Anup Srivastava

Prevention of diabetic nephropathy in Ins2(+/)⁻(AkitaJ) mice by the mitochondria-targeted therapy MitoQ.

Mitochondrial production of ROS (reactive oxygen species) is thought to be associated with the cellular damage resulting from chronic exposure to high glucose in long-term diabetic patients. We hypothesized that a mitochondria-targeted antioxidant would prevent kidney damage in the Ins2+/−AkitaJ mouse model (Akita mice) of Type 1 diabetes. To test this we orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period and assessed tubular and glomerular function. Fibrosis and pro-fibrotic signalling pathways were determined by immunohistochemical analysis, and mitochondria were isolated from the kidney for functional assessment. MitoQ treatment improved tubular and glomerular function in the Ins2+/−AkitaJ mice. MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Consistent with previous studies, renal mitochondrial function showed no significant change between any of the diabetic or wild-type groups. Importantly, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and β-catenin showed a nuclear accumulation in the Ins2+/−AkitaJ mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy. They also highlight a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis.

Mitochondria‐targeted ubiquinone (MitoQ) decreases ethanol‐dependent micro and macro hepatosteatosis

Chronic alcohol‐induced liver disease results in inflammation, steatosis, and increased oxidative and nitrosative damage to the mitochondrion. We hypothesized that targeting an antioxidant to the mitochondria would prevent oxidative damage and attenuate the steatosis associated with alcoholic liver disease. To test this we investigated the effects of mitochondria‐targeted ubiquinone (MitoQ) (5 and 25 mg/kg/day for 4 weeks) in male Sprague‐Dawley rats consuming ethanol using the Lieber‐DeCarli diet with pair‐fed controls. Hepatic steatosis, 3‐nitrotyrosine (3‐NT), 4‐hydroxynonenal (4‐HNE), hypoxia inducible factor α (HIF1α), and the activity of the mitochondrial respiratory chain complexes were assessed. As reported previously, ethanol consumption resulted in hepatocyte ballooning, increased lipid accumulation in the form of micro and macrovesicular steatosis, and induction of cytochrome P450 2E1 (CYP2E1). MitoQ had a minor effect on the ethanol‐dependent decrease in mitochondrial respiratory chain proteins and their activities; however, it did decrease hepatic steatosis in ethanol‐consuming animals and prevented the ethanol‐induced formation of 3‐NT and 4‐HNE. Interestingly, MitoQ completely blocked the increase in HIF1α in all ethanol‐fed groups, which has previously been demonstrated in cell culture models and shown to be essential in ethanol‐dependent hepatosteatosis. Conclusion: These results demonstrate the antioxidant capacity of MitoQ in alleviating alcohol‐associated mitochondrial reactive oxygen species (ROS) and several downstream effects of ROS/RNS (reactive nitrogen species) production such as inhibiting protein nitration and protein aldehyde formation and specifically ROS‐dependent HIF1α stabilization. (HEPATOLOGY 2011;)

Mycobacterium tuberculosis WhiB4 regulates oxidative stress response to modulate survival and dissemination in vivo.

Host‐generated oxidative stress is considered one of the main mechanisms constraining Mycobacterium tuberculosis (Mtb) growth. The redox‐sensing mechanisms in Mtb are not completely understood. Here we show that WhiB4 responds to oxygen (O2) and nitric oxide (NO) via its 4Fe‐4S cluster and controls the oxidative stress response in Mtb. The WhiB4 mutant (MtbΔwhiB4) displayed an altered redox balance and a reduced membrane potential. Microarray analysis demonstrated that MtbΔwhiB4 overexpresses the antioxidant systems including alkyl hydroperoxidase (ahpC‐ahpD) and rubredoxins (rubA‐rubB). DNA binding assays showed that WhiB4 [4Fe‐4S] cluster is dispensable for DNA binding. However, oxidation of the apo‐WhiB4 Cys thiols induced disulphide‐linked oligomerization, DNA binding and transcriptional repression, whereas reduction reversed the effect. Furthermore, WhiB4 binds DNA with a preference for GC‐rich sequences. Expression analysis showed that oxidative stress repressed whiB4 and induced antioxidants in Mtb, while their hyper‐induction was observed in MtbΔwhiB4. MtbΔwhiB4 showed increased resistance to oxidative stress in vitro and enhanced survival inside the macrophages. Lastly, MtbΔwhiB4 displayed hypervirulence in the lungs of guinea pigs, but showed a defect in dissemination to their spleen. These findings suggest that WhiB4 systematically calibrates the activation of oxidative stress response in Mtb to maintain redox balance, and to modulate virulence.

Reductive stress in microbes: implications for understanding Mycobacterium tuberculosis disease and persistence.

Mycobacterium tuberculosis (Mtb) is a remarkably successful pathogen that is capable of persisting in host tissues for decades without causing disease. Years after initial infection, the bacilli may resume growth, the outcome of which is active tuberculosis (TB). In order to establish infection, resist host defences and re-emerge, Mtb must coordinate its metabolism with the in vivo environmental conditions and nutrient availability within the primary site of infection, the lung. Maintaining metabolic homeostasis for an intracellular pathogen such as Mtb requires a carefully orchestrated series of oxidation-reduction reactions, which, if unbalanced, generate oxidative or reductive stress. The importance of oxidative stress in microbial pathogenesis has been appreciated and well studied over the past several decades. However, the role of its counterpart, reductive stress, has been largely ignored. Reductive stress is defined as an aberrant increase in reducing equivalents, the magnitude and identity of which is determined by host carbon source utilisation and influenced by the presence of host-generated gases (e.g. NO, CO, O(2) and CO(2)). This increased reductive power must be dissipated for bacterial survival. To recycle reducing equivalents, microbes have evolved unique electron sinks that are distinct for their particular environmental niche. In this review, we describe the specific mechanisms that some microbes have evolved to dispel reductive stress. The intention of this review is to introduce the concept of reductive stress, in tuberculosis research in particular, in the hope of stimulating new avenues of investigation.

Antioxidant and cytoprotective properties of 2-(hydroxymethyl)-3-methoxybenzaldehyde.

Currently there is a great deal of interest in the study of natural compounds with free radical scavenging activity because of their potential role in maintaining human health and preventing diseases. In this paper, we report the antioxidant and cytoprotective properties of 2-(hydroxymethyl)-3-methoxybenzaldehyde (HMMB) isolated from the aqueous extract of Decalepis hamiltonii roots. Our results show that HMMB is a potent scavenger of superoxide (O2(-)), hydroxyl (OH), nitric oxide (NO), and lipid peroxide (LOO) physiologically relevant free radicals with IC50 values in the nmolar (5-214) range. HMMB also exhibited concentration dependent secondary antioxidant activities, such as reducing power, metal chelating activity, and inhibition of protein carbonylation. Further, HMMB at nmolar concentration prevented CuSO4-induced human LDL oxidation. Apart from the in vitro free radical scavenging activity, HMMB demonstrated cytoprotective activity in primary hepatocytes and Ehrlich Ascites Tumour (EAT) cells against oxidative stress inducing xenobiotics. The mechanism of cytoprotective action involved maintaining the intracellular glutathione (GSH), scavenging of reactive oxygen species (ROS), and inhibition of lipid peroxidation (LPO). Based on the results it is suggested that HMMB is a novel bioactive molecule with health implications in both prevention and amelioration of diseases involving oxidative stress, as well as in the general well being.

Differential cholinesterase inhibition in the rat brain regions by dichlorvos and protective effect of Decalepis hamiltonii roots

Dichlorvos (DDVP) causes neurotoxicity primarily by inhibiting cholinesterase (ChE) which is the characteristic feature of organophosphate pesticides. In this study, we found for the first time that DDVP shows differential inhibition of ChE (acetylcholinesterase+butyrylcholinesterase) in various rat brain regions. A single dose of DDVP (1/3 LD(50)) after 16 h of treatment elicited ChE inhibition in the brain regions which was highest in striatum and lowest in cerebellum. The inhibition of ChE by DDVP has been shown to be accompanied with induction of oxidative stress. Further, we investigated the protective potential of the aqueous extract of the roots of Decalepis hamiltonii (DHA), having potent antioxidant constituents, against DDVP-induced ChE inhibition in various rat brain regions. Pretreatment of rats with multiple doses of DHA, 50 and 100mg/kg b.w., for 7 consecutive days did not produce any significant change in ChE activity. Pretreatment of rats with DHA, at high dose, significantly protected against DDVP-induced ChE inhibition in all the brain regions except cerebellum. Pretreatment of rats with DHA, at low dose, showed significant protection in striatum, cortex, and pons against DDVP-induced ChE inhibition. The protective activity of DHA can be attributed to the characterized potent antioxidant constituents which could have an important role in preventing ChE inhibition by inducing the DDVP detoxifying enzymes. We strongly believe that these antioxidant constituents are prospective novel nutriceuticals.

Stereospecificity in the cytotoxic action of hexachlorocyclohexane isomers.

Hexachlorocyclohexane (HCH) is a highly recalcitrant organochlorine insecticide known for its chronic toxicity. In spite of many isolated studies a clear mechanism of cytotoxic action of HCH and the structure-toxicity relationship of its isomers is not well understood. We have investigated the toxicity of HCH isomers and its mechanism in Ehrlich Ascites tumor (EAT) cells. Our studies show differential cytotoxicity of HCH isomers (alpha, beta, gamma, and delta), delta isomer being most toxic and beta the least. HCH-induced cell death was associated with induction of reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and depletion of glutathione (GSH). The increase in oxidative stress was linked with increased NAD(P)H oxidase activity. HCH inhibited Na(+),K(+)-ATPase, which could be involved in raising the intracellular calcium and increased Ca(2+),Mg(2+)-ATPase activity. HCH lead to apoptotic as well as necrotic cell death as it was marked by increased caspase-3 activity and lactate dehydrogenase (LDH) leakage, respectively. Based on the results it is concluded that the HCH isomers inflict differential cytotoxicity which was highest by delta and lowest by beta. Further, this study demonstrates for the first time a clear link between Na(+),K(+)-ATPase, i[Ca(2+)] level, and oxidative stress in HCH-induced cytotoxicity.