to the absorbance decrease at 510 and 551 nm. Furthermore, the fluorescence at 504 nm was restored due to the quenching effect of oxDPD on decreased PEI-AgNCs. Under the catalysis of GST, GSH and1-chloro-2,4-dinitrobenzo (CDNB) conjugate to generate an adduct, initiating the occurrence of the oxidation of the chromogenic substrate DPD, thereby inducing a distinct colorimetric response again and the significant quenching of PEI-AgNCs. The detection limits for GST determination were 0.04 and 0.21 U/L for fluorometric and colorimetric modes, respectively. The sensing platform illustrated reliable applicability in detecting GST in real samples.

Erythrocyte Glutathione S-Transferase Activity as a Sensitive Marker of Kidney Function Impairment in Children with IgA Vasculitis.

IgA vasculitis (IgAV) is the most common childhood vasculitis. The main cause of morbidity and mortality in children with IgAV is nephritis (IgAVN), but the risk of its development, severity, and chronicity remain unclear. Erythrocyte glutathione S-transferase (e-GST) activity has been previously detected as a sensitive marker of kidney function impairment in several diseases. We spectrophotometrically assessed and correlated e-GST activity between 55 IgAV patients without nephritis (IgAVwN), 42 IgAVN patients, and 52 healthy controls. At disease onset, e-GST activity was significantly higher in IgAVN patients (median (interquartile range)) (5.7 U/gHb (4.4-7.5)) than in IgAVwN patients (3.1 U/gHb (2.2-4.2); p < 0.001), and controls (3.1 U/gHb (1.9-4.2); p < 0.001). Therewithal, there were no differences between the IgAVwN patients and controls (p = 0.837). e-GST activity was also significantly higher in the IgAVN patients than in the IgAVwN patients after 3 months (5.0 U/gHb (4.2-6.2) vs. 3.3 U/gHb (2.3-4.1); p < 0.001) and 6 months (4.2 U/gHb (3.2-5.8) vs. 3.3 U/gHb (2.1-4.1); p < 0.001) since the disease onset. Consistent correlations between e-GST activity and serum creatinine, estimated glomerular filtration rate (eGFR), and proteinuria levels were not detected. In conclusion, increased e-GST activity can serve as a subtle indicator of kidney function impairment in children with IgAV.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.

Afriplex GRTTM extract attenuates hepatic steatosis in an in vitro model of NAFLD.

BACKGROUND: Currently, it is acknowledged that vitamin E, insulin sensitizers and anti-diabetic drugs are used to manage non-alcoholic fatty liver disease (NAFLD), however, these therapeutic interventions harbour adverse side effects. Pioglitazone, an anti-diabetic drug, is currently the most effective therapy to manage NAFLD. The use of natural medicines is widely embraced due to the lack of evidence of their negative side effects. Rooibos has been previously shown to decrease inflammation and oxidative stress in experimental models of diabetes, however, this is yet to be explored in a setting of NAFLD. This study was aimed at investigating the effects of an aspalathin-rich green rooibos extract (Afriplex GRTTM) against markers of hepatic oxidative stress, inflammation and apoptosis in an in vitro model of NAFLD. METHODS: Oleic acid [1 mM] was used to induce hepatic steatosis in C3A liver cells. Thereafter, the therapeutic effect of Afriplex GRTTM, with or without pioglitazone, was determined by assessing its impact on cell viability, changes in mitochondrial membrane potential, intracellular lipid accumulation and the expression of genes and proteins (ChREBP, SREBF1, FASN, IRS1, SOD2, Caspase-3, GSTZ1, IRS1 and TNF-α) that are associated with the development of NAFLD. RESULTS: Key findings showed that Afriplex GRTTM added to the medium alone or combined with pioglitazone, could effectively block hepatic lipid accumulation without inducing cytotoxicity in C3A liver cells exposed oleic acid. This positive outcome was consistent with effective regulation of genes involved in insulin signaling, as well as carbohydrate and lipid metabolism (IRS1, SREBF1 and ChREBP). Interestingly, in addition to reducing protein levels of an inflammatory marker (TNF-α), the Afriplex GRTTM could ameliorate oleic acid-induced hepatic steatotic damage by decreasing the protein expression of oxidative stress and apoptosis related markers such as GSTZ1 and caspase-3. CONCLUSION: Afriplex GRTTM reduced hepatic steatosis in oleic acid induced C3A liver cells by modulating SREBF1, ChREBP and IRS-1 gene expression. The extract may also play a role in alleviating inflammation by reducing TNF-α expression, suggesting that additional experiments are required for its development as a suitable therapeutic option against NAFLD. Importantly, further research is needed to explore its antioxidant role in this model.

The influence of genetic predisposition to oxidative stress on painful diabetic peripheral neuropathy: A mendelian randomization study.

Genetic predisposition to oxidative stress (OS) may influence the risk of Painful Diabetic Peripheral Neuropathy (PDPN). This study employed a Mendelian Randomization (MR) approach to investigate the causal relationship between genetic predisposition to OS and PDPN. Genetic instruments associated with OS biomarkers were selected as exposures. Summary-level data on PDPN was obtained from the largest available genome-wide association study (GWAS). MR analyses were conducted using the inverse-variance weighted (IVW) method, with sensitivity analyses employing the MR-Egger, weighted median, and MR-PRESSO approaches. Genetic predisposition to increased glutathione S-transferase (GST) activity was associated with a reduced risk of PDPN (OR=0.66, 95%CI: 0.49-0.89, P=0.006). Higher ascorbate levels conferred a protective effect against PDPN (OR=0.83, 95%CI: 0.71-0.97, P=0.018). No significant association was observed between genetic predisposition to OS biomarkers and PDPN severity. Genetic predisposition to increased GST activity and higher ascorbate levels protect against the development of PDPN, suggesting a causal relationship.

Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

Effects of Glutathione S-Transferases (GSTM1, GSTT1 and GSTP1) gene variants in combination with smoking or drinking on cancers: A meta-analysis.

BACKGROUND: This meta-analysis aimed to systematically summarize the association between cancer risks and glutathione s-transferases (GSTs) among smokers and drinkers. METHODS: Literature was searched through PubMed, Web of Science, CNKI, and WANFANG published from 2001 to 2022. Stata was used with fixed-effect model or random-effect model to calculate pooled odds ratios (ORs) and the 95% confidence interval (95% CI). Sensitivity and heterogeneity calculations were performed, and publication bias was analyzed by Begg and Egger's test. Regression analysis was performed on the correlated variables about heterogeneity, and the false-positive report probabilities (FPRP) and the Bayesian False Discovery Probability (BFDP) were calculated to assess the confidence of a statistically significant association. RESULTS: A total of 85 studies were eligible for GSTs and cancer with smoking status (19,604 cases and 23,710 controls), including 14 articles referring to drinking status (4409 cases and 5645 controls). GSTM1-null had significant associations with cancer risks (for smokers: OR = 1.347, 95% CI: 1.196-1.516, P < .001; for nonsmokers: OR = 1.423, 95% CI: 1.270-1.594, P < .001; for drinkers: OR = 1.748, 95% CI: 1.093-2.797, P = .02). GSTT1-null had significant associations with cancer risks (for smokers: OR = 1.356, 95% CI: 1.114-1.651, P = .002; for nonsmokers: OR = 1.103, 95% CI: 1.011-1.204, P = .028; for drinkers: OR = 1.423, 95% CI: 1.042-1.942, P = .026; for nondrinkers: OR = 1.458, 95% CI: 1.014-2.098, P = .042). Negative associations were found between GSTP1rs1695(AG + GG/AA) and cancer risks among nondrinkers (OR = 0.840, 95% CI: 0.711-0.985, P = .032). CONCLUSIONS: GSTM1-null and GSTT1-null might be related cancers in combination with smoking or drinking, and GSTP1rs1695 might be associated with cancers among drinkers.

Association of Oral Submucous Fibrosis Risk with GSTM1 and GSTT1 Gene Polymorphisms.

OBJECTIVE: To determine the association of GSTM1 and GSTT1 polymorphisms with oral submucous fibrosis (OSF). STUDY DESIGN: A case-control study. Place and Duration of the Study: Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore and Oral and Maxillofacial Surgery Department, de Montmorency, College of Dentistry/ Punjab Dental Hospital, Lahore, Pakistan, from 1st April 2019 to 31st April 2020. METHODOLOGY: OSF patients were diagnosed with different clinical staging of mouth opening by Vernier caliper with the help of a professional dentist in the Department of Oral and Maxillofacial, de Montmorency, College of Dentistry, Lahore. One hundred and eight blood samples of OSF patients and 108 samples of normal controls were collected. Genomic DNA was obtained from whole-blood extraction. Multiplex PCR amplification using GSTM1, GSTT1, and ß -Globin gene primers was performed. RESULTS: GSTM1 and GSTT1 null genotypes frequencies were found in 43.5% (47/108) and 13.9% (15/108) of controls, whereas 54.6% (59/108) and 25.9% (28/108) of OSF patients, respectively. OSF patients had a greater frequency rate of GSTM1 and GSTT1 null genotypes than controls [OR 1.56, 95% CI 0.91-2.67 (p=0.13)] and [OR 2.17, 95% CI 1.08-4.34 (p=0.04)], respectively. The GSTT1 genotype was found statistically significant with OSF (p=0.05), and risk was also determined. The cumulative effect of null genotypes of GSTM1/GSTT1 did not show any association with the controls and in OSF patients. Proportions of active and null alleles of the patient group were; 86.1%/13.9%; and in control, it was 92.6%/7.4% (OR = 2.01; CI: 0.82-4.97; p=0.18), respectively. CONCLUSION: The study determined a statistically significant association of GSTT1 gene polymorphism with OSF. KEY WORDS: Oral submucous fibrosis, GSTM1, GSTT1, Gene polymorphisms, Genetic risk.

The micro-743a-3p-GSTM1 pathway is an endogenous protective mechanism against alcohol-related liver disease in mice.

BACKGROUND AND AIMS: Epidemiological evidence suggests that the phenotype of glutathione S-transferase mu 1 (GSTM1), a hepatic high-expressed phase II detoxification enzyme, is closely associated with the incidence of alcohol-related liver disease (ALD). However, whether and how hepatic GSTM1 determines the development of ALD is largely unclear. This study was designed to elucidate the role and potential mechanism(s) of hepatic GSTM1 in the pathological process of ALD. METHODS: GSTM1 was detected in the liver of various ALD mice models and cultured hepatocytes. Liver-specific GSTM1 or/and micro (miR)-743a-3p deficiency mice were generated by adenoassociated virus-8 delivered shRNA, respectively. The potential signal pathways involving in alcohol-regulated GSTM1 and GSTM1-associated ALD were explored via both genetic manipulation and pharmacological approaches. RESULTS: GSTM1 was significantly upregulated in both chronic alcohol-induced mice liver and ethanol-exposed murine primary hepatocytes. Alcohol-reduced miR-743a-3p directly contributed to the upregulation of GSTM1, since liver specific silencing miR-743a-3p enhanced GSTM1 and miR-743a-3p loss protected alcohol-induced liver dysfunctions, which was significantly blocked by GSTM1 knockdown. GSTM1 loss robustly aggravated alcohol-induced hepatic steatosis, oxidative stress, inflammation, and early fibrotic-like changes, which was associated with the activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38. GSTM1 antagonized ASK1 phosphorylation and its downstream JNK/p38 signaling pathway upon chronic alcohol consumption via binding with ASK1. ASK1 blockage significantly rescued hepatic GSTM1 loss-enhanced disorders in alcohol-fed mice liver. CONCLUSIONS: Chronic alcohol consumption-induced upregulation of GSTM1 in the liver provides a feedback protection against hepatic steatosis and liver injury by counteracting ASK1 activation. Down-regulation of miR-743a-3p improves alcohol intake-induced hepatic steatosis and liver injury via direct targeting on GSTM1. The miR-743a-3p-GSTM1 axis functions as an innate protective pathway to defend the early stage of ALD.

Toxicity assays and in silico approach to assess the impacts of chlorine dioxide on survival, respiration and some biochemical markers of marine zooplankton.

Chlorination is the common antifouling method in desalination and power plant water intake structures to control microbial and macrofouling growth. In this study, the impacts of chlorine dioxide on toxicity, metabolic activity and biochemical markers like glutathione S-transferase and catalase enzyme activity were tested using four zooplankton species (Centropages sp., Acartia sp., Oncaea sp., and Calanus sp.) collected from the Red Sea. The zooplankton species were treated with different concentrations (0.02, 0.05, 0.1, 0.2, and 0.5 mg L-1) of chlorine dioxide. Further, chlorite, the main decomposition product of chlorine dioxide, was used for molecular docking studies against glutathione S-transferase and catalase enzymes. The results indicated the LC50 range of 0.552-1.643 mg L-1 for the studied zooplankton species. The respiration rate of the zooplankton increased due to the chlorine dioxide treatment with a maximum of 0.562 µg O2 copepod h-1 in Acartia. The glutathione S-transferase and catalase enzyme activities showed elevated values in zooplankton treated with chlorine dioxide. Molecular docking of chlorite with enzymes involved in antioxidant defense activity, such as glutathione S-transferase and catalase enzyme showed weak interactions. Overall, this study yielded significant insights for understanding the effects of chlorine dioxide on the survival, metabolism, and biochemical composition of marine zooplankton.

Can environmental factors increase oxidative responses in fish exposed to polycyclic aromatic hydrocarbons (PAHs)?

Relations among polycyclic aromatic hydrocarbons (PAHs), biomarkers of oxidative stress (lipid peroxidation, glutathione, and glutathione S-transferase activity), and the possible influence of environmental factors (temperature, pH, and salinity) were assessed in situ for specimens of Ramnogaster arcuata, a native estuarine fish. PAH levels found in the muscular tissue of R. arcuata ranged from 0.7 to 293.4 ng g-1 wet weight with petrogenic and pyrolytic inputs. Lipid peroxidation in the liver showed positive correlations (P < 0.05) with total PAHs (r = 0.66), 3-ring (r = 0.66) and 4-ring PAHs (r = 0.52) and glutathione in muscle (r = 0.58). Significant positive correlations (P < 0.05) were also evidenced between muscular glutathione with total (r = 0.62) and 3-ring PAHs (r = 0.75). Hepatic glutathione S-transferase negatively correlated with 4-ring PAHs (r = -0.58). These correlations suggest that lipid peroxidation and muscular glutathione could be good biomarkers for complex mixtures of PAHs, and hepatic glutathione S-transferase could be a suitable biomarker for 4-ring PAHs. Furthermore, significant correlations (P < 0.05) of environmental factors with PAH levels and biomarkers were observed, especially pH with 3-ring PAHs (r = -0.65), lipid peroxidation (r = -0.6), glutathione in the liver (r = -0.73) and muscle (r = -0.75); and temperature with 2-ring PAHs (r = -0.75) and glutathione in muscle (r = 0.51). The data suggest an influence of physicochemical parameters which could be driving a shift in PAH toxicity in R. arcuata. These results are essential for an integrated understanding of ecotoxicology and could help to predict environmental effects in present and future scenarios of ocean warming and acidification.

Assessing combined effects of long-term exposure to copper and marine heatwaves on the reef-forming serpulid Ficopomatus enigmaticus through a biomarker approach.

Sessile benthic organisms can be affected by global changes and local pressures, such as metal pollution, that can lead to damages at different levels of biological organization. Effects of exposure to marine heatwaves (MHWs) alone and in combination with environmentally relevant concentration of copper (Cu) were evaluated in the reef-forming tubeworm Ficopomatus enigmaticus using a multi-biomarker approach. Biomarkers of cell membrane damage, enzymatic antioxidant defences, metabolic activity, neurotoxicity, and DNA integrity were analyzed. The exposure to Cu alone did not produce any significant effect. Exposure to MHWs alone produced effects only on metabolic activity (increase of glutathione S-transferase) and energy reserves (decrease in protein content). MHWs in combination with copper was the condition that most influenced the status of cell homeostasis of exposed F. enigmaticus. The combination of MHWs plus Cu exposure induced increase of protein carbonylation and glutathione S-transferase activity, decrease in protein/carbohydrate content and carboxylesterase activity. This study on a reef-forming organism highlighted the additive effect of a climate change-related stressor to metals pollution of marine and brackish waters.

Pesticides compromise health: a comparison between lizards collected within and outside an agricultural area.

Reptiles are the least studied vertebrates regarding the impact of pesticides on their health, despite being good models for ecotoxicological studies given their abundance and easy handling. Salvator merianae is widely distributed in South America and often found in agricultural cultivation areas. Here, we compared the morphological, biochemical, and physiological parameters of S. merianae from an exposed area (EA) to pesticides and a reference area (RA) or control. These parameters were measured in plasma (albumin, alanine transaminase, alkaline phosphatase, gamma-glutamyl transpeptidase, glucose, total proteins, uric acid, triglycerides, VLDL, and corticosterone) and in erythrocytes (TBARS, glutathione S-transferase, superoxide dismutase, and catalase activity). Blood samples were collected from 28 lizards (EA: three juveniles, three adult females, and three adult males; RA: nine juveniles, four females, and five males) in southern Brazil during the reproductive period. We observed a decrease in body mass, the ratio between body mass and total length and snout-vent length in juvenile lizards collected at EA. The levels of TBARS, glutathione S-transferase, triglycerides, VLDL, and uric acid were altered for juveniles in EA. When comparing the two areas, females differed in superoxide dismutase activity and total proteins, while males differed in superoxide dismutase, catalase, and glutathione S-transferase activity. This set of results shows that S. merianae, especially juveniles, suffers a negative impact when inserted in an agricultural area. The analyzed biomarkers proved suitable for monitoring these lizards and the quality of this environment.

Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly).

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 104-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.

In Vitro Low-Bortezomib Doses Induce Apoptosis and Independently Decrease the Activities of Glutathione S-Transferase and Glutathione Peroxidase in Multiple Myeloma, Taking into Account the GSTT1 and GSTM1 Gene Variants.

BACKGROUND: Multiple myeloma (MM) is a malignancy derived from plasma cells. Bortezomib affects the concentration of reduced glutathione (GSH) and the activity of glutathione enzymes. The aim of our study was to analyze deletion (null/present) variants of GSTT1 and GSTM1 genes and their association with the levels of glutathione and its enzymes in bortezomib-treated cell cultures derived from MM patients. MATERIALS AND METHODS: This study included 180 individuals (80 MM patients and 100 healthy blood donors) who were genotyped via multiplex PCR (for the GSTT1/GSTM1 genes). Under in vitro conditions, MM bone marrow cells were treated with bortezomib (1-4 nM) to determine apoptosis (via fluorescence microscopy), GSH concentration, and activity of glutathione enzymes (via ELISA). RESULTS: Bortezomib increased the number of apoptotic cells and decreased the activity of S-glutathione transferase (GST) and glutathione peroxidase (GPx). We found significant differences in GST activity between 1 nM (GSTT1-null vs. GSTT1-present), 2 nM (GSTT1-null vs. GSTT1-present), and 4 nM (GSTM1-null vs. GSTM1-present) bortezomib: 0.07 vs. 0.12, p = 0.02; 0.06 vs. 0.10, p = 0.02; and 0.03 vs. 0.08, p = 0.01, respectively. CONCLUSIONS: Bortezomib affects the activities of GST and GPx. GST activity was associated with GSTT1 and GSTM1 variants but only at some bortezomib doses.

Survival analysis in association with GST gene polymorphism and Treatment outcomes of Gemcitabine and Cisplatin/Carboplatin-based chemotherapy among patients with Gallbladder Carcinoma.

PURPOSE: Majority of the gallbladder cancer (GBC) cases are diagnosed at an advanced stage where chemotherapy alone (or in combination with other treatment methods) is mainly opted as therapeutic approach. However, success or failure of this approach largely depends on the interindividual genetic differences. Careful consideration on the genetic association could assist in the evaluation of patient's treatment response and survival rate. Hence, the present study aims to investigate the survival of patients with GBC and their treatment response to gemcitabine and cisplatin/carboplatin-based chemotherapy in association with Glutathione S-transferase (GSTs) gene polymorphism. MATERIAL AND METHODS: A total of 216 histologically confirmed cases of gallbladder cancer were recruited. A total of 180 patients were treated with gemcitabine and cisplatin/carboplatin-based chemotherapy. GSTM1, GSTT1, and GSTP1 genotypes were determined by multiplex PCR and by PCR restriction fragment length polymorphism (PCR-RFLP), respectively. The influence of genetic polymorphism on overall survival was analyzed by Kaplan-Meier method, survival rate difference was analyzed by log-rank test, and hazard ratio for mortality outcomes was estimated using Cox regression method. RESULTS: GBC patients having genotype GSTP1 (AG + GG) showed poor 3-year survival rate of 0.8% compared to 10.9% of GSTP1 (AA) genotype (χ2 = 6.456, P = 0.011). The multivariate Cox regression results showed that the death risk was significantly higher in GSTP1 (AG + GG) genotype (HR = 3.858, P = 0.050). We found no association of GSTM1 and GSTT1 gene polymorphism with the survival; however, the combined genotypes of GSM1/GSTP1, GSTT1/GSTP1, and GSTM1/GSTT1/GSTP1 were associated with survival (P = 0.053, 0.006, and 0.058, respectively). Increased death hazard was noted by the genotype combinations of GSTM1+/GSTP1AG + GG (HR = 3.484, P = 0.024), GSTM1-/GSTP1AG + GG (HR = 2.721, P = 0.014), GSTT1+/GSTP1AG + GG (HR = 20.690, P = 0.001), and GSTT1-/GSTP1AA (HR = 26.111, P < 0.0001). Our findings indicate that chemotherapy treatment response of GSTP1 (AG + GG) has 1.62-fold increased risk for progression compared to GSTP1 (AA) genotype (p = 0.018); however, none of the genotypes showed association with overall survival and death risk after chemotherapeutic treatment. CONCLUSION: We found that the presence of GSTP1 (AG + GG) genotype showed survival disadvantage and poor treatment outcomes in response to gemcitabine and cisplatin/carboplatin-based chemotherapy. This could serve as biomarker, and future research in pharmacogenomics will definitely pave the way for the development of better treatment approach for GBC.

Environmental-related doses of afidopyropen induced toxicity effects in earthworms (Eisenia fetida).

Afidopyropen has high activity against pests. However, it poses potential risks to the soil ecology after entering the environment. The toxicity of afidopyropen to earthworms (Eisenia fetida) was studied for the first time in this study. The results showed that afidopyropen had low level of acute toxicity to E. fetida. Under the stimulation of chronic toxicity, the increase of reactive oxygen species (ROS) level activated the antioxidant and detoxification system, which led to the increase of superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. Lipid peroxidation and DNA damage were characterized by the increase of malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents. Meanwhile, the functional genes SOD, CAT, GST, heat shock protein 70 (HSP70), transcriptionally controlled tumor protein (TCTP), and annetocin (ANN) played a synergistic role in antioxidant defense. However, the comprehensive toxicity of high concentration still increased on the 28th day. In addition, strong histopathological damage in the body wall and intestine was observed, accompanied by weight loss, which indicated that afidopyropen inhibited the growth of E. fetida. The molecular docking revealed that afidopyrene combined with the surface structure of SOD and GST proteins, which made SOD and GST become sensitive biomarkers reflecting the toxicity of afidopyropen to E. fetida. Summing up, afidopyropen destroys the homeostasis of E. fetida through chronic toxic. These results provide theoretical data for evaluating the environmental risk of afidopyropen to soil ecosystem.

Whole transcriptomic analysis reveals overexpression of salivary gland and cuticular proteins genes in insecticide-resistant Anopheles arabiensis from Western Kenya.

BACKGROUND: Effective vector control is key to malaria prevention. However, this is now compromised by increased insecticide resistance due to continued reliance on insecticide-based control interventions. In Kenya, we have observed heterogenous resistance to pyrethroids and organophosphates in Anopheles arabiensis which is one of the most widespread malaria vectors in the country. We investigated the gene expression profiles of insecticide resistant An. arabiensis populations from Migori and Siaya counties in Western Kenya using RNA-Sequencing. Centers for Disease Control and Prevention (CDC) bottle assays were conducted using deltamethrin (DELTA), alphacypermethrin (ACYP) and pirimiphos-methyl (PMM) to determine the resistance status in both sites. RESULTS: Mosquitoes from Migori had average mortalities of 91%, 92% and 58% while those from Siaya had 85%, 86%, and 30% when exposed to DELTA, ACYP and PMM, respectively. RNA-Seq analysis was done on pools of mosquitoes which survived exposure ('resistant'), mosquitoes that were not exposed, and the insecticide-susceptible An. arabiensis Dongola strain. Gene expression profiles of resistant mosquitoes from both Migori and Siaya showed an overexpression mainly of salivary gland proteins belonging to both the short and long form D7 genes, and cuticular proteins (including CPR9, CPR10, CPR15, CPR16). Additionally, the overexpression of detoxification genes including cytochrome P450s (CYP9M1, CYP325H1, CYP4C27, CYP9L1 and CYP307A1), 2 carboxylesterases and a glutathione-S-transferase (GSTE4) were also shared between DELTA, ACYP, and PMM survivors, pointing to potential contribution to cross resistance to both pyrethroid and organophosphate insecticides. CONCLUSION: This study provides novel insights into the molecular basis of insecticide resistance in An. arabiensis in Western Kenya and suggests that salivary gland proteins and cuticular proteins are associated with resistance to multiple classes of insecticides.