Altered Cellular Redox Status Research Articles

Redox state and altered pyruvate metabolism contribute to a dose-dependent metformin-induced lactate production of human myotubes.

Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 μM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, p < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, p < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, p < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (p < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state, shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.

Abstract 405: A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance by macropinocytosis-mediated internalization and redox changes

Abstract Drug resistance are responsible for most relapses and up to 90% of cancer related death. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. In tumor microenvironment, intratumoral extracellular ATP (eATP) levels are 1,000 times or more higher than those in corresponding normal tissues. Our previous studies have shown that eATP can be taken up by cancer cell via macropinocytosis, leading to substantial increase in intracellular ATP levels. We found that this high intracellular ATP levels contribute to drug resistance in several ways: first, it directly fueled and enhanced drug-pumping activity of major ATP-binding cassette (ABC) multidrug transporters, which are located in plasma membrane and can pump out a wide range of substrates including anticancer drugs, leading to reduced intracellular drug retention. Secondly, ATP treatment upregulated gene expression of the major ABC transporters, and this gene regulatory effect of ATP was comparable to TGF-beta, which is a well-established epithelial-mesenchymal transition (EMT) inducer, and EMT is known to induce ABC transporter expression and drug resistance. Moreover, eATP treatment led to an increase in intracellular glutathione (GSH) levels, probably by enhancing GSH synthesis. Increased GSH levels have been described to significantly contribute to drug resistance. We also found eATP altered cellular redox state, which potentially stimulates EMT. Furthermore, we performed RNA-sequencing and identified several genes that may serve as downstream factors of eATP in promoting EMT and drug resistance. We are currently investigating the roles of these genes by knocking down gene expression or knocking out these genes. Citation Format: Haiyun Zhang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance by macropinocytosis-mediated internalization and redox changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 405.

A Clinical Perspective of the Multifaceted Mechanism of Metformin in Diabetes, Infections, Cognitive Dysfunction, and Cancer.

In type 2 diabetes, ecological and lifecourse factors may interact with the host microbiota to influence expression of his/her genomes causing perturbation of interconnecting biological pathways with diverse clinical course. Metformin is a plant-based or plant-derived medicinal product used for the treatment of type 2 diabetes for over 60 years and is an essential drug listed by the World Health Organization. By reducing mitochondrial oxidative phosphorylation and adenosine triphosphate (ATP) production, metformin increased AMP (adenosine monophosphate)-activated protein kinase (AMPK) activity and altered cellular redox state with reduced glucagon activity, endogenous glucose production, lipogenesis, and protein synthesis. Metformin modulated immune response by directly reducing neutrophil to lymphocyte ratio and improving the phagocytic function of immune cells. By increasing the relative abundance of mucin-producing and short-chain-fatty-acid-producing gut microbes, metformin further improved the host inflammatory and metabolic milieu. Experimentally, metformin promoted apoptosis and reduced proliferation of cancer cells by reducing their oxygen consumption and modulating the microenvironment. Both clinical and mechanistic studies support the pluripotent effects of metformin on reducing cardiovascular–renal events, infection, cancer, cognitive dysfunction, and all-cause death in type 2 diabetes, making this low-cost medication a fundamental therapy for individualization of other glucose-lowering drugs in type 2 diabetes. Further research into the effects of metformin on cognitive function, infection and cancer, especially in people without diabetes, will provide new insights into the therapeutic value of metformin in our pursuit of prevention and treatment of ageing-related as well as acute and chronic diseases beyond diabetes.

What Role do Mitochondria Have in Diastolic Dysfunction? Implications for Diabetic Cardiomyopathy and Heart Failure With Preserved Ejection Function.

Diastolic dysfunction is common to both diabetic cardiomyopathy and heart failure with preserved ejection fraction (HFpEF). Although commonly attributed to increased fibrosis, alterations in mitochondrial function and associated Ca2+ handling may contribute to impaired cardiac function. With mitochondrial dysfunction, increased reactive oxygen species (ROS), inflammation, and decreased adenosine triphosphate/adenosine diphosphate ratio may lead to increased extracellular matrix and diminished contractile relaxation. In this article, we discuss recent research implicating deficient mitochondria-associated endoplasmic reticulum membranes (MAMs) as it relates to impaired metabolic function and what role that may have in diastolic dysfunction in diabetic cardiomyopathy. The contribution of mitochondrial dysfunction to diastolic dysfunction in HFpEF is less established, but equally credible based on preclinical studies. However, there are notable differences between diabetic cardiomyopathy and HFpEF. Recent evidence implicates impaired endoplasmic reticulum signaling, in particular the unfolded protein response (UPR), in the pathogenesis of HFpEF. With HFpEF, enhanced pressure on the mitochondrial "gas pedal" because of increased cytosolic Ca2+ may perturb mitochondrial homeostasis. For diabetic cardiomyopathy and HFpEF, a better understanding of how altered cellular ion and redox status affect mitochondrial function is needed. Finally, we discuss the implications that mitochondrial dysfunction may have in devising novel therapeutic strategies for diabetic cardiomyopathy and HFpEF.

Metformin: A Growing Journey from Glycemic Control to the Treatment of Alzheimer's Disease and Depression.

Metabolic stress, transduced as an altered cellular redox and energy status, presents as the main culprit in many diseases, including diabetes. However, its role in the pathology of neurological disorders is still not fully elucidated. Metformin, a biguanide compound, is an FDA approved antidiabetic drug generally used for the treatment of type 2 diabetes. The recently described wide spectrum of action executed by this drug suggests a potential therapeutic benefit in a panoply of disorders. Current studies imply that metformin could play a neuroprotective role by reversing hallmarks of brain injury (metabolic dysfunction, neuronal dystrophy and cellular loss), in addition to cognitive and behavioral alterations that accompany the onset of certain brain diseases such as Alzheimer's disease (AD) and depression. However, the mechanisms by which metformin exerts its protective effect in neurodegenerative disorders are not yet fully elucidated. The aim of this review is to reexamine the mechanisms through which metformin performs its function while concentrating on its effect on reestablishing homeostasis in a metabolically disturbed milieu. We will also highlight the importance of metabolic stress, not only as a component of many neurological disorders, but also as a primary driving force for neural insult. Of interest, we will explore the involvement of metabolic stress in the pathobiology of AD and depression. The derangement in major metabolic pathways, including AMPK, insulin and glucose transporters, will be explored and the potential therapeutic effects of metformin administration on the reversal of brain injury in such metabolism dependent diseases will be exposed.

Ascorbate sensitizes human osteosarcoma cells to the cytostatic effects of cisplatin.

Osteosarcoma (OS) is the most common malignant bone tumor and a leading cause of cancer‐related deaths in children and adolescents. Current standard treatments for OS are a combination of preoperative chemotherapy, surgical resection, and adjuvant chemotherapy. Cisplatin is used as the standard chemotherapeutic for OS treatment, but it induces various adverse effects, limiting its clinical application. Improving treatment efficacy without increasing the cisplatin dosage is desirable. In the present study, we assessed the combined effect of ascorbate on cisplatin treatment using cultured human OS cells. Co‐treatment with ascorbate induced greater suppression of OS cell but not nonmalignant cell proliferation. The chemosensitizing effect of ascorbate on cisplatin treatment was tightly linked to ROS production. Altered cellular redox state due to increased ROS production modified glycolysis and mitochondrial function in OS cells. In addition, OS cell sphere formation was markedly decreased, suggesting that ascorbate increased the treatment efficacy of cisplatin against stem‐like cells in the cancer cell population. We also found that enhanced MYC signaling, ribosomal biogenesis, glycolysis, and mitochondrial respiration are key signatures in OS cells with cisplatin resistance. Furthermore, cisplatin resistance was reversed by ascorbate. Taken together, our findings provide a rationale for combining cisplatin with ascorbate in therapeutic strategies against OS.

Conserved redox regulation of kinases

Biochemistry The effects of pathological oxidative stress are partially mediated through functional modification of various proteins. A pair of papers show that the oxidation of kinases at evolutionarily conserved cysteine residues regulates cell metabolism and mitosis. Shrestha et al. found that oxidation of the diabetes-associated metabolic kinase FN3K promoted its functional oligomerization and altered cellular redox status. Byrne et al. found that oxidation of mitotic kinases in human cells and yeast suppressed kinase catalytic activity and impaired cellular division in yeast. Sci. Transl. Med. 12 , eaaz6313, eaax2713 (2020).

Chronic dietary administration of lower levels of diethyl phthalate induces murine testicular germ cell inflammation and sperm pathologies: Involvement of oxidative stress

The wide occurrence of male infertility is a matter of grave concern. One of the major causes being exposure to endocrine disrupting chemicals (EDCs) many of which are known reproductive toxicants but the molecular mechanisms of action remain much unexplored. Diethyl phthalate (DEP) is ubiquitous in the environment due to its extensive use as plasticizer in myriad consumer products. In the present study, we sought to find out whether chronic DEP exposure affects reproductive function in sexually mature adult male mice. For this, 8-week old Swiss albino mice were treated with DEP (1 mg and 10 mg kg−1 body weight day−1) in diet for three months, mirroring the relevant doses of human exposure, and various analyses were carried out in the testicular germ cells and epididymal spermatozoa. We found that altered testicular histoarchitecture was accompanied with disturbed prooxidant: antioxidant balance in the germ cells. Involvement of Nrf2-HO-1 pathway was crucial in this altered cellular redox state. Besides, NFκB mediated inflammatory response was triggered in the germ cells leading to enhanced levels of proinflammatory cytokines. DEP adversely affected sperm count, motility, viability and morphology. Numerous structural anomalies were found in DEP treated mice spermatozoa reflecting decline in sperm function. Our results revealed overactivation of PARP-1 and subsequent cleavage in spermatozoa with induction of apoptosis as a key mechanism in DEP mediated sperm pathology. Given the indiscriminate use of plasticizers and long term low level human exposure, the present study highlights the undesirable male reproductive outcomes following chronic DEP exposure.

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b+ mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation.

KIF1B\u03b2 increases ROS to mediate apoptosis and reinforces its protein expression through O2\u2212 in a positive feedback mechanism in neuroblastoma

Relapse-prone, poor prognosis neuroblastoma is frequently characterized by deletion of chr1p36 where tumor suppressor gene KIF1Bβ resides. Interestingly, many 1p36-positive patients failed to express KIF1Bβ protein. Since altered cellular redox status has been reported to be involved in cell death and protein modification, we investigated the relationship between reactive oxygen species (ROS) and KIF1Bβ. Here, we showed that wild-type KIF1Bβ protein expression positively correlates with superoxide (O2−) and total ROS levels in neuroblastoma cells, unlike apoptotic loss-of-function KIF1Bβ mutants. Overexpression of KIF1Bβ apoptotic domain variants increases total ROS and, specifically O2−, whereas knockdown of endogenous KIF1Bβ decreases ROS and O2−. Interestingly, O2− increases KIF1Bβ protein expression, independent of the proteasomal degradation pathway. Scavenging O2− or ROS decreases KIF1Bβ protein expression and subsequent apoptosis. Moreover, treatment with investigational redox compound Gliotoxin increases O2−, KIF1Bβ protein expression, apoptosis and colony formation inhibition. Overall, our findings suggest that ROS and O2− may be important downstream effectors of KIF1Bβ-mediated apoptosis. Subsequently, O2− produced may increase KIF1Bβ protein expression in a positive feedback mechanism. Therefore, ROS and, specifically O2−, may be critical regulators of KIF1Bβ-mediated apoptosis and its protein expression in neuroblastoma.

Abstract B20: Modulation of a cancer lipogenic phenotype by dietary n-6/n-3 fatty acids in rat liver and colon cancer models

Abstract Cancer development is a multistage process involving a breakdown of controlled processes such as cell differentiation, proliferation and apoptosis and regulatory feed-back mechanisms resulting in the characteristic rapid growth and metastases of cancer cells. The deregulation of cell growth and survival in cancer development has been associated with a distinct lipogenic phenotype involving cholesterol, phospholipids and fatty acids (FA). This phenotype is associated with changes in membrane structure and cellular oxidative status resulting in changes to membrane function, activity of enzymes and signal transduction pathways. Dietary components are associated with a protective effect against the development of certain cancers. Amongst these, dietary fat has been linked to either the promotion or prevention of cancer involving various biological mechanisms, depending on the type of fat and its FA content. The present study characterized the lipogenic phenotype in the liver and colon utilizing rat cancer models and the resultant modulation of membrane lipid components by dietary oils with specific fatty acid content. Sunflower oil (S) in combination with borage oil (B) and/or fish oil (F) was used to generate different n-6/n-3 FA ratios: SB (n-6/n-3: 38:1), SF (n-6/n-3: 13:1) and SBF (n-6/n-3: 10:1), while canola and olive oil were included as individual oil sources. In separate colon and liver in vivo cancer models, rats were fed the diets in respective groups and terminated whereupon liver and colon cancerous and respective adjacent normal tissue samples were collected for lipid and oxidative PCR micro-array analyses. The characteristic lipogenic phenotype associated with the growth and development of preneoplastic lesions involved a decrease in the phospholipid phosphatidylcholine / phosphatidylethanolamine (PC/PE) ratio, C20:4n-6 PC/PE ratio, n-3 polyunsaturated FA (PUFA) content and oxidative status. An interactive role of saturated FA, monounsaturated FA and C20:4n-6 appear to be a driving force sustaining altered growth characteristics in these lesions with a diet high in n-6 PUFA playing an underlying role. This characteristic phenotype was modulated by the dietary oils with varying n-6/n-3 FA ratios resulting in an increased n-3 PUFA tissue content, a higher lipid peroxidation level and altered growth of preneoplastic lesions. Depending on the specific stage of cancer development, dietary oil may either inhibit or stimulate the growth of preneoplastic lesions which appear to be related to differential changes in proliferative cell survival responses associated with an altered cellular redox status. Challenges in utilizing fatty acids in cancer modulation exist not only with regards to dietary sources and intake but also the effect on cell structure and modulation of signal transduction pathways governing altered growth responses in the prevention of cancer development. Therefore, the modulation of the lipid profile and oxidative status through dietary PUFA with a specific n-6/n-3 ratio may be an important chemopreventive tool in altering the growth characteristics of cancer cells. Citation Format: Stefan Abel, Celeste H. Abrahams, Maryna De Kock, Wentzel CA Gelderblom. Modulation of a cancer lipogenic phenotype by dietary n-6/n-3 fatty acids in rat liver and colon cancer models [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B20.

Resveratrol stimulates mitochondrial fusion by a mechanism requiring mitofusin-2

Resveratrol (RES) is a plant-derived stilbene associated with a wide range of health benefits. Mitochondria are a key downstream target of RES, and in some cell types RES promotes mitochondrial biogenesis, altered cellular redox status, and a shift toward oxidative metabolism. Mitochondria exist as a dynamic network that continually remodels via fusion and fission processes, and the extent of fusion is related to cellular redox status and metabolism. We investigated RES's effects on mitochondrial network morphology in several cell lines using a quantitative approach to measure the extent of network fusion. 48 h continuous treatment with 10–20 μM RES stimulated mitochondrial fusion in C2C12 myoblasts, PC3 cancer cells, and mouse embryonic fibroblasts stimulated significant increases in fusion in all instances, resulting in larger and more highly branched mitochondrial networks. Mitofusin-2 (Mfn2) is a key protein facilitating mitochondrial fusion, and its expression was also stimulated by RES. Using Mfn2-null cells we demonstrated that RES's effects on mitochondrial fusion, cellular respiration rates, and cell growth are all dependent upon the presence of Mfn2. Taken together, these results demonstrate that Mfn2 and mitochondrial fusion are affected by RES in ways that appear to relate to RES's known effects on cellular metabolism and growth.

Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices

Some of the main electron carriers in the metabolism are mono- or dinucleotides and they play crucial roles in maintaining a balanced redox homeostasis of cells, and in coupling many anabolic and catabolic reactions. Altered cellular redox status can be an indicator of various metabolic disorders such as obesity, the metabolic syndrome, or type 2 diabetes and of other pathological conditions, which involve oxidative stress, such as cardiovascular diseases. Adequate NAD + /NADH and NADP + /NADPH ratios are fundamental for normal cellular functions, thus accurate measurement of these pyridine dinucleotides is essential in biochemical research. Liquid chromatography coupled to tandem mass spectrometry has become the leading analytical technology in (targeted) state-of-the-art metabolic profiling. Main difficulties that hamper quantification of metabolites are chemical similarities, high polarity, and chemical and biological instability of the molecules to be measured. In this review, some critical steps of studying cellular redox status are described, in particular, different techniques of sample preparation and challenges in chromatographic separation.

Proline Coordination with Fatty Acid Synthesis and Redox Metabolism of Chloroplast and Mitochondria.

Proline (Pro) accumulation is one of the most prominent changes in plant metabolism during drought and low water potential; however, the regulation and function of Pro metabolism remain unclear. We used a combination of forward genetic screening based on a Proline Dehydrogenase1 (PDH1) promoter-luciferase reporter (PDH1pro:LUC2) and RNA sequencing of the Pro synthesis mutant p5cs1-4 to identify multiple loci affecting Pro accumulation in Arabidopsis (Arabidopsis thaliana). Two mutants having high PDH1pro:LUC2 expression and increased Pro accumulation at low water potential were found to be alleles of Cytochrome P450, Family 86, Subfamily A, Polypeptide2 (CYP86A2) and Long Chain Acyl Synthetase2 (LACS2), which catalyze two successive steps in very-long-chain fatty acid (VLCFA) synthesis. Reverse genetic experiments found additional VLCFA and lipid metabolism-related mutants with increased Pro accumulation. Altered cellular redox status is a key factor in the coordination of Pro and VLCFA metabolism. The NADPH oxidase inhibitor diphenyleneiodonium (DPI) induced high levels of Pro accumulation and strongly repressed PDH1pro:LUC2 expression. cyp86a2 and lacs2 mutants were hypersensitive to diphenyleneiodonium but could be reverted to wild-type Pro and PDH1pro:LUC2 expression by reactive oxygen species scavengers. The coordination of Pro and redox metabolism also was indicated by the altered expression of chloroplast and mitochondria electron transport genes in p5cs1-4 These results show that Pro metabolism is both influenced by and influences cellular redox status via previously unknown coordination with several metabolic pathways. In particular, Pro and VLCFA synthesis share dual roles to help buffer cellular redox status while producing products useful for stress resistance, namely the compatible solute Pro and cuticle lipids.

Altered cellular redox status, sirtuin abundance and clock gene expression in a mouse model of developmentally primed NASH

BackgroundWe have previously shown that high fat (HF) feeding during pregnancy primes the development of non-alcoholic steatohepatits (NASH) in the adult offspring. However, the underlying mechanisms are unclear. AimsSince the endogenous molecular clock can regulate hepatic lipid metabolism, we investigated whether exposure to a HF diet during development could alter hepatic clock gene expression and contribute to NASH onset in later life. MethodsFemale mice were fed either a control (C, 7%kcal fat) or HF (45%kcal fat) diet. Offspring were fed either a C or HF diet resulting in four offspring groups: C/C, C/HF, HF/C and HF/HF. NAFLD progression, cellular redox status, sirtuin expression (Sirt1, Sirt3), and the expression of core clock genes (Clock, Bmal1, Per2, Cry2) and clock-controlled genes involved in lipid metabolism (Rev-Erbα, Rev-Erbβ, RORα, and Srebp1c) were measured in offspring livers. ResultsOffspring fed a HF diet developed NAFLD. However HF fed offspring of mothers fed a HF diet developed NASH, coupled with significantly reduced NAD+/NADH (p<0.05, HF/HF vs C/C), Sirt1 (p<0.001, HF/HF vs C/C), Sirt3 (p<0.01, HF/HF vs C/C), perturbed clock gene expression, and elevated expression of genes involved lipid metabolism, such as Srebp1c (p<0.05, C/HF and HF/HF vs C/C). ConclusionOur results suggest that exposure to excess dietary fat during early and post-natal life increases the susceptibility to develop NASH in adulthood, involving altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression.

Role of Nuclear Factor Erythroid 2-Like 2 in the Induction of Cytochrome P450 2a5 in Vivo of Nonalcoholic Fatty Liver Disease

Mouse cytochrome P450 2A5 (Cyp2a5) is upregulated in various pathophysiological liver disease and induced by structurally variable hepatotoxic chemicals. A putative common feature for all of these conditions is altered cellular redox status. Nuclear factor erythroid 2-like 2 (Nrf2) is a transcription factor that is post-translationally regulated by oxidative stress and controls the transcription of numerous protective target genes. In order to determine if upregulation of Cyp2a5 by Nonalcoholic fatty liver disease (NAFLD) was mediated through Nrf2, we applied Nrf2 knockout mice (Nrf2-/-) to examine the Cyp2a5 mRNA and protein levels compared to wild type mice. Furthermore, the interaction of Nrf2 with Cyp2a5 was confirmed by co-immunoprecipitation (co-IP) in vivo. The NAFLD increased the Nrf2 mRNA and protein levels in wild type mice and also increased the Cyp2a5 mRNA and protein levels resulted from translocation of Nrf2 into the nucleus. However, the expression and activity of Cyp2a5 were no difference in the Nrf2 knockout mice. Nuclear immunohistochemical staining of Nrf2, an indicator of activation of the transcription factor, was evident in NAFLD. Co-IP experiments showed that Nrf2 binds to the Cyp2a5. Our current results unequivocally show that expression of Cyp2a5 is tightly controlled by Nrf2 in liver. Nrf2 is needed for constitutive expression of Cyp2a5, and Cyp2a5 is also sensitively upregulated by an increased level of Nrf2 protein. Therefore, Cyp2a5 upregulation could be a useful indicator for hepatic activation of the Nrf2 pathway.

Deletion of Pim kinases elevates the cellular levels of reactive oxygen species and sensitizes to K-Ras-induced cell killing.

The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-RasG12V) and undergo cell death. Transduction of K-RasG12V into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuates ROS production and reversed the cytotoxic effects of K-RasG12V in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-RasG12V-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-RasG12V-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases play an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth.

Copper: toxicological relevance and mechanisms.

Copper (Cu) is a vital mineral essential for many biological processes. The vast majority of all Cu in healthy humans is associated with enzyme prosthetic groups or bound to proteins. Cu homeostasis is tightly regulated through a complex system of Cu transporters and chaperone proteins. Excess or toxicity of Cu, which is associated with the pathogenesis of hepatic disorder, neurodegenerative changes and other disease conditions, can occur when Cu homeostasis is disrupted. The capacity to initiate oxidative damage is most commonly attributed to Cu-induced cellular toxicity. Recently, altered cellular events, including lipid metabolism, gene expression, alpha-synuclein aggregation, activation of acidic sphingomyelinase and release of ceramide, and temporal and spatial distribution of Cu in hepatocytes, as well as Cu-protein interaction in the nerve system, have been suggested to play a role in Cu toxicity. However, whether these changes are independent of, or secondary to, an altered cellular redox state of Cu remain to be elucidated.

Matriptase Autoactivation Is Tightly Regulated by the Cellular Chemical Environments

The ability of cells to rapidly detect and react to alterations in their chemical environment, such as pH, ionic strength and redox potential, is essential for cell function and survival. We present here evidence that cells can respond to such environmental alterations by rapid induction of matriptase autoactivation. Specifically, we show that matriptase autoactivation can occur spontaneously at physiological pH, and is significantly enhanced by acidic pH, both in a cell-free system and in living cells. The acid-accelerated autoactivation can be attenuated by chloride, a property that may be part of a safety mechanism to prevent unregulated matriptase autoactivation. Additionally, the thio-redox balance of the environment also modulates matriptase autoactivation. Using the cell-free system, we show that matriptase autoactivation is suppressed by cytosolic reductive factors, with this cytosolic suppression being reverted by the addition of oxidizing agents. In living cells, we observed rapid induction of matriptase autoactivation upon exposure to toxic metal ions known to induce oxidative stress, including CoCl2 and CdCl2. The metal-induced matriptase autoactivation is suppressed by N-acetylcysteine, supporting the putative role of altered cellular redox state in metal induced matriptase autoactivation. Furthermore, matriptase knockdown rendered cells more susceptible to CdCl2-induced cell death compared to control cells. This observation implies that the metal-induced matriptase autoactivation confers cells with the ability to survive exposure to toxic metals and/or oxidative stress. Our results suggest that matriptase can act as a cellular sensor of the chemical environment of the cell that allows the cell to respond to and protect itself from changes in the chemical milieu.

Correlation of Glycemic Status with Oxidative/Antioxidant Status in Diabetic Retinopathy

Context : Diabetes is a chronic disease and sustained hyperglycemia attacks both micro vessels and macro vessels throughout the body. It is the leading cause of retinopathy, nephropathy, end stage renal disease and cardiovascular diseases. In Diabetes and its complication, oxidative stress is caused by both increased production of reactive oxygen species, sharp reduction in antioxidant defenses and altered cellular redox status. Aims: The aim of the present study was to assess the correlation of glycemic status with lipid peroxidation and antioxidant vitamin levels in patient with diabetic retinopathy. Setting and design: 50 patients with diabetic retinopathy and 50 age and sex matched healthy controls were included in the study. Fasting blood glucose levels, post prandial blood glucose levels were estimated to assess the severity of Diabetes. Glycated haemoglobin was estimated to assess glycemic control. The degree of lipid peroxidation in terms of serum malondialdehyde (MDA) by Thiobarbituric acid method along with antioxidant defenses, vitamin A, vitamin C and vitamin E were estimated in cases and controls. Statistical analysis used: Student t test (two tailed, independent) has been used to find the significance of study parameters on continuous scale between two groups (Inter group analysis) on metric parameters. Pearson correlation between HbA1c and other parameters was performed to assess the relationship in each group. Results: The serum FBS, PPBS, HbA1c and MDA were found to be increased significantly (p<0.001) in diabetic retinopathy patients while plasma Vitamin A, C and E levels were significantly reduced (p<0.001) as compared to controls. Correlation study conducted between HbA1c and other parameters revealed that there was statistically significant negative correlation between HbA1c and vitamin A and vitamin C. Conclusions: The present study concludes that sustained hyperglycemia leads to excessive peroxidation and antioxidant deficiency in diabetic retinopathy. Therapeutic measures to increase the antioxidant levels and to control lipid peroxidation are warranted for effective control of diabetic complications.