Using Sensors and Generators of H2O2 to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

Aims: Chemotherapeutics target vital functions that ensure survival of cancer cells, including their increased reliance on defense mechanisms against oxidative stress compared to normal cells. Many chemotherapeutics exploit this vulnerability to oxidative stress by elevating the levels of intracellular reactive oxygen species (ROS). A quantitative understanding of the oxidants generated and how they induce toxicity will be important for effective implementation and design of future chemotherapeutics. Molecular tools that facilitate measurement and manipulation of individual chemical species within the context of the larger intracellular redox network present a means to develop this understanding. In this work, we demonstrate the use of such tools to elucidate the roles of H[subscript 2]O[subscript 2] and glutathione (GSH) in the toxicity mechanism of two ROS-based chemotherapeutics, piperlongumine and phenethyl isothiocyanate. Results: Depletion of GSH as a result of treatment with these compounds is not an important part of the toxicity mechanisms of these drugs and does not lead to an increase in the intracellular H[subscript 2]O[subscript 2] level. Measuring peroxiredoxin-2 (Prx-2) oxidation as evidence of increased H[subscript 2]O[subscript 2], only piperlongumine treatment shows elevation and it is GSH independent. Using a combination of a sensor (HyPer) along with a generator (D-amino acid oxidase) to monitor and mimic the drug-induced H[subscript 2O[subscript 2] production, it is determined that H[subscript 2]O[subscript 2] produced during piperlongumine treatment acts synergistically with the compound to cause enhanced cysteine oxidation and subsequent toxicity. The importance of H[subscript 2]O[subscript 2] elevation in the mechanism of piperlongumine promotes a hypothesis of why certain cells, such as A549, are more resistant to the drug than others. Innovation and Conclusion: The approach described herein sheds new light on the previously proposed mechanism of these two ROS-based chemotherapeutics and advocates for the use of both sensors and generators of specific oxidants to isolate their effects. Antioxid. Redox Signal. 24, 924–938.National Science Foundation (U.S.). Graduate Research Fellowship ProgramBurroughs Wellcome Fund (Career Award at the Scientific Interface

γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor

Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine—the immediate glutathione precursor—to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor

Development of an invasively monitored porcine model of acetaminophen-induced acute liver failure

Background: The development of effective therapies for acute liver failure (ALF) is limited by our knowledge of the pathophysiology of this condition, and the lack of suitable large animal models of acetaminophen toxicity. Our aim was to develop a reproducible invasively-monitored porcine model of acetaminophen-induced ALF. Method: 35kg pigs were maintained under general anaesthesia and invasively monitored. Control pigs received a saline infusion, whereas ALF pigs received acetaminophen intravenously for 12 hours to maintain blood concentrations between 200-300 mg/l. Animals surviving 28 hours were euthanased. Results: Cytochrome p450 levels in phenobarbital pre-treated animals were significantly higher than non pre-treated animals (300 vs 100 pmol/mg protein). Control pigs (n=4) survived 28-hour anaesthesia without incident. Of nine pigs that received acetaminophen, four survived 20 hours and two survived 28 hours. Injured animals developed hypotension (mean arterial pressure; 40.8+/-5.9 vs 59+/-2.0 mmHg), increased cardiac output (7.26+/-1.86 vs 3.30+/-0.40 l/min) and decreased systemic vascular resistance (8.48+/-2.75 vs 16.2+/-1.76 mPa/s/m3). Dyspnoea developed as liver injury progressed and the increased pulmonary vascular resistance (636+/-95 vs 301+/-26.9 mPa/s/m3) observed may reflect the development of respiratory distress syndrome. Liver damage was confirmed by deterioration in pH (7.23+/-0.05 vs 7.45+/-0.02) and prothrombin time (36+/-2 vs 8.9+/-0.3 seconds) compared with controls. Factor V and VII levels were reduced to 9.3 and 15.5% of starting values in injured animals. A marked increase in serum AST (471.5+/-210 vs 42+/-8.14) coincided with a marked reduction in serum albumin (11.5+/-1.71 vs 25+/-1 g/dL) in injured animals. Animals displayed evidence of renal impairment; mean creatinine levels 280.2+/-36.5 vs 131.6+/-9.33 mumol/l. Liver histology revealed evidence of severe centrilobular necrosis with coagulative necrosis. Marked renal tubular necrosis was also seen. Methaemoglobin levels did not rise >5%. Intracranial hypertension was not seen (ICP monitoring), but there was biochemical evidence of encephalopathy by the reduction of Fischer's ratio from 5.6 +/- 1.1 to 0.45 +/- 0.06. Conclusion: We have developed a reproducible large animal model of acetaminophen-induced liver failure, which allows in-depth investigation of the pathophysiological basis of this condition. Furthermore, this represents an important large animal model for testing artificial liver support systems

Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.We thank T. Jacks (Kras^LSL-G12D), A. Berns (p53^Fx) and the NIH Mouse repository for mice. We also thank Sam Kleeman and Patricia Ogger for assistance with redox cell profiling and cell viability assays, respectively. We are very thankful to CRUK CI BRU staff for support with in vivo work and all the members of the Martins lab for critical comments and advice. This work was supported by the Medical Research Council.This is the author accepted manuscript. The final version is available at http://www.nature.com/nature/journal/v531/n7592/full/nature16967.html

Effects of Long-Term Space Flight on Erythrocytes and Oxidative Stress of Rodents

Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions; these observations have been designated as “space anemia”. Erythrocytes exposed to microgravity have a modified rheology and undergo hemolysis to a greater extent. Cell membrane composition plays an important role in determining erythrocyte resistance to mechanical stress and it is well known that membrane composition might be influenced by external events, such as hypothermia, hypoxia or gravitational strength variations. Moreover, an altered cell membrane composition, in particular in fatty acids, can cause a greater sensitivity to peroxidative stress, with increase in membrane fragility. Solar radiation or low wavelength electromagnetic radiations (such as gamma rays) from the Earth or the space environment can split water to generate the hydroxyl radical, very reactive at the site of its formation, which can initiate chain reactions leading to lipid peroxidation. These reactive free radicals can react with the non-radical molecules, leading to oxidative damage of lipids, proteins and DNA, etiologically associated with various diseases and morbidities such as cancer, cell degeneration, and inflammation. Indeed, radiation constitutes on of the most important hazard for humans during long-term space flights. With this background, we participated to the MDS tissue-sharing program performing analyses on mice erythrocytes flown on the ISS from August to November 2009. Our results indicate that space flight induced modifications in cell membrane composition and increase of lipid peroxidation products, in mouse erythrocytes. Moreover, antioxidant defenses in the flight erythrocytes were induced, with a significant increase of glutathione content as compared to both vivarium and ground control erythrocytes. Nonetheless, this induction was not sufficient to prevent damages caused by oxidative stress. Future experiments should provide information helpful to reduce the effects of oxidative stress exposure and space anemia, possibly by integrating appropriate dietary elements and natural compounds that could act as antioxidants

Role of oxidative stress and intracellular glutathione in the sensitivity to apoptosis induced by proteasome inhibitor in thyroid cancer cells

<p>Abstract</p> <p>Background</p> <p>The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the treatment of multiple myeloma (MM) and some solid cancers. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance are poorly understood.</p> <p>Methods</p> <p>Proteasome activity, intracellular glutathione (GSH) and ROS levels, as well as activities of GSH synthesis enzymes were measured using spectrophotometric methods. Cell death was analyzed using flow cytometry and caspase activity assay. The expression level of GSH synthesis enzymes were measured using real-time RT-PCR.</p> <p>Results</p> <p>At concentrations that effectively inhibited proteasome activity, bortezomib induced apoptosis in FRO cells, but not in ARO cells. Bortezomib elevated the amount of glutathione (GSH) and the treatment with bortezomib increased the level of mRNA for GCL, a rate-limiting enzyme in glutathione synthesis. Furthermore, depletion of GSH increases apoptosis induced by bortezomib, in contrast, repletion of GSH decreases bortezomib-mediated cell death.</p> <p>Conclusion</p> <p>GSH protects cells from proteasome inhibition-induced oxidative stress and glutathione-dependent redox system might play an important role in the sensitivity to proteasome inhibition-induced apoptosis.</p

Experimental Assessment of the Role of Acetaldehyde in Alcoholic Cardiomyopathy

Alcoholism is one of the major causes of non-ischemic heart damage. The myopathic state of the heart due to alcohol consumption, namely alcoholic cardiomyopathy, is manifested by cardiac hypertrophy, compromised ventricular contractility and cardiac output. Several mechanisms have been postulated for alcoholic cardiomyopathy including oxidative damage, accumulation of triglycerides, altered fatty acid extraction, decreased myofilament Ca(2+ )sensitivity, and impaired protein synthesis. Despite intensive efforts to unveil the mechanism and ultimate toxin responsible for alcohol-induced cardiac toxicity, neither has been clarified thus far. Primary candidates for the specific toxins are ethanol, its first and major metabolic product - acetaldehyde (ACA) and fatty acid ethyl esters. Evidence from our lab suggests that ACA directly impairs cardiac function and promotes lipid peroxidation resulting in oxidative damage. The ACA-induced cardiac contractile depression may be reconciled with inhibitors of Cytochrome P-450 oxidase, xanthine oxidase and lipid peroxidation Unfortunately, the common methods to investigate the toxicity of ACA have been hampered by the fact that direct intake of ACA is toxic and unsuitable for chronic study, which is unable to provide direct evidence of direct cardiac toxicity for ACA. In order to overcome this obstacle associated with the chemical properties of ACA, our laboratory has used the chronic ethanol feeding model in transgenic mice with cardiac over-expression of alcohol dehydrogenase (ADH) and an in vitro ventricular myocyte culture model. The combination of both in vivo and in vitro approaches allows us to evaluate the role of ACA in ethanol-induced cardiac toxicity and certain cellular signaling pathways leading to alcoholic cardiomyopathy

Glucose-6-Phosphate Dehydrogenase Protects Escherichia coli from Tellurite-Mediated Oxidative Stress

The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress

Evaluation of the functional efficacy of an antioxidative probiotic in healthy volunteers

BACKGROUND: In persons without clinical symptom it is difficult to assess an impact of probiotics regarding its effect on health. We evaluated the functional efficacy of the probiotic Lactobacillus fermentum ME-3 in healthy volunteers by measuring the influence of two different formulations on intestinal lactoflora, fecal recovery of the probiotic strain and oxidative stress markers of blood and urine after 3 weeks consumption. METHODS: Two 3-week healthy volunteer trials were performed. Open placebo controlled (OPC) study participants (n = 21) consumed either goat milk or by L. fermentum ME-3 fermented goat milk (daily dose 11.8 log CFU (Colony Forming Units). Double blind randomised placebo controlled (DBRP) study participants (n = 24) received either capsules with L. fermentum ME-3 (daily of dose 9.2 CFU) or placebo capsules. The faecal lactoflora composition, faecal ME-3 recovery, effect of the consumption on intestinal lactoflora, and oxidative stress markers of blood (total antioxidative activity; total antioxidative status and glutathione red-ox ratio) was measured. RESULTS: ME-3 was well tolerated and a significant increase in total faecal lactobacilli yet no predominance of ME-3 was detected in all study groups. Faecal recovery of ME-3 was documented by molecular methods only in fermented milk group, however the significant improvement of blood TAA (Total Antioxidative Activity) and TAS (Total Antioxidative Status) indices was seen both in case of fermented goat milk and capsules", yet glutathione re-ox ratio values decreased only in case of fermented by ME-3 goat milk. CONCLUSION: The functional efficacy of both consumed formulations of an antioxidative probiotic L. fermentum ME-3 is proved by the increase of the intestinal lactobacilli counts providing putative defence against enteric infections and by reduction of the oxidative stress indices of blood and urine of healthy volunteers. In non-diseased host the probiotic health claims can be assessed by improvement of some measurable laboratory indices of well-established physiological functions of host, e.g. markers of antioxidative defence system