Causative Role Of Oxidative Stress Research Articles

Oxidative Stress, Cytotoxic and Inflammatory Effects of Azoles Combinatorial Mixtures in Sertoli TM4 Cells.

Triazole and imidazole fungicides are an emerging class of contaminants with an increasing and ubiquitous presence in the environment. In mammals, their reproductive toxicity has been reported. Concerning male reproduction, a combinatorial activity of tebuconazole (TEB; triazole fungicide) and econazole (ECO; imidazole compound) in inducing mitochondrial impairment, energy depletion, cell cycle arrest, and the sequential activation of autophagy and apoptosis in Sertoli TM4 cells (SCs) has recently been demonstrated. Given the strict relationship between mitochondrial activity and reactive oxygen species (ROS), and the causative role of oxidative stress (OS) in male reproductive dysfunction, the individual and combined potential of TEB and ECO in inducing redox status alterations and OS was investigated. Furthermore, considering the impact of cyclooxygenase (COX)-2 and tumor necrosis factor-alpha (TNF-α) in modulating male fertility, protein expression levels were assessed. In the present study, we demonstrate that azoles-induced cytotoxicity is associated with a significant increase in ROS production, a drastic reduction in superoxide dismutase (SOD) and GSH-S-transferase activity levels, and a marked increase in the levels of oxidized (GSSG) glutathione. Exposure to azoles also induced COX-2 expression and increased TNF-α production. Furthermore, pre-treatment with N-acetylcysteine (NAC) mitigates ROS accumulation, attenuates COX-2 expression and TNF-α production, and rescues SCs from azole-induced apoptosis, suggesting a ROS-dependent molecular mechanism underlying the azole-induced cytotoxicity.

IL‐10 Attenuates ER‐Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes

Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox‐induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein‐kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X‐box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress‐initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase‐12 activation, phosphorylation of c‐JUN NH2‐terminal kinase (JNK) and pro‐apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning.In this study on isolated rat cardiomyocytes, we analyzed ER‐stress markers, spliced XBP1 mRNA, and ER‐apoptotic proteins. We also assessed protective effects of Interleukin‐10 (IL‐10) against Dox initiated ER‐induced apoptosis and overall ER‐stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL‐10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER‐stress markers as well as ER‐related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL‐10 treatment were recorded. IL‐10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox‐treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL‐10 in Dox treated cells. Dox also initiated activation of procaspase‐12, which is known to activate caspase‐3 and apoptosis. IL‐10 significantly reduced the activation of procaspase‐12 and cell death. These data suggest that IL‐10 provides protection against Dox‐induced cardiotoxicity by restoring ER homeostasis.

Role of Oxidative Stress in Heart Failure: Insights from Gene Transfer Studies.

Under physiological circumstances, there is an exquisite balance between reactive oxygen species (ROS) production and ROS degradation, resulting in low steady-state ROS levels. ROS participate in normal cellular function and in cellular homeostasis. Oxidative stress is the state of a transient or a persistent increase of steady-state ROS levels leading to disturbed signaling pathways and oxidative modification of cellular constituents. It is a key pathophysiological player in pathological hypertrophy, pathological remodeling, and the development and progression of heart failure. The heart is the metabolically most active organ and is characterized by the highest content of mitochondria of any tissue. Mitochondria are the main source of ROS in the myocardium. The causal role of oxidative stress in heart failure is highlighted by gene transfer studies of three primary antioxidant enzymes, thioredoxin, and heme oxygenase-1, and is further supported by gene therapy studies directed at correcting oxidative stress linked to metabolic risk factors. Moreover, gene transfer studies have demonstrated that redox-sensitive microRNAs constitute potential therapeutic targets for the treatment of heart failure. In conclusion, gene therapy studies have provided strong corroborative evidence for a key role of oxidative stress in pathological remodeling and in the development of heart failure.

Oxidative stress and its impact on skin, scalp and hair.

Oxidative stress is an unbalanced condition in which the tissues of the body are not sufficiently able to counteract either exogenous or endogenous sources of reactive oxygen species. Oxidative stress is strongly associated with ageing, both local and systemic, as well as a wide range of local health conditions. This review focuses on the oxidative stress data known for skin, scalp and hair. This oxidative stress may be the 'currency' by which an unhealthy scalp leads to deleterious consequences to the hair. The ramifications of this scalp oxidative stress to normal hair elongation, retention and replacement are discussed.

Low doses of the neonicotinoid insecticide imidacloprid induce ROS triggering neurological and metabolic impairments in Drosophila

Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.

Role of Inositol Hexakisphosphate Kinase‐2 (IP6K2) in regulating mitochondrial brain functions

Inositol hexakisphosphate kinase (IP6K) isoform ‐ IP6K2, selectively controls cellular apoptosis. To explore the possible role of IP6K2 in degenerative brain damage, we elucidated its protein interactome in mouse brain to identify its functional partners. IP6K2‐immunoprecipitates from wild‐type mouse brain lysates were subjected to co‐immunoprecipitation followed by liquid chromatography mass spectrometric analysis. This revealed robust association of IP6K2 with creatine kinase‐B (CK‐B), an enzyme responsible for synthesis of phosphocreatine and ATP. Notably, both IP6K2 and CK‐B were highly expressed in the mouse cerebellum. IP6K2‐knockout mice (IP6K2‐KO) produced less phosphocreatine and ATP and generated higher levels of ROS and consequent protein oxidative damage in their cerebellum compared to wild‐type counterparts. As mitochondrial respiratory chains regulate energy metabolism/ATP production through glycolysis and TCA cycle coupled to oxidative phosphorylation and electron transfer, functional abrogation of any event that controls these processes can lead to abnormal electron transfer, increased ROS and consequent mitochondrial oxidative stress/damage and cell death as observed in the IP6K2‐KO mice. IP6K2‐KO mice also showed significant impairment of locomotive and cognitive functions. We thus propose to identify the precise metabolic events that are controlled by IP6K2‐CK‐B interaction and examine the possible causal role of oxidative stress and determine whether antioxidant treatment can restore normal brain functions. We plan to explore mitochondria‐targeted antioxidants like CoQ10/Mito Q along with derivatives of higher inositol phosphates (IP7‐product of IP6K2) in the IP6K2‐KO mice to reinforce the therapeutic potential of such anti‐oxidative treatment for the clinical management of diseases associated with mitochondrial dysfunction of the brain. Overall, our proposed study would help to establish the precise role of IP6K2‐CK‐B interaction in controlling cellular degeneration in the brain.

Oxidative stress pathways in pancreatic β-cells and insulin-sensitive cells and tissues: importance to cell metabolism, function, and dysfunction.

It is now accepted that nutrient abundance in the blood, especially glucose, leads to the generation of reactive oxygen species (ROS), ultimately leading to increased oxidative stress in a variety of tissues. In the absence of an appropriate compensatory response from antioxidant mechanisms, the cell, or indeed the tissue, becomes overwhelmed by oxidative stress, leading to the activation of intracellular stress-associated pathways. Activation of the same or similar pathways also appears to play a role in mediating insulin resistance, impaired insulin secretion, and late diabetic complications. The ability of antioxidants to protect against the oxidative stress induced by hyperglycemia and elevated free fatty acid (FFA) levels in vitro suggests a causative role of oxidative stress in mediating the latter clinical conditions. In this review, we describe common biochemical processes associated with oxidative stress driven by hyperglycemia and/or elevated FFA and the resulting clinical outcomes: β-cell dysfunction and peripheral tissue insulin resistance.

Abstract # 3164 Memory impairment following high-fat diet refeeding is related to caspase-1 activation and oxidative stress in the amygdala

As obesity prevalence increases year-over-year the neurological comorbidities that present with it are increasing as well. Over consumption of calories is a significant causal factor in the development of obesity, and induces a pro-inflammatory state in the brain. Importantly, brain-based IL-1 β appears to be crucial to obesity-related cognitive dysfunction, and several fasting paradigms have been shown to be effective at ameliorating both obesity and IL-1 β -induced cognitive dysfunction. However, these fasting programs can induce binge eating following fasting and it remains unknown whether this type of acute overconsumption can result in negative inflammatory and cognitive sequela. Here, we show that 2hr of high-fat diet (HFD) refeeding reduces novel object recognition (NOR) and novel location recognition (NLR) memory by 13% and 14%, respectively. Additionally, HFD refeeding increases the IL-1 β activating enzyme caspase-1 in the amygdala by ∼ 100%. Calorie intake was significantly correlated with both cognitive performance and caspase-1 activity. Additionally, knockout of either caspase-1 or IL-1 receptor 1 (IL1R1) eliminated recognition memory impairments due to HFD. The antioxidant n-acetylcysteine (NAC; 50 mg/kg) improved NOR and NLR performance by 18% and 14% respectively, and reduced caspase-1 in HFD fed animals by 169%, suggesting a causal role for oxidative stress. Taken together, these data suggest that acute overconsumption during refeeding activates caspase-1 in the amygdala which leads to cognitive impairment via a mechanism mediated by oxidative stress.

Supplementation with Triptolide Increases Resistance to Environmental Stressors and Lifespan in C. elegans.

Triptolide is a major active compound found in Tripterygium wilfordii., also known as Thunder God Vine. Triptolide has been shown to have anti-inflammatory and anticancer activities. In this study, we examined the effect of dietary supplementation with triptolide on response to environmental stressors and lifespan in vivo using Caenorhabditis elegans as a model system. Treatment with 50 mg/L of triptolide in the growth media increased resistance to oxidative stress and reduced the generation of intracellular reactive oxygen species. We also observed a lifespan-extending activity for triptolide. Both mean and maximum lifespans were significantly increased by supplementation with triptolide. Response to other environmental stressors was modulated by triptolide. The survival after heat shock or UV irradiation was markedly increased in worms treated with triptolide. Unlike many lifespan-extending genetic or nutritional interventions, the longevity phenotype conferred by triptolide did not have the trade-off of a reduction in fertility or a delay in the gravid period. The expressions of hsp-16.2 and sod-3, known to positively correlate with a long lifespan, were significantly upregulated by supplementation with triptolide. These findings suggest that triptolide can exhibit antistress and lifespan-extending effects in vivo, possibly through its antioxidant activity and support the free radical theory of aging, which emphasizes the causative role of oxidative stress in aging.

Beyond Diabetes: Does Obesity-Induced Oxidative Stress Drive the Aging Process?

Despite numerous correlative data, a causative role for oxidative stress in mammalian longevity has remained elusive. However, there is strong evidence that increased oxidative stress is associated with exacerbation of many diseases and pathologies that are also strongly related to advanced age. Obesity, or increased fat accumulation, is one of the most common chronic conditions worldwide and is associated with not only metabolic dysfunction but also increased levels of oxidative stress in vivo. Moreover, obesity is also associated with significantly increased risks of cardiovascular disease, neurological decline and cancer among many other diseases as well as a significantly increased risk of mortality. In this review, we investigate the possible interpretation that the increased incidence of these diseases in obesity may be due to chronic oxidative stress mediating segmental acceleration of the aging process. Understanding how obesity can alter cellular physiology beyond that directly related to metabolic function could open new therapeutic areas of approach to extend the period of healthy aging among people of all body composition.

Oxidative Stress-Mediated Skeletal Muscle Degeneration: Molecules, Mechanisms, and Therapies.

Oxidative stress is a loss of balance between the production of reactive oxygen species during cellular metabolism and the mechanisms that clear these species to maintain cellular redox homeostasis. Increased oxidative stress has been associated with muscular dystrophy, and many studies have proposed mechanisms that bridge these two pathological conditions at the molecular level. In this review, the evidence indicating a causal role of oxidative stress in the pathogenesis of various muscular dystrophies is revisited. In particular, the mediation of cellular redox status in dystrophic muscle by NF-κB pathway, autophagy, telomere shortening, and epigenetic regulation are discussed. Lastly, the current stance of targeting these pathways using antioxidant therapies in preclinical and clinical trials is examined.

Direct effect of chronic hypoxia in suppressing large conductance Ca(2+)-activated K(+) channel activity in ovine uterine arteries via increasing oxidative stress.

Chronic hypoxia has a direct effect in down-regulating the BKCa channel β1 subunit and inhibiting the BKCa channel activity in uterine arteries of pregnant sheep. Oxidative stress plays a causal role in hypoxia-mediated suppression of BKCa channel function. The steroid hormone-induced effect on BKCa channels is a target of hypoxia-mediated oxidative stress. Inhibition of oxidative stress ameliorates the adverse effect of hypoxia both ex vivo and in vivo in pregnant sheep exposed to long-term high-altitude hypoxia. Our findings provide novel evidence of a causative role of oxidative stress in hypoxia-mediated inhibition of the BKCa channel activity in uterine arteries and new insights in understanding and alleviating pregnancy complications associated with gestational hypoxia such as pre-eclampsia and fetal growth restriction. Uterine arteries of pregnant sheep acclimatized to long-term high-altitude hypoxia were associated with a decrease in large-conductance Ca(2+)-activated K(+) (BKCa) channel activity. The present study tested the hypothesis that prolonged hypoxia has a direct effect in suppressing BKCa channel activity by increasing oxidative stress. Uterine arteries were isolated from non-pregnant and near-term (∼142 days) pregnant sheep, and were treated ex vivo with 21.0 or 10.5% O2 for 48 h. The hypoxia treatment significantly increased the production of reactive oxygen species in uterine arteries, which was blocked by N-acetylcysteine. In uterine arteries of pregnant sheep, hypoxia significantly inhibited BKCa channel current density, decreased NS1619-induced relaxations and increased pressure-dependent tone, which were annulled by N-acetylcysteine. In accordance, hypoxia resulted in down-regulation of BKCa channel β1 subunit, which was restored in the presence of N-acetylcysteine. In addition, the N-acetylcysteine treatment significantly increased BKCa channel β1 subunit abundance and BKCa channel current density in uterine arteries from pregnant sheep exposed to high-altitude hypoxia (3801 m, PaO2: 60 mmHg) for 110 days. In uterine arteries of non-pregnant animals, hypoxia inhibited steroid hormone-induced up-regulation of BKCa channel current density and NS1619-mediated relaxations, which were reversed by N-acetylcysteine. Furthermore, the synthetic superoxide dismutase and catalase mimetic EUK-134 also ablated the effects of hypoxia on BKCa channel currents in uterine arteries. The results demonstrate a direct effect of hypoxia in inhibiting the BKCa channel activity in uterine arteries via increased oxidative stress.

The Immp2l mutation causes age‐dependent degeneration of cerebellar granule neurons prevented by antioxidant treatment

Reactive oxygen species are implicated in age-associated neurodegeneration, although direct invivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2-like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age-associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (>16months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria-targeted antioxidant SkQ1 from 6weeks to 21months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age-matched normal control mice or SkQ1-treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4-hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age-associated neurodegeneration.

The role of oxidative stress in amiodaron toxicity, in left ventricular dysfunction of hypertensive patients and in heart failure with preserved ejection fraction

The author briefly summarizes his scientific work investigating the role of oxidative stress in cardiovascular disorders. Using in vitro biochemical, biophysical and in vivo animal research it was found that oxidative stress plays a substantial role in the pathogenesis of amiodarone toxicity and antioxidants co-administered with amiodarone exert at least partial protective effect on amiodarone toxicity, while antioxidants did not diminish and perhaps even enhanced the antiarrhythmic action of amiodarone. Thus, co-administration of antioxidants with amiodarone may lead to the more widespread application of amiodarone, which is currently the most potent available antiarrhythmic agent, but its clinical use is limited due the potentially severe toxic effect In hypertensive patients with normal ejection fraction, the most common precursor condition of heart failure with preserved ejection fraction, the potential primary causal role of oxidative stress and inflammation in the left ventricular systolic, diastolic and atrial dysfunction, which are important determinants of the transition of hypertensive heart disease to heart failure with preserved ejection fraction was verified.

Age modifies respiratory complex I and protein homeostasis in a muscle type‐specific manner

Changes in mitochondrial function with age vary between different muscle types, and mechanisms underlying this variation remain poorly defined. We examined whether the rate of mitochondrial protein turnover contributes to this variation. Using heavy label proteomics, we measured mitochondrial protein turnover and abundance in slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) from young and aged mice. We found that mitochondrial proteins were longer lived in EDL than SOL at both ages. Proteomic analyses revealed that age-induced changes in protein abundance differed between EDL and SOL with the largest change being increased mitochondrial respiratory protein content in EDL. To determine how altered mitochondrial proteomics affect function, we measured respiratory capacity in permeabilized SOL and EDL. The increased mitochondrial protein content in aged EDL resulted in reduced complex I respiratory efficiency in addition to increased complex I-derived H2 O2 production. In contrast, SOL maintained mitochondrial quality, but demonstrated reduced respiratory capacity with age. Thus, the decline in mitochondrial quality with age in EDL was associated with slower protein turnover throughout life that may contribute to the greater decline in mitochondrial dysfunction in this muscle. Furthermore, mitochondrial-targeted catalase protected respiratory function with age suggesting a causal role of oxidative stress. Our data clearly indicate divergent effects of age between different skeletal muscles on mitochondrial protein homeostasis and function with the greatest differences related to complex I. These results show the importance of tissue-specific changes in the interaction between dysregulation of respiratory protein expression, oxidative stress, and mitochondrial function with age.

Causal role of oxidative stress in unfolded protein response development in the hyperthyroid state

l-3,3′,5-Triiodothyronine (T3)-induced liver oxidative stress underlies significant protein oxidation, which may trigger the unfolded protein response (UPR). Administration of daily doses of 0.1mg T3 for three consecutive days significantly increased the rectal temperature of rats and liver O2 consumption rate, with higher protein carbonyl and 8-isoprostane levels, glutathione depletion, and absence of morphological changes in liver parenchyma. Concomitantly, liver protein kinase RNA-like endoplasmic reticulum (ER) kinase and eukaryotic translation initiator factor 2α were phosphorylated in T3-treated rats compared to controls, with increased protein levels of binding immunoglobulin protein and activating transcription factor 4. In addition, higher mRNA levels of C/EBP homologous protein, growth arrest and DNA damage 34, protein disulfide isomerase, and ER oxidoreductin 1α were observed, changes that were suppressed by N-acetylcysteine (0.5g/kg) given before each dose of T3. In conclusion, T3-induced liver oxidative stress involving higher protein oxidation status has a causal role in UPR development, a response that is aimed to alleviate ER stress and promote cell survival.

Tempol treatment reduces anxiety-like behaviors induced by multiple anxiogenic drugs in rats.

We have published that pharmacological induction of oxidative stress (OS) causes anxiety-like behavior in rats. Using animal models, we also have established that psychological stress induces OS and leads to anxiety-like behaviors. All evidence points towards the causal role of OS in anxiety-like behaviors. To fully ascertain the role of OS in anxiety-like behaviors, it is reasonable to test whether the pro-anxiety effects of anxiogenic drugs caffeine or N-methyl-beta-carboline-3-carboxamide (FG-7142) can be mitigated using agents that minimize OS. In this study, osmotic pumps were either filled with antioxidant tempol or saline. The pumps were attached to the catheter leading to the brain cannula and inserted into the subcutaneous pocket in the back pocket of the rat. Continuous i.c.v. infusion of saline or tempol in the lateral ventricle of the brain (4.3mmol/day) was maintained for 1 week. Rats were intraperitoneally injected either with saline or an anxiogenic drug one at a time. Two hours later all groups were subjected to behavioral assessments. Anxiety-like behavior tests (open-field, light-dark and elevated plus maze) suggested that tempol prevented anxiogenic drug-induced anxiety-like behavior in rats. Furthermore, anxiogenic drug-induced increase in stress examined via plasma corticosterone and increased oxidative stress levels assessed via plasma 8-isoprostane were prevented with tempol treatment. Protein carbonylation assay also suggested preventive effect of tempol in the prefrontal cortex brain region of rats. Antioxidant protein expression and pro-inflammatory cytokine levels indicate compromised antioxidant defense as well as an imbalance of inflammatory response.

Hepatoprotection by curcumin and sulforaphane against tBHP-induced oxidative damage

Background and aim: Antioxidants have recently been of keen therapeutic interest, due to the causative role of oxidative stress in induction and progression of liver, pancreatic and neurodegenerative diseases. Most plant-derived antioxidants are key components of vegetables and spices which are part of daily meals. Curcumin (from Asian spice turmeric) and sulforaphane (abundant in cruciferous vegetables such as broccoli), are reported to have antioxidant potential [1, 2] and were assessed in the current study for their cytoprotective activity in HepG2 liver cells. Methods: Radical scavenging activities of curcumin and sulforaphane were investigated against 2,2-diphenyl-1-picrylhydrazyl (DPPH). Using HepG2 liver cells, cytoprotection against tert-butyl hydroperoxide (tBHP)-induced cytotoxicity was evaluated in 5h co-exposure and 20h pre-exposure protocols with phytochemicals. Cell viability was determined in Neutral Red assay. Quercetin served as a positive phyto-antioxidant standard [3]. All phytochemicals were dissolved in DMSO to final solution of 10 mg/ml. Results and Discussion: Radical scavenging activity of quercetin was stronger than curcumin, but not significantly more potent in 5h co-exposure with tBHP. Sulforaphane was not cytoprotective in 5h protocol, this it lacked antiradical activity. Unlike quercetin, both phytochemicals were cytotoxic at high concentrations (Table 1). Conclusion: Sulforaphane, curcumin and quercetin could protect HepG2 cells via indirectly inducing intrinsic cytoprotective mechanisms during 20h pre-exposure protocol. Sulforophane does not act as a free radical scavenger, whilst curcumin and quercetin have antiradical properties, acting as direct and indirect antioxidants. The cytoprotective activities of quercetin, curcumin and sulforaphane suggest that these may be strong therapeutic phyto-antioxidants in preventing oxidative damage. Keywords: Curcumin, Antioxidant, Quercetin, Sulforaphane, HepG2

Vascular oxidative stress upregulates angiotensin II type I receptors via mechanisms involving nuclear factor kappa B

The association of oxidative stress with hypertension is well known. However, a causal role of oxidative stress in hypertension is unclear. Vascular angiotensin II type 1 receptor (AT1R) upregulation is a prominent contributor to pathogenesis of hypertension. However, the mechanisms causing this upregulation are unknown. Oxidative stress is an important regulator of protein expression via activation of transcription factors such as nuclear factor kappa B (NFκB). The present study was carried out to test the hypothesis that oxidative stress contributes to vascular AT1R upregulation via NFκB in human aortic smooth muscle cells (HASMC) and spontaneously hypertensive rats (SHR). HASMC exposed to oxidative stress exhibited a robust increase in AT1R mRNA in HASMC. Furthermore, oxidative stress failed to upregulate AT1Rs in the presence of either an antioxidant catalase or siRNA against p65 subunit of NFκB. To test the role of oxidative stress and NFκB in hypertension, prehypertensive SHR were treated with NFκB inhibitor pyrrolidine dithiocarbamate from 5 weeks to 11–12 weeks of age. At 11–12 weeks of age, SHR exhibited increased NFκB expression, AT1R upregulation and exaggerated Ang II-induced vasoconstriction as compared to age-matched Wistar Kyoto (WKY) rats. PDTC treatment of SHR lowered NFκB expression, normalized AT1R expression and Ang II-induced vasoconstriction. More importantly, PDTC treatment significantly attenuated hypertension development in SHR. In conclusion, vascular oxidative can upregulate AT1R, via mechanisms involving NFκB, and contribute to the development of hypertension.

Cellular accumulation of Cys326-OGG1 protein complexes under conditions of oxidative stress

The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents β-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes.In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 protein accumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress.