Inhibition Of Glutathione Reductase Activity Research Articles

Quercetin-Induced Glutathione Depletion Sensitizes Colorectal Cancer Cells to Oxaliplatin.

Quercetin is an antioxidant phytochemical which belongs to the natural flavonoids group. Recently, the compound has been reported to inhibit glutathione reductase responsible for replenishing reduced forms of glutathione and thus leads to glutathione depletion, triggering cell death. In this study, we examined if quercetin sensitizes tumors to oxaliplatin by inhibiting glutathione reductase activity in human colorectal cancer cells, and thereby facilitates apoptotic cell death. A combined treatment with quercetin and oxaliplatin was found to synergistically inhibit glutathione reductase activity, lower intracellular glutathione level, increase reactive oxygen species production, and reduce cell viability, compared to treatment with oxaliplatin alone in human colorectal HCT116 cancer cells. Furthermore, the incorporation of sulforaphane, recognized for its ability to scavenge glutathione, in combination with quercetin and oxaliplatin, substantially suppressed tumor growth in an HCT116 xenograft mouse model. These findings suggest that the depletion of intracellular glutathione by quercetin and sulforaphane could strengthen the anti-cancer efficacy of oxaliplatin.

The Early Oxidative Stress Induced by Mercury and Cadmium Is Modulated by Ethylene in Medicago sativa Seedlings.

Cadmium (Cd) and mercury (Hg) are ubiquitous soil pollutants that promote the accumulation of reactive oxygen species, causing oxidative stress. Tolerance depends on signalling processes that activate different defence barriers, such as accumulation of small heat sock proteins (sHSPs), activation of antioxidant enzymes, and the synthesis of phytochelatins (PCs) from the fundamental antioxidant peptide glutathione (GSH), which is probably modulated by ethylene. We studied the early responses of alfalfa seedlings after short exposure (3, 6, and 24 h) to moderate to severe concentration of Cd and Hg (ranging from 3 to 30 μM), to characterize in detail several oxidative stress parameters and biothiol (i.e., GSH and PCs) accumulation, in combination with the ethylene signalling blocker 1-methylcyclopropene (1-MCP). Most changes occurred in roots of alfalfa, with strong induction of cellular oxidative stress, H2O2 generation, and a quick accumulation of sHSPs 17.6 and 17.7. Mercury caused the specific inhibition of glutathione reductase activity, while both metals led to the accumulation of PCs. These responses were attenuated in seedlings incubated with 1-MCP. Interestingly, 1-MCP also decreased the amount of PCs and homophytochelatins generated under metal stress, implying that the overall early response to metals was controlled at least partially by ethylene.

Pleiotropic effects of a mitochondrion-targeted glutathione reductase inhibitor on restraining tumor cells

Mitochondria has been identified as a target for tumor therapy. Agents preferentially concentrated in mitochondria may exert more potent antitumor effects by interfering with the normal function of mitochondria. Glutathione reductase (GR) in mitochondria is a crucial antioxidant enzyme to maintain mitochondrial function, and has been recognized as an important target for the development of anticancer drugs. Herein, we present a triphenylphosphonium-modified anticancer agent, MT-1, which can preferentially accumulate in mitochondria and bind to GR by covalent binding manner.As a result, morphology and function of mitochondria were severely damaged, as well as cellular energy supply was severely impeded due to the simultaneously inhibition against mitochondrial respiration and glycolysis. Moreover, MT-1 was found to bind to a completely new site of GR (C278) that has never considered as binding site of inhibitors before. This new binding mode led to the change of GR structure, which affected the stability of the transition state of the catalytic process, and finally led to the inhibition of GR activity. Thus, current study provided a potentially novel tumor therapeutic strategy by targeting novel sites of GR in mitochondrion.

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. For the experiment, semen extender was supplemented with Mito TEMPO (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p<0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p<0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

GSH Levels Serve As a Biological Redox Switch Regulating Sulforaphane-Induced Cell Fate in Human Lens Cells.

PurposeSulforaphane (SFN) is a therapeutic phytochemical agent for many health conditions. SFN-induced cytotoxicity is shown to have promise in preventing posterior capsule opacification (PCO). In the current study, we aimed to elucidate key processes and mechanisms linking SFN treatment to lens cell death.MethodsThe human lens epithelial cell line FHL124 and central anterior epithelium were used as experimental models. Cell death was assessed by microscopic observation and cell damage/viability assays. Gene or protein levels were assessed by TaqMan RT-PCR or immunoblotting. Mitochondrial networks and DNA damage were assessed by immunofluorescence. Mitochondrial membrane potential, activating transcription factor 6 (ATF6) activity, ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and glutathione reductase (GR) activity were measured using different light reporter assays. SFN metabolites were analyzed by LC-MS/MS.ResultsTreatment with N-acetylcysteine (NAC), a reactive oxygen species scavenger, prevented SFN-induced cell death in both models. NAC also significantly protected FHL124 cells from SFN-induced mitochondrial dysfunctions, endoplasmic reticulum stress (ERS), DNA damage and autophagy. SFN significantly depleted GSH, the major antioxidant in the eye, and reduced GR activity, despite doubling its protein levels. The most abundant SFN conjugate detected in lens cells following SFN application was SFN–GSH. The addition of GSH protected lens cells from all SFN-induced cellular events.ConclusionsSFN depletes GSH levels in lens cells through conjugation and inhibition of GR activity. This leads to increased reactive oxygen species and oxidative stress that trigger mitochondrial dysfunction, ERS, autophagy, and DNA damage, leading to cell death. In summary, the work presented provides a mechanistic understanding to support the therapeutic application of SFN for PCO and other disorders.

An in vivo and in vitro comparison of the effects of amoxicillin, gentamicin, and cefazolin sodium antibiotics on the mouse hepatic and renal glutathione reductase enzyme.

Despite the fact that the use of antibiotics is increasing worldwide, it is clear that antibiotics can lead to oxidative stress. This is the first study to make a comparison of the impact of frequently prescribed antibiotics, including amoxicillin, gentamicin, and cefazolin sodium, on the gene, protein, and activity of glutathione reductase (GR), which is one of the primary antioxidant enzymes, in mouse liver and kidney tissues. First, the GR enzyme was purified by the 2',5'-ADP Sepharose 4B affinity chromatography with a specific activity of 84.615 EU/mg protein and 9.63 EU/mg protein from the mouse liver and kidney, respectively. The in vitro inhibitory effects of the antibiotics in question was determined. While cefazolin sodium did not exhibit any inhibitory effect, gentamicin and amoxicillin inhibited GR activity in both tissues. Furthermore, the in vivo effects of these drugs were investigated, and amoxicillin and cefazolin sodium-inhibited GR activity in both liver and kidney tissues, while gentamicin did not have any effect on the kidney. Besides, while gentamicin downregulated and cefazolin sodium upregulated Gr gene expression, amoxicillin did not alter it. Protein expression was only affected by the administration of cefazolin sodium in the kidney. This study is important as it demonstrates that while amoxicillin and gentamicin showed parallel effects on the GR activity in liver and kidney tissues both in vitro and in vivo, cefazolin sodium had a very strong effect on hepatic and renal GR in vivo. Furthermore, the antibiotics used in this study induced oxidative stress in both tissues.

Toxicity of mercury and post-exposure recovery in Corbicula fluminea: Neurotoxicity, oxidative stress and oxygen consumption

The toxicity of mercury to the invasive species Corbicula fluminea and the post-exposure recovery were investigated in relation to previous developmental exposure to distinct environmental conditions. Bivalves were collected in the estuaries of Minho River (M-est) and Lima River (L-est) that have several abiotic differences, including in environmental contamination, with the former being generally less contaminated. After 14 days of acclimation to laboratory conditions, two 14-day bioassays were performed simultaneously: one with bivalves from the M-est and the other with bivalves from the L-est. In each bioassay, the treatments were: dechlorinated tap water (clean medium) for 8 days, clean medium for 14 days, 31 µg/L of mercury for 8 days, 31 µg/L of mercury for 14 days and 31 µg/L of mercury for 8 days followed by 6 days in clean medium (post-exposure recovery). The effect criteria were the oxygen consumption rate (OCR), the activity of the enzymes cholinesterases (ChE), NADP-dependent isocitrate dehydrogenase (IDH), octopine dehydrogenase (ODH), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferases (GST) and lipid peroxidation (LPO) levels. Exposure to mercury for 8 days caused significant (p ≤ 0.05) inhibition of GR activity in M-est bivalves, whereas no significant adverse effects were observed in L-est animals. Moreover, evidences of delayed toxicity caused by 8-day exposure to mercury in OCR, IDH activity and LPO levels were found in M-est individuals but not in those of the L-est. Exposure to mercury for 14 days caused significant (p ≤ 0.05) depression of the OCR and of IDH activity in animals from both estuaries, indicating reduced individual fitness and hypoxia conditions. Moreover, oxidative stress and lipid peroxidation damage were observed in bivalves from the M-est exposed to mercury for 14 days but not in L-est animals. Differences in M-est and L-est environmental conditions to which animals were exposed in the wild likely contributed to the differences of sensitivity to mercury between M-est and L-est bivalves. The results of this study highlight the importance of investigating delayed toxicity, post-exposure recovery, and of taking into consideration the background contamination and other abiotic conditions of the original habitats when assessing the effects of environmental contaminants on animals from wild populations.

Target identification reveals protein arginine methyltransferase 1 is a potential target of phenyl vinyl sulfone and its derivatives.

Phenyl vinyl sulfone (PVS) and phenyl vinyl sulfonate (PVSN) inactivate protein tyrosine phosphatases (PTPs) by mimicking the phosphotyrosine structure and providing a Michael addition acceptor for the active-site cysteine residue of PTPs, thus forming covalent adducts between PVS (or PVSN) and PTPs. We developed a specific antiserum against PVS. This antiserum can be used in general antibody-based assays such as immunoblotting, immunofluorescence staining, and immunoprecipitation. Target identification through immunoprecipitation and mass spectrometry analysis reveals potential targets of PVS, mostly proteins with reactive cysteine residues or low-pKa cysteine residues that are prone to reversible redox modifications. Target identification of PVSN has been conducted because the anti-PVS antiserum can also recognize PVSN. Among the targets, protein arginine methyltransferase 1 (PRMT1), inosine-5′-monophosphate dehydrogenase 1, vimentin, and glutathione reductase (GR) were further confirmed by immunoprecipitation followed by immunoblotting. In addition, PVSN and Bay11-7082 inhibited GR activity, and PVS, PVSN, and Bay 11-7082 inhibited PRMT1 activity in in vitro assays. In addition, treatment of PVSN, Bay11-7082, or Bay 11-7085 in cultured HeLa cells can cause the quick decline in the levels of protein asymmetric dimethylarginine. These results indicate that the similar moiety among PVS, PVSN, Bay 11-7082, and Bay 11-7085 can be the key structure of lead compounds of PRMT1. Therefore, we expect to use this approach in the identification of potential targets of other covalent drugs.

Тканиноспецифічність впливу фізичних та фармакологічних чинників на антиоксидантний захист у тварин з ерозивно-виразковою патологією гастродуоденальної зони

&lt;p&gt;&lt;span lang="EN"&gt;Influence of the natural magnetic field (MP) is an integral part of the existence of all living systems on Earth. Artificial MPS appeared precisely due to human activity. It is established that the interaction of MP with biosystems, its biological activity depends on the biotropic parameters of this field, such as frequency, intensity, gradient (rate of growth), vector, pulse shape, exposure time and localization of influence. &lt;/span&gt;&lt;span lang="EN"&gt;There is now no single point of view regarding the mechanisms of interaction of the MP with biological objects. Experimental investigations have documented the influence of MP on the reactions occurring in the free radical type. Free radical pathologies include diseases such as gastroesophageal reflux disease and peptic ulcer disease. Therefore, the search for factors that can affect the components of the system of oxidative homeostasis remains. &lt;/span&gt;&lt;span lang="EN"&gt;The purpose of the work is to evaluate the isolated effect of the magnetic field and in combination with injections of L-arginine-L-glutamate into lipoperoxidation processes and antioxidant defense system in rat tissues with experimental pathology of the gastroduodenal zone. The modeling of erosive-ulcerative lesions of the gastroduodenal zone in rats was accompanied by an imbalance in the functioning of antioxidant defense systems and the intensification of lipoeroxidation LPO processes in the blood, liver, brain and stomach tissues of experimental animals. Applications of a magnetic field to animals with simulated pathology of the gastroduodenal zone resulted in inhibition of the processes of LPO in the blood of experimental rats at the activation of free radical reactions in the liver, brain and stomach tissues, on the background of partial decontamination of the glutathione system enzymes against the backdrop of an increase in the amount of reduced glutathione. With the combined action of glutargin and magnetic field in the blood, liver and brain tissues of experimental rats with erosive-ulcerative defeat of the gastroduodenal zone, activation of anti-radical, anti-peroxide protection enzymes, increased levels of reduced glutathione against the inhibition of glutathione reductase activity and a decrease in the intensity of the LPO processes with simultaneous normalization &lt;/span&gt;&lt;span lang="EN-US"&gt;of &lt;/span&gt;&lt;span lang="EN"&gt;processes of lipoperoxidation in the stomach tissue took place.&lt;/span&gt;&lt;/p&gt;

Alterations of mitochondrial electron transport chain and oxidative stress induced by alkaloid-like α-aminonitriles on Aedes aegypti larvae

Aedes aegypti mosquitoes are responsible for dengue, chikungunya, and Zika virus transmission in tropical and subtropical areas around the world. Due to the absence of vaccines or antiviral drugs for human treatment, the majority of control strategies are targeted at Ae. aegypti elimination. Our research on mosquito control insecticidal agents has previously shown that the alkaloid girgensohnine and its analogues (α-aminonitriles) present in vitro acetylcholinesterase inhibition and in vivo insecticidal activity against Ae. aegypti. However, acetylcholinesterase inhibition may not be the only mechanism of action behind these effects. On this basis, the principal aim of this study was to elucidate the possible action mode of four α-aminonitriles on Ae. aegypti by studying other important enzymatic targets, such as mitochondrial electron transport chain complexes, catalase, and superoxide dismutase, key oxidative stress enzymes. Mitochondria were isolated from Ae. aegypti larvae by differential centrifugation, stored at −70°C, and fragmented using ultrasound for 10min. The effects of α-aminonitriles (1 to 4) over enzymatic activities were evaluated using concentrations of 8nM, 2μM, 8μM, and 40μM. Results indicated that α-aminonitriles caused significant NADH dehydrogenase and succinate oxidase inhibition (~44% at the highest concentration tested). Succinate dehydrogenase and cytochrome c oxidase activities were found to increase (162% and 106% at 40μM, respectively). It was also observed that these compounds produced catalase inhibition and thus prevented H2O2 reduction, which induced the formation of reactive oxygen species (ROS). Moreover, NBT assay showed that compounds 3 and 4 (with 2-(pyrrolidin-1-yl) acetonitrile as substituent) increased by approximately 50% the O2●- concentration in the mitochondrial respiratory chain. It was concluded that the tested compounds act as complex I inhibitors by blocking electron transport and causing electron leak, possibly between complex I and III. Furthermore, α-aminonitriles inhibited catalase activity; compounds 1 and 2 (with piperidine fragment) inhibited glutathione reductase activity and further promoted the accumulation of ROS, which probably induced oxidative stress.

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M.hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100µg MC-LR l-1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100µgl-1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-L-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

Characterization of nineteen antimony(III) complexes as potent inhibitors of photosystem II, carbonic anhydrase, and glutathione reductase.

Nineteen antimony(III) complexes were obtained and examined as possible herbicides. Six of these were synthesized for the first time, and their structures were identified using elemental analyses, 1H-NMR, 13C-NMR, FTIR, LCMS, magnetic susceptibility, and conductivity measurement techniques. For the nineteen examined antimony(III) complexes their most-stable forms were determined by DFT/B3LYP/LanL2DZ calculation method. These compounds were examined for effects on photosynthetic electron transfer and carbonic anhydrase activity of photosystem II, and glutathione reductase from chloroplast as well were investigated. Our results indicated that all antimony(III) complexes inhibited glutathione reductase activity of chloroplast. A number of these also exhibited good inhibitory efficiency of the photosynthetic and carbonic anhydrase activity of Photosystem II.

Automated evaluation of the inhibition of glutathione reductase activity: application to the prediction of ionic liquids' toxicity

An automated bioassay based on glutathione reductase was developed and applied to the evaluation of the toxicity of ionic liquids.

A biophysically based mathematical model for the catalytic mechanism of glutathione reductase

Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and thereby maintains a constant GSH level in the system. GSH scavenges superoxide (O2−) and hydroxyl radicals (OH) nonenzymatically or by serving as an electron donor to several enzymes involved in reactive oxygen species (ROS) detoxification. In either case, GSH oxidizes to GSSG and is subsequently regenerated by the catalytic action of GR. Although the GR kinetic mechanism has been extensively studied under various experimental conditions with variable substrates and products, the catalytic mechanism has not been studied in terms of a mechanistic model that accounts for the effects of the substrates and products on the reaction kinetics. The aim of this study is therefore to develop a comprehensive mathematical model for the catalytic mechanism of GR. We use available experimental data on GR kinetics from various species/sources to develop the mathematical model and estimate the associated model parameters. The model simulations are consistent with the experimental observation that GR operates via both ping–pong and sequential branching mechanisms based on relevant concentrations of its reaction substrate GSSG. Furthermore, we show the observed pH-dependent substrate inhibition of GR activity by GSSG and bimodal behavior of GR activity with pH. The model presents a unique opportunity to understand the effects of products on the kinetics of GR. The model simulations show that under physiological conditions, where both substrates and products are present, the flux distribution depends on the concentrations of both GSSG and NADP+, with ping–pong flux operating at low levels and sequential flux dominating at higher levels. The kinetic model of GR may serve as a key module for the development of integrated models for ROS-scavenging systems to understand protection of cells under normal and oxidative stress conditions.

Fluorescence detection of glutathione reductase activity based on deoxyribonucleic acid-templated silver nanoclusters

Fluorescent silver nanoclusters stabilized by DNA (DNA-AgNCs) exhibit distinct response rates to thiol and disulfide. Glutathione reductase can catalyze the reduction of the oxidized glutathione (GSSG) quickly to reduced glutathione (GSH) in the presence of β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH). Consequently, DNA-AgNCs can serve as a new fluorescent platform for assaying the glutathione reductase (GR) activity. This newly proposed assay has a high sensitivity and a good selectivity toward GR. The GR activity can be detected in the range of 0.2–2.0mUmL−1 with a minimum detectable concentration of 0.2mUmL−1. Pepsin, lysozyme, trypsin, avidin, thrombin, myoglobin, and BSA have little effect on the fluorescence intensity of DNA-AgNCs. The GR activity assay is successfully used to monitor the inhibition of GR activity by a typical inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea.

Investigation of 7-benzylidenenaltrexone derivatives as resistance reverser for chloroquine-resistant Plasmodium chabaudi

Derivatives of 7-benzylidenenaltrexone (BNTX), which was recently reported to be an effective chloroquine (CQ)-resistance reverser, were synthesized and evaluated for their CQ-resistance reversing activities. The synthesized derivatives showed CQ-resistance reversing effects. They also reacted with glutathione (GSH) both enzymatically and chemically, and inhibited glutathione reductase activity. 7-Benzyl derivative, which was obtained by reduction of the olefin group in α,β-unsaturated ketone structure of BNTX, also exhibited CQ-resistance reversing effect, but its potency was significantly lower than that of BNTX. These outcomes suggested that the decrease in GSH level could be one of the mechanisms of CQ-resistance reversing effects induced by BNTX derivatives.

Metabolism of two new benzodiazepine-type anti-leishmanial agents in rat hepatocytes and hepatic microsomes and their interaction with glutathione in macrophages

Abstract Objectives To measure the metabolism and toxicity of 7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. Methods The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. Key findings BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 μM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. Conclusions The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.

Metabolism of two new benzodiazepine-type anti-leishmanial agents in rat hepatocytes and hepatic microsomes and their interaction with glutathione in macrophages

To measure the metabolism and toxicity of 7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 microM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.

Profiling patterns of glutathione reductase inhibition by the natural product illudin S and its acylfulvene analogues.

Acylfulvenes (AFs) are a class of antitumor agents with favorable cytotoxic selectivity profiles compared to their natural product precursor, illudin S. Like many alkylating agents, illudin S and AFs readily react with thiol-containing small molecules such as cysteine, glutathione and cysteine-containing peptides; reduced cellular glutathione levels can affect illudin S toxicity. Glutathione reductase (GR) is a critical cellular antioxidant enzyme that regulates the intracellular ratio of reduced-oxidized glutathione. In this study, we found that acylfulvene analogues are GR inhibitors, and evaluated aspects of the drug-enzyme interactions as compared with the structurally related natural product illudin S and the known irreversible GR inhibitor, carmustine. Acylfulvene analogues exhibited concentration-dependent GR inhibitory activity with micromolar IC(50)s; however, up to 2 mM illudin S did not inhibit GR activity. The absence of NADPH attenuates GR inhibition by AFs and the presence of glutathione disulfide (GSSG), the natural GR substrate, which binds to the enzyme active site, has a minimal effect in protecting GR from AFs. Furthermore, each compound can induce GR conformation changes independent of the presence of NADPH or GSSG. These results, together with gel-filtration analysis results and mass spectrometry data, indicate AF is a reversible inhibitor and HMAF an irreversible inhibitor that can form a bis-adduct with GR by reacting with active site cysteines. Finally in a cell-based assay, illudin S and HMAF were found to inhibit GR activity, but this inhibition was not associated with the reduction of GR levels in the cell. A model accounting for differences in mechanisms of GR inhibition by the series of compounds is discussed.

Generation of reactive oxygen species during mouse hepatic microsomal metabolism of cannabidiol and cannabidiol hydroxy-quinone

We investigated whether cannabidiol (CBD) and cannabidiol hydroxy-quinone (CBDHQ) generate reactive oxygen species (ROS) during metabolism with mouse hepatic microsomes. CBD and CBDHQ (91.5 μM) significantly suppressed lipid peroxidation in the mouse hepatic microsomes. CBDHQ also significantly decreased NADH-cytochrome b 5 reductase (fp 1) activity by 25% of the control activity in the hepatic microsomes, and tended to increase NADPH-cytochrome c (P450) reductase (fp 2) activity. CBDHQ also significantly inhibited superoxide dismutase and catalase activities in mouse hepatic 105,000 × g supernatant. Moreover, CBDHQ significantly increased glutathione reductase activity and significantly inhibited NAD(P)H-quinone reductase activity. CBD exhibited similar effects on these enzymes, except that cannabinoid significantly inhibited glutathione reductase activity in mouse hepatic 105,000 × g supernatant. These results suggest that CBDHQ is easily converted to the semiquinone form rather than the hydroquinone form. It was also suggested that CBDHQ and CBD were capable of generating ROS as superoxide anion radicals during their metabolism with mouse hepatic microsomes or with purified fp 2 by electron spin resonance spin trapping methods with 5,5-dimethyl-1-pyrroline- N-oxide. The present results suggest that CBDHQ formed during hepatic microsomal metabolism of CBD is capable of generating ROS and inducing cell toxicity.