Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway in cultured C2C12 cells.

Oxidative stress, an imbalance of redox homeostasis, contributes to the pathogenesis and progress of muscle atrophy. However, it is debated whether oxidative stress is a cause or consequence of muscle atrophy. In this study, we investigated the relationship between menadione-induced oxidative stress and serum starvation-induced muscle atrophy in C2C12 myotubes. We found that atrophic phenotypes including myotube diameter decrease, protein ubiquitination, and the expression of atrogenes were detected under oxidative stress as well as during serum starvation. Oxidative stress during serum starvation was assessed to confirm the correlation. Both intracellular reactive oxygen species (ROS) and protein oxidation were increased in atrophic myotubes. These results indicate that menadione-induced oxidative stress triggers muscle atrophy and vice versa. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular response to oxidative stress and it is considered to have a cytoprotective role in the mitigation of muscle atrophy. Transcription of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase-1, target genes of Nrf2, was decreased during serum starvation, which is related to decreased nuclear translocation of Nrf2. Pre-treatment of sulforaphane (SFN), a known Nrf2 inducer, before serum starvation showed a protective effect via Nrf2/HO-1 upregulation. SFN can liberate Nrf2 from Keap1, enabling the nuclear translocation of Nrf2. Consequently, the expression of HO-1 increased and intracellular ROS was significantly reduced by SFN pre-treatment. These results demonstrate that oxidative stress mediates the pathophysiology of muscle atrophy, which can be improved via upregulation of the Nrf2-mediated antioxidant response.

Neuronal Metabolism and Neuroprotection: Neuroprotective Effect of Fingolimod on Menadione-Induced Mitochondrial Damage.

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.

Protective effect of 1950 MHz electromagnetic field in human neuroblastoma cells challenged with menadione.

This study aims to assess whether a 1950 MHz radiofrequency (RF) electromagnetic field could protect human neuroblastoma SH-SY5Y cells against a subsequent treatment with menadione, a chemical agent inducing DNA damage via reactive oxygen species formation. Cells were pre-exposed for 20 h to specific absorption rate of either 0.3 or 1.25 W/kg, and 3 h after the end of the exposure, they were treated with 10 µM menadione (MD) for 1 h. No differences were observed between sham- and RF-exposed samples. A statistically significant reduction in menadione-induced DNA damage was detected in cells pre-exposed to either 0.3 or 1.25 W/kg (P < 0.05). Moreover, our analyses of gene expression revealed that the pre-exposure to RF almost inhibited the dramatic loss of glutathione peroxidase-based antioxidant scavenging efficiency that was induced by MD, and in parallel strongly enhanced the gene expression of catalase-based antioxidant protection. In addition, RF abolished the MD-dependent down-regulation of oxoguanine DNA glycosylase, which is a critical DNA repairing enzyme. Overall, our findings suggested that RF pre-exposure reduced menadione-dependent DNA oxidative damage, most probably by enhancing antioxidant scavenging efficiency and restoring DNA repair capability. Our results provided some insights into the molecular mechanisms underlying the RF-induced adaptive response in human neuroblastoma cells challenged with menadione.

Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells.

Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.

Mitochondrial reactive oxygen species production mediates ursolic acid-induced mitochondrial uncoupling and glutathione redox cycling, with protection against oxidant injury in H9c2 cells.

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is a ubiquitous compound widely distributed in many plants, fruits and medicinal herbs worldwide. A previous study in our laboratory has shown that UA can increase the mitochondrial ATP generation capacity (ATP-GC) and a glutathione-dependent antioxidant response, thereby protecting against oxidant injury in H9c2 cells in vitro and rat hearts ex vivo. However, the mechanism underlying the cellular protective effects induced by UA remains largely unknown. The present study has shown that pre-incubation with UA produces a transient increase in the mitochondrial membrane potential in H9c2 cells, which was accompanied by increases in mitochondrial reactive oxygen species (ROS) production. Studies using an antioxidant (dimethylthiourea) indicated that the suppression of mitochondrial ROS completely abrogated the UA-induced enhancement of mitochondrial uncoupling and glutathione reductase (GR)-mediated glutathione redox cycling, as well as protection against menadione cytotoxicity in H9c2 cells. Co-incubation with specific inhibitors of uncoupling proteins and GR almost completely prevented the cytoprotection afforded by UA against menadione-induced cytotoxicity in H9c2 cells. The results obtained so far suggest that UA-induced mitochondrial ROS production can elicit mitochondrial uncoupling and glutathione-dependent antioxidant responses, which offer cytoprotection against oxidant injury in H9c2 cells.

Responsiveness of entomopathogenic fungi to menadione-induced oxidative stress.

Entomopathogenic fungi are predisposed to ROS induced by heat and UV-A radiation when outside the insect host. When inside the host, they are subject to phagocytic cells that generate ROS to eliminate invading pathogens. The oxidative stress tolerance of the entomopathogenic fungi Aschersonia aleyrodis (ARSEF 430 and 10276), Aschersonia placenta (ARSEF 7637), Beauveria bassiana (ARSEF 252), Isaria fumosorosea (ARSEF 3889), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium acridum (ARSEF 324), Metarhizium anisopliae (ARSEF 5749), Metarhizium brunneum (ARSEF 1187 and ARSEF 5626), Metarhizium robertsii (ARSEF 2575), Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), and Simplicillium lanosoniveum (ARSEF 6430 and ARSEF 6651) was studied based on conidial germination on a medium supplemented with menadione. Conidial germination was evaluated 24 h after inoculation on potato dextrose agar (PDA) (control) or PDA supplemented with menadione. The two Aschersonia species (ARSEF 430, 7637, and 10276) were the most susceptible fungi, followed by the two Tolypocladium species (ARSEF 3392 and 4877) and the M. acridum (ARSEF 324). Metarhizium brunneum (ARSEF 5626) and M. anisopliae (ARSEF 5749) were the most tolerant isolates with MIC 0.28 mM. All fungal isolates, except ARSEF 5626 and ARSEF 5749, were not able to germinate at 0.20 mM.

Effects of quercetin and menadione on intestinal calcium absorption and the underlying mechanisms.

Quercetin (QT) could be considered as a potential therapeutic agent for different diseases due to its antioxidant, anti-inflammatory, antiviral and anticancer properties. This study was designed to investigate the ability of QT to protect the chick intestine against menadione (MEN) induced injury in vivo and in vitro. Four-week old chicks (Gallus gallus) were treated i.p. with 2.5µmol of MEN/kg b.w. or with i.l. 50µM QT or both. QT protected the intestinal Ca(2+) absorption against the inhibition caused by MEN, but QT alone did not modify. Glutathione (GSH) depletion provoked by MEN in chick enterocytes was abolished by QT treatment, whereas QT alone did not modify the intestinal GSH content. The enhancement of GSH peroxidase activity produced by MEN was blocked by QT treatment. In contrast, superoxide dismutase activity remained high after simultaneous treatment of enterocytes with MEN and QT. The flavonol also avoided changes in the mitochondrial membrane permeability (swelling) produced by MEN. The FasL/Fas/caspase-3 pathway was activated by MEN, effect that was abrogated by QT. In conclusion, QT may be useful in preventing inhibition of chick intestinal Ca(2+) absorption caused by MEN or other substances that deplete GSH, by blocking the oxidative stress and the FasL/Fas/caspase-3 pathway activation.

Genome-wide association for sensitivity to chronic oxidative stress in Drosophila melanogaster.

Reactive oxygen species (ROS) are a common byproduct of mitochondrial energy metabolism, and can also be induced by exogenous sources, including UV light, radiation, and environmental toxins. ROS generation is essential for maintaining homeostasis by triggering cellular signaling pathways and host defense mechanisms. However, an imbalance of ROS induces oxidative stress and cellular death and is associated with human disease, including age-related locomotor impairment. To identify genes affecting sensitivity and resistance to ROS-induced locomotor decline, we assessed locomotion of aged flies of the sequenced, wild-derived lines from the Drosophila melanogaster Genetics Reference Panel on standard medium and following chronic exposure to medium supplemented with 3 mM menadione sodium bisulfite (MSB). We found substantial genetic variation in sensitivity to oxidative stress with respect to locomotor phenotypes. We performed genome-wide association analyses to identify candidate genes associated with variation in sensitivity to ROS-induced decline in locomotor performance, and confirmed the effects for 13 of 16 mutations tested in these candidate genes. Candidate genes associated with variation in sensitivity to MSB-induced oxidative stress form networks of genes involved in neural development, immunity, and signal transduction. Many of these genes have human orthologs, highlighting the utility of genome-wide association in Drosophila for studying complex human disease.

Genome-wide association analysis of oxidative stress resistance in Drosophila melanogaster.

BACKGROUND: Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress. METHODS AND FINDINGS: We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67-79% and 56-66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis. CONCLUSIONS: We identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease.

Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death.

Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

Investigations in pet birds (Agapornis spp.) fed different vitamin K contents in the diet.

In the past many discussions about possible toxic effects of vitamin K(3) fed to pet birds arose frequently, and were published also in magazines for pet bird fanciers, in the internet as well as in veterinary journals. Therefore, the aim of this study was an evaluation of effects of different dosages of vitamin K(3) on birds' health when given orally for a longer period. These investigations were carried out with adult lovebirds (Agapornis spp.) fed a pelleted diet with different levels of vitamin K(3) (menadione-sodium-bisulphite): control group 0 mg, group V1 20 mg and group V2 200 mg/kg diet dry matter. General condition and well being of the lovebirds were checked daily. Body weight gains as well as feed and water intakes were examined once a week. Every 2-month blood samples of each lovebird were collected and analysed. After a period of 6 and 10 months, respectively, four birds of each group were necropsied in order to carry out a pathological and histological examination. In general, the behaviour, feed and water intakes as well as quality of excreta were not influenced by ingestion of diets with different levels of vitamin K(3). All variations were in physiological ranges. Individuals of all groups showed positive body weight gains and an active reproduction status. However, the best body mass (BM) development and egg laying activity could be observed in lovebirds of group V2 with the highest vitamin K(3) supplementation. In the haemotogram some time-depending variations could be observed; however, a systematic influence of vitamin K(3) could not be determined in any group or at any time. All analysed biochemical values in plasma and the activities of enzymes were within normal ranges. Only few birds of every group showed aberrant histological findings, but none of these could be related to the vitamin K(3) intake. Moreover, no forced accumulation of vitamin K(3) in the liver depending on vitamin K(3) intake was found. This result suggests a rapid metabolism of the absorbed vitamin K(3). All in all, the application of pelleted diets with addition of 20 or 200 mg vitamin K(3)/kg diet over a period of several months did not affect pet birds' health. Given these results, any doubts about the compatibility of usual doses of vitamin K(3) in diets for lovebirds must be considered as absolutely groundless.