Diamide Exposure Research Articles

Identification of differentially expressed miRNAs associated with diamide detoxification pathways in Spodoptera frugiperda.

The fall armyworm (FAW) Spodoptera frugiperda is a severe economic pest of multiple crops globally. Control of this pest is often achieved using insecticides; however, over time, S. frugiperda has developed resistance to new mode of action compounds, including diamides. Previous studies have indicated diamide resistance is a complex developmental process involving multiple detoxification genes. Still, the mechanism underlying the possible involvement of microRNAs in post-transcriptional regulation of resistance has not yet been elucidated. In this study, a global screen of microRNAs (miRNAs) revealed 109 known and 63 novel miRNAs. Nine miRNAs (four known and five novel) were differentially expressed between insecticide-resistant and -susceptible strains. Gene Ontology analysis predicted putative target transcripts of the differentially expressed miRNAs encoding significant genes belonging to detoxification pathways. Additionally, miRNAs are involved in response to diamide exposure, indicating they are probably associated with the detoxification pathway. Thus, this study provides comprehensive evidence for the link between repressed miRNA expression and induced target transcripts that possibly mediate diamide resistance through post-transcriptional regulation. These findings highlight important clues for further research to unravel the roles and mechanisms of miRNAs in conferring diamide resistance.

Mycobacterium tuberculosis PPE44 (Rv2770c) is involved in response to multiple stresses and promotes the macrophage expression of IL-12 p40 and IL-6 via the p38, ERK, and NF-κB signaling axis

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable threat to global public health. The successful intracellular persistence of M. tuberculosis significantly contributes to the intractability of tuberculosis. Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are mycobacterial exclusive protein families that widely reported to be involved in the bacterial virulence, physiology and interaction with host. Rv2770c (PPE44), a predicted virulence factor, was up-regulated upon the infected guinea pig lungs. To investigate the role of Rv2770c, we heterologously expressed the PPE44 in the nonpathogenic fast-growing M. smegmatis strain. Subcellular location analysis demonstrated that Rv2770c is a cell wall associated protein, suggestive of a potential candidate involved in host-pathogen interaction. The Rv2770c can enhance M. smegmatis survival within macrophages and under stresses such as H2O2, SDS, diamide exposure, and low pH condition. M. smegmatis expressing Rv2770c is more virulent as testified by the increased death of macrophages and the increased expression of interlukin-6 (IL-6) and interlukin-12p40 (IL-12p40). Moreover, Rv2770c altered the secretion of IL-6 and IL-12p40 of macrophages via NF-κB, ERK1/2 and p38 MAPK axis. Taken together, this study implicated that Rv2770c was a virulent factor actively engaged in the interaction with host macrophage.

Overexpression of Rv2788 increases mycobacterium stresses survival

Mycobacterium tuberculosis, the causative agent of tuberculosis-one of the most devastating infectious diseases, is a successful intracellular pathogen capable of surviving diverse stresses. Unveiling the molecular mechanisms governing this superior adaptation will inspire better control measures against tuberculosis. To define the role of Rv2788, a manganese-dependent transcriptional repressor, M.smegmatis was used as the host strain for heterologous expression Rv2788. Rv2788 can significantly change the colony morphology and fatty acids and permeability of cell wall, enhance the growth of the recombinants and resistance to diverse stresses, such as hydrogen peroxide (H₂O₂), diamide exposure, surface stress, acidic condition, multiple antibiotics treatment including chloramphenicol, vancomycin and amikacin. The dysregulation of the target genes of Rv2788, such as whiB1 and lexA, might underpin such phenotypes. The results implicate important roles of Rv2788 in the survival of Mycobacterium under stresses, and might represent ideal novel antibiotics target candidate.

Quantification of Global Protein Disulfides and Thiol-Protein Mixed Disulfides to Study the Protein Dethiolation Mechanisms

The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ←→ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman’s reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 μmoles/g and after diamide exposure they were significantly augmented of about 65%-70% in liver and heart, but not in the kidney. Normal XSSP, that were -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.

Effect of diamide‐induced oxidative stress on Ca2+ signaling in cultured bovine aortic endothelial cells (BAEC)

Diamide is a thiol‐oxidizing agent commonly used to rapidly and reversibly oxidize intracellular glutathione (GSH) to its disulfide (GSSG) and to increase the formation of protein‐glutathione mixed disulfides (P‐SSG). In the present study we examined the acute effects of diamide on free cytosolic Ca2+ ([Ca2+]i) of cultured BAECs loaded with fura‐2. Diamide increased spontaneous Ca2+ oscillations and elevated basal [Ca2+]i when compared to vehicle‐treated controls. Low concentrations (50, 100 μM) progressively increased the number of cells spontaneously oscillating as well as the oscillation frequency of individual cells, whereas high concentrations (250, 500 μM) caused a synchronized oscillation of almost all cells immediately upon diamide exposure followed by a significant rise in basal [Ca2+]i. Similar dose‐ and time‐dependent effects of diamide were observed in the absence of extracellular Ca2+. Pretreatment with the phospholipase C inhibitor U73122, but not the inactive analog U73343, prevented the observed changes in [Ca2+]i. These results indicate that diamide promotes Ca2+ release from the inositol 1,4,5‐trisphosphate (IP3)‐sensitive internal Ca2+ store and is capable of elevating basal [Ca2+]i in the absence of extracellular Ca2+, effects which may be related to a diamide‐induced glutathionylation of the IP3 receptor and the plasmalemmal Ca2+ pump, respectively. Supported in part by HL097355

Cardiac arrhythmias induced by glutathione oxidation can be inhibited by preventing mitochondrial depolarization

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (ΔΨm) during ischemia and reperfusion contributes to arrhythmogenesis through the formation of metabolic sinks in the myocardium, wherein clusters of myocytes with uncoupled mitochondria and high KATP current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide ΔΨm depolarization, through a reactive oxygen species (ROS)-induced ROS release mechanism, can be triggered by global depletion of the antioxidant pool with diamide, a glutathione oxidant. Here we examine whether diamide causes mitochondrial depolarization and promotes arrhythmias in normoxic isolated perfused guinea pig hearts. We also investigate whether stabilization of ΔΨm with a ligand of the mitochondrial benzodiazepine receptor (4′-chlorodiazepam; 4-ClDzp) prevents the formation of metabolic sinks and, consequently, precludes arrhythmias. Oxidation of the GSH pool was initiated by treatment with 200 μM diamide for 35 min, followed by washout. This treatment increased GSSG and decreased both total GSH and the GSH/GSSG ratio. All hearts receiving diamide transitioned from sinus rhythm into ventricular tachycardia and/or ventricular fibrillation during the diamide exposure: arrhythmia scores were 5.5±0.5; n=6 hearts. These arrhythmias and impaired LV function were significantly inhibited by co-administration of 4-ClDzp (64 μM): arrhythmia scores with diamide+4-ClDzp were 0.4±0.2 (n=5; P<0.05 vs. diamide alone). Imaging ΔΨm in intact hearts revealed the heterogeneous collapse of ΔΨm beginning 20 min into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of ΔΨm was prevented by 4-ClDzp treatment, as was the increase in myocardial GSSG. These findings show that oxidative stress induced by oxidation of GSH with diamide can cause electromechanical dysfunction under normoxic conditions. Analogous to ischemia–reperfusion injury, the dysfunction depends on the mitochondrial energy state. Targeting the mitochondrial benzodiazepine receptor can prevent electrical and mechanical dysfunction in both models of oxidative stress.

Diamide-induced cytotoxicity and thermotolerance in CHO cells.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro.

Direct oxidation of embryonic reduced glutathione (GSH) by a thiol oxidant, diamide, has been demonstrated to result in increased glutathione disulfide (GSSG) and protein-glutathione mixed disulfide (protein-S-SG) formation, which is accompanied by embryotoxicity and reductions in amniotic fluid volume. The altered functions of critical proteins or enzymes caused by the formation of protein-S-SG perturb cellular metabolism and may be involved in the embryotoxicity produced by GSH oxidation. The present study investigates changes in the metabolism of glucose through glycolysis and the pentose phosphate shunt pathways (PPP) and their related enzymes under the oxidative conditions produced by diamide exposure in organogenesis-stage rat conceptus (gestational day 10) in vitro. The metabolism of glucose via the PPP, measured as amounts of CO2 production from D-[1-14C]-glucose, was significantly increased in the conceptus exposed to 100-500 microM diamide to levels 2.5-3-fold those of controls. It was found that these substantial increases in the PPP activity did not correlate well with a moderate activation of glucose 6-phosphate dehydrogenase (G6PD) activity, the key enzyme in the PPP pathway. Changes in glycolysis due to diamide treatment were also determined by measurements of lactate production from D-[U-14C]-glucose. Production of lactate by the conceptus exposed to 250-500 microM diamide for 60 min was reduced (to approximately 54% of control values) concomitantly with a significant inhibition of the glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GPD) and phosphofructokinase (PFK), indicating an overall decrease in glycolysis. Diamide was found to produce a differential effect on the enzymatic activities determined in this study, with greater degrees of inhibition seen in the tissue supernatants from the visceral yolk sac (VYS) compared to those from the embryo. Activities of GPD and PFK were decreased to approximately 22% and 43% control values, respectively, when determined in the supernatants from the VYS of the conceptus exposed to 500 microM diamide for 60 min. In addition, more than 90% of the GPD activity in the VYS, but not the embryo, was rapidly inhibited by the thiol alkylating agent N-ethylmaleimide (NEM, 100 microM) within 15 min of the exposure. In contrast to diamide and NEM, no alterations in lactate production were seen in the conceptus treated with the GSH depletor L-buthionine-S,R-sulfoximine (1 mM) for 5 hr in the culture media. Further experiments demonstrated that the activity of the GPD, inhibited by a 30-min incubation with 500 microM diamide, can be reversed after removal of diamide and that this effect was potentiated by subsequent treatment with dithiothreitol (30 mM), a thiol reducing agent. These results indicated the involvement of thiol/disulfide status in regulation of the metabolism of glucose in the developing conceptus and support the hypothesis that GSH oxidation and protein-S-SG formation could be a critical event associated with mechanisms of embryotoxicity elicited by oxidative stress. It was suggested in this study that, under these experimental conditions, embryotoxicity induced by diamide is primarily mediated via altered VYS functions, including disrupted energy production (glycolysis).

Reversible S-thiolation of Human Endothelial Cell Actin Accompanies a Structural Reorganisation of the Cytoskeleton

Exposure of human umbilical vein endothelial cells to oxidants such as hydrogen peroxide, tert-butyl hydroperoxide and diamide has been shown to induce oxidant-specific S-thiolation of cellular proteins with glutathione. In this study we show that the diamide-induced S-thiolation of a 42 KDa protein is accompanied by cell shrinkage and the loss of cell to cell contact due to a structural reorganization of the cytoskeleton, as assessed by cytofluorometric visualization by confocal microscopy. Both events are simultaneously and rapidly reversed upon the removal of the stimulus. The 42 KDa protein was identified as actin by sequence analysis of the excised and electro-blotted protein band from SDS-PAGE gels, by immunochemical detection and by binding to immobilized deoxyribonuclease I. Additionally, we have shown that diamide exposure of the cells resulted in a S-thiolation of both polymerized F-actin and monomeric G-actin.

Sensory neuropeptide-mediated bronchoconstriction of the guinea pig lung by diamide; a comparison to hydrogen peroxide

The effect of the thiol oxidizing agent diamide on airway conductance, dynamic compliance and perfusion flow of isolated perfused and ventilated guinea pig lungs was investigated. When infused in the pulmonary circulation, diamide (100 μM) induced bronchoconstriction, but no effect on perfusion flow was observed. Although diamide exposure induced the formation of thromboxane A 2, the thromboxane/prostaglandin endoperoxide receptor antagonist L-670,596 did not affect the decrease in conductance and compliance induced by diamide. Diamide induced the release of the sensory neuropeptide calcitonin gene-related peptide. The bronchoconstriction and the release of calcitonin gene-related peptide induced by diamide were abolished by capsaicin pretreatment of the guinea pigs. Combined pretreatment with the NK 1 and NK 2 receptor antagonists, CP-96,345 and SR-48968, attenuated the effect of diamide. Hydrogen peroxide-induced vaso- and bronchoconstriction was not affected by capsaicin-pretreatment, nor did hydrogen peroxide induce detectable release of calcitonin gene-related peptide. The results indicate that diamide activates sensory nerves and induces neuropeptide release and neurokinin receptor-mediated bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

Induction of thermotolerance by chemical agents in enucleate erythrocytes.

Erythrocytes treated with various chemical agents for 1 h at 37 degrees C showed resistance to a subsequent 1 h heat treatment at 53 degrees C. Maximal thermotolerance was observed 6 h after 3 mM DNP and 0.03 mM disulfiram treatment and 4 h after diamide exposure at 0.3 mM. Our results suggest that chemically induced thermotolerance to heat treatment in erythrocytes was similar to heat-induced thermotolerance.

Diamide stimulates calcium-sodium exchange in dog red blood cells.

Calcium influx can be stimulated in dog red blood cells by preexposure to diamide under certain conditions. Diamide-activated calcium influx resembles swelling-induced Ca2+-Na+ exchange in several respects. These include saturation of calcium influx at external calcium levels greater than 0.5 mM, suppression of calcium influx by external sodium, and inhibition by quinidine. The ability of diamide to stimulate this transport pathway depends critically on the ionic composition of the medium in which the cells are bathed at the time of diamide exposure. The effect is greatest if the diamide preincubation is conducted in a hypotonic lithium chloride medium containing at least 1 microM calcium. Stimulation of Ca2+-Na+ exchange is seen at diamide concentrations (0.10-0.33 mM) that are lower than those reported to cause major spectrin cross-linking, glutathione depletion, Ca2+-ATPase inhibition, or ion channel formation. The results suggest that dog red cells have a large latent capacity for Ca2+-Na+ exchange.

Diamide exposure, thermal resistance, and synthesis of stress (heat shock) proteins

Chinese hamster ovary (CHO) cells were treated with the thiol oxidant diamide for 1 hr at 37°, incubated in diamide-free medium for 4 hr at 37°, and then exposed to hyperthermic treatment (43°) or assayed for the presence of 110, 90 and 66 kilodalton (kD) stress (heat shock) proteins. Cellular inactivation produced by the hyperthermic treatment was measured using colony formation as the end point. Low concentrations of diamide, which did not result in depletion of intracellular GSH, induced a moderate degree of protection against thermal toxicity but did not affect the pattern of protein synthesis. Exposure to 0.4 mM diamide, which reduced intracellular GSH concentrations by 50–60%, significantly reduced the rate of hyperthermic cellular inactivation. This occurred coincidentally with the synthesis of stress proteins of approximate molecular weights of 110, 90 and 66 kD. Furthermore, this concentration of diamide protected cells from thermal inhibition of protein synthesis. These results indicate that thiol oxidation by diamide can induce both the development of thermal resistance to cellular inactivation and the synthesis of stress proteins.