Menadione

Menadione, a synthetic naphthoquinone, can be converted to active vitamin K2 in vivo. Target: Others Menadione (Vitamin K3) is a synthetic analogue of of 1,4-naphthoquinone with a methyl group in the 2-position. Menadione is used as a phosphatase inhibitor and an inhibitor of mitochondrial DNA polymerase γ (pol γ). Menadione can be used as an oxidative injury (free radical generator) inducing agent [1].

References:
[1]. http://en.wikipedia.org/wiki/Menadione

Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD(+) supports substrate-level phosphorylation during respiratory inhibition.[Pubmed:29746824]

Biochim Biophys Acta Bioenerg. 2018 Sep;1859(9):909-924.

Provision of NAD(+) for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD(+), provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1(-/-) and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD(+) to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, Menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.

THE ROLE OF PROTEIN CHAPERONES IN THE SURVIVAL FROM ANTHRACYCLINE-INDUCED OXIDATIVE STRESS IN SACCHAROMYCES CEREVISIAE.[Pubmed:29657945]

Int J Adv Res (Indore). 2018 Mar;6(3):144-152.

Several S. cerevisiae deletion strains involving heat-shock response factors were among the most sensitive mutants identified in a previous genetic screen for doxorubicin hypersensitivity. These strains included ydj1Delta, ssz1Delta and zuo1Delta mutants. In addition, new1Delta, whose function was unknown, also displayed significant sensitivity to anthracyclines. We further investigated the basis for the sensitivity of these mutants. We determined that heat-shock could partially rescue the sensitivity of the strains to doxorubicin, including the homologous recombination mutant rad52Delta, which is sensitive to doxorubicin-mediated DNA double strand breaks (DSBs). However, none of the heat-shock response mutants were sensitive to DSBs, but were highly sensitive to reactive oxygen species (ROS) generated by quinone-ring-containing agents, such as anthracyclines and Menadione. A fluorescent-based assay indicates that doxorubicin causes protein aggregation. Interestingly, the disaggregase mutant hsp104Delta is not sensitive to anthracyclines or Menadione suggesting that Hsp104p does not play a role in disaggregating doxorubicin-induced protein aggregates. However New1p, which has been recently shown to be a novel disaggregase, is essential for cell viability after exposure to anthracyclines and Menadione and it is not involved in thermotolerance. Our data suggest that in S. cerevisiae, doxorubicin produces protein aggregation through ROS and requires Ydj1p and New1p for resolution.

Glucose Acutely Reduces Cytosolic and Mitochondrial H2O2 in Rat Pancreatic Beta Cells.[Pubmed:29756464]

Antioxid Redox Signal. 2018 Jun 14.

AIMS: Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic beta cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 (reduction/oxidation-sensitive enhanced green fluorescent protein fused to oxidant receptor peroxidase 1) and HyPer (hydrogen peroxide sensor) with its pH-control SypHer, to test the acute effects of glucose, monomethyl succinate, leucine with glutamine, and alpha-ketoisocaproate on beta cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and Menadione concentrations on insulin secretion. RESULTS: RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10 muM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15 muM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase, or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5 mM glucose in the cytosol and 10 mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15 muM) did not affect insulin secretion. By contrast, Menadione (1-5 muM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 mM glucose. INNOVATION: Subcellular changes in beta cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in beta cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. CONCLUSION: The GSIS occurs independently of a detectable increase in beta cell cytosolic or mitochondrial H2O2 levels. Antioxid. Redox Signal. 00, 000-000.

Differential correlations between changes to glutathione redox state, protein ubiquitination, and stress-inducible HSPA chaperone expression after different types of oxidative stress.[Pubmed:29754332]

Cell Stress Chaperones. 2018 Sep;23(5):985-1002.

In primary bovine fibroblasts with an hspa1b/luciferase transgene, we examined the intensity of heat-shock response (HSR) following four types of oxidative stress or heat stress (HS), and its putative relationship with changes to different cell parameters, including reactive oxygen species (ROS), the redox status of the key molecules glutathione (GSH), NADP(H) NAD(H), and the post-translational protein modifications carbonylation, S-glutathionylation, and ubiquitination. We determined the sub-lethal condition generating the maximal luciferase activity and inducible HSPA protein level for treatments with hydrogen peroxide (H2O2), UVA-induced oxygen photo-activation, the superoxide-generating agent Menadione (MN), and diamide (DA), an electrophilic and sulfhydryl reagent. The level of HSR induced by oxidative stress was the highest after DA and MN, followed by UVA and H2O2 treatments, and was not correlated to the level of ROS production nor to the extent of protein S-glutathionylation or carbonylation observed immediately after stress. We found a correlation following oxidative treatments between HSR and the level of GSH/GSSG immediately after stress, and the increase in protein ubiquitination during the recovery period. Conversely, HS treatment, which led to the highest HSR level, did not generate ROS nor modified or depended on GSH redox state. Furthermore, the level of protein ubiquitination was maximum immediately after HS and lower than after MN and DA treatments thereafter. In these cells, heat-induced HSR was therefore clearly different from oxidative stress-induced HSR, in which conversely early redox changes of the major cellular thiol predicted the level of HSR and polyubiquinated proteins.

Inhibitory effects of drugs on the metabolic activity of mouse and human aldehyde oxidases and influence on drug-drug interactions.[Pubmed:29678521]

Biochem Pharmacol. 2018 Aug;154:28-38.

As aldehyde oxidase (AOX) plays an emerging role in drug metabolism, understanding its significance for drug-drug interactions (DDI) is important. Therefore, we tested 10 compounds for species-specific and substrate-dependent differences in the inhibitory effect of AOX activity using genetically engineered HEK293 cells over-expressing human AOX1, mouse AOX1 or mouse AOX3. The IC50 values of 10 potential inhibitors of the three AOX enzymes were determined using phthalazine and O(6)-benzylguanine as substrates. 17beta-Estradiol, Menadione, norharmane and raloxifene exhibited marked differences in inhibitory effects between the human and mouse AOX isoforms when the phthalazine substrate was used. Some of the compounds tested exhibited substrate-dependent differences in their inhibitory effects. Docking simulations with human AOX1 and mouse AOX3 were conducted for six representative inhibitors. The rank order of the minimum binding energy reflected the order of the corresponding IC50 values. We also evaluated the potential DDI between an AOX substrate (O(6)-benzylguanine) and an inhibitor (hydralazine) using chimeric mice with humanized livers. Pretreatment of hydralazine increased the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-24) of O(6)-benzylguanine compared to single administration. Our in vitro data indicate species-specific and substrate-dependent differences in the inhibitory effects on AOX activity. Our in vivo data demonstrate the existence of a DDI which may be of relevance in the clinical context.

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift.[Pubmed:29626097]

J Biol Chem. 2018 May 25;293(21):8032-8047.

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca(2+)-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1-10 mum), whereas higher levels (0.5-1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD(+) redox ratio and DeltaPsim in a concentration-dependent manner (10 mum to 1 mm H2O2), with maximal effects at 500 mum H2O2 H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 mum H2O2 However, higher H2O2 levels (>/=50 mum) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of DeltaPsim, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.

Daily melatonin protects the endothelial lineage and functional integrity against the aging process, oxidative stress, and toxic environment and restores blood flow in critical limb ischemia area in mice.[Pubmed:29570854]

J Pineal Res. 2018 Sep;65(2):e12489.

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide-induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that Menadione-induced oxidative stress (NOX-1/NOX-2), inflammation (TNF-alpha/NF-kB), apoptosis (cleaved caspase-3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3-siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C-kit/CD31+/Sca-1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow-derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age-match mice receiving melatonin (all P < .01). ED-induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide-induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging-, oxidative stress-, lipopolysaccharide-, and ischemia-induced damage probably through upregulating the SIRT signaling pathway.

Simultaneous evaluation of superoxide content and mitochondrial membrane potential in stallion semen samples provides additional information about sperm quality.[Pubmed:29622352]

Anim Reprod Sci. 2018 May;192:290-297.

An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoechst 33258 exclusion were measured and correlated to superoxide production and MMP. Both bivariate correlations between measured parameters as well as multivariate analysis were performed. Measured values in the superoxide/MMP assay did not correlate with other parameters. However, there was a strong negative correlation (r=0.96 after 0h, r=0.95 after 24h) between membrane integrity and chromatin integrity. Moderate positive correlations were found between motility parameters and membrane integrity, as well as moderate negative correlations between motility parameters and chromatin integrity. The multivariate analysis revealed that membrane integrity, chromatin integrity and motility contributed to the first principal component, while the second was influenced by superoxide/MMP parameters as well as sperm kinematics. Storage of samples for 24h decreased motility, chromatin integrity and membrane integrity. In conclusion, combined measurement of superoxide and MMP provides additional information not obtained by other assays of sperm quality.