Uday Bandyopadhyay

A Novel Antioxidant and Antiapoptotic Role of Omeprazole to Block Gastric Ulcer through Scavenging of Hydroxyl Radical

The mechanism of the antiulcer effect of omeprazole was studied placing emphasis on its role to block oxidative damage and apoptosis during ulceration. Dose-response studies on gastroprotection in stress and indomethacin-induced ulcer and inhibition of pylorus ligation-induced acid secretion indicate that omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent mechanism for its antiulcer effect. Time course studies on gastroprotection and acid reduction also indicate that omeprazole almost completely blocks lesions at 1 h when acid inhibition is partial. The severity of lesions correlates well with the increased level of endogenous hydroxyl radical (⋅OH), which when scavenged by dimethyl sulfoxide causes around 90% reduction of the lesions, indicating that ⋅OH plays a major role in gastric damage. Omeprazole blocks stress-induced increased generation of ⋅OH and associated lipid peroxidation and protein oxidation, indicating that its antioxidant role plays a major part in preventing oxidative damage. Omeprazole also prevents stress-induced DNA fragmentation, suggesting its antiapoptotic role to block cell death during ulceration. The oxidative damage of DNA by ⋅OH generated in vitro is also protected by omeprazole or its analogue, lansoprazole. Lansoprazole when incubated in a ⋅OH-generating system scavenges⋅OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak atm/z 385, which is 16 mass units higher than that of lansoprazole (m/z 369). The product shows no additional aromatic proton signal for aromatic hydroxylation in 1H NMR. The product absorbing at 278 nm shows no alkaline shift for phenols, thereby excluding the formation of hydroxylansoprazole. The product is assigned to lansoprazole sulfone formed by the addition of one oxygen atom at the sulfur center following attack by the ⋅OH. Thus, omeprazole plays a significant role in gastroprotection by acting as a potent antioxidant and antiapoptotic molecule.

Apoptosis in liver during malaria: role of oxidative stress and implication of mitochondrial pathway

Hepatic dysfunction is a common clinical complication in malaria, although its pathogenesis remains largely unknown. Using a variety of in vivo and ex vivo approaches, we have shown for the first time that malarial infection induces hepatic apoptosis through augmentation of oxidative stress. Apoptosis in hepatocyte has been confirmed by terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin‐nickend labeling assay (TUNEL) and caspase‐3 activation. Gene expression analysis using RT‐PCR indicates the significant down‐regulation of Bcl‐2 and up‐regulation of Bax expression in liver of malaria infected mice suggesting the involvement of mitochondrial pathway of apoptosis. The levels of Fas expression and caspase‐8 activity in infected liver were same as that of uninfected control mice indicating death receptor (Fas) pathway did not contribute to liver apoptosis during malarial infection. Moreover, evidence has been presented by confocal microscopy to show the translocation of Bax from cytosol to mitochondria in apoptotic hepatocyte, resulting in opening of permeability transition pores, which in turn decreases mitochondrial membrane potential and induces cytochrome c release into cytosol. Malarial infection induces the generation of hydroxyl radical (·OH) in liver, which may be responsible for the induction of oxidative stress and apoptosis as administration of ·OH specific antioxidant as well as spin trap, alpha‐phenyl‐tert‐butyl‐nitrone in malaria‐infected mice significantly inhibits the development of oxidative stress as well as induction of apoptosis. Thus, results suggest the implication of oxidative stress induced‐mitochondrial pathway of apoptosis in the pathophysiology of hepatic dysfunction in malaria.—Guha, M., Kumar, S., Choubey, V., Maity, P., Bandyopadhyay, U. Apoptosis in liver during malaria: Role of oxidative stress and implication of mitochondrial pathway. FASEB J. 20, E439–E449 (2006)

Gastroprotective effect of Neem (Azadirachta indica) bark extract: Possible involvement of H+-K+-ATPase inhibition and scavenging of hydroxyl radical

The antisecretory and antiulcer effects of aqueous extract of Neem (Azadirachta indica) bark have been studied along with its mechanism of action, standardisation and safety evaluation. The extract can dose dependently inhibit pylorus-ligation and drug (mercaptomethylimidazole)-induced acid secretion with ED(50) value of 2.7 and 2 mg Kg(-1) b.w. respectively. It is highly potent in dose-dependently blocking gastric ulcer induced by restraint-cold stress and indomethacin with ED(50) value of 1.5 and 1.25 mg Kg(-1) b.w. respectively. When compared, bark extract is equipotent to ranitidine but more potent than omeprazole in inhibiting pylorus-ligation induced acid secretion. In a stress ulcer model, it is more effective than ranitidine but almost equipotent to omeprazole. Bark extract inhibits H(+)-K(+)-ATPase activity in vitro in a concentration dependent manner similar to omeprazole. It offers gastroprotection against stress ulcer by significantly preventing adhered mucus and endogenous glutathione depletion. It prevents oxidative damage of the gastric mucosa by significantly blocking lipid peroxidation and by scavenging the endogenous hydroxyl radical ((z.rad;)OH)-the major causative factor for ulcer. The (z.rad;)OH-mediated oxidative damage of human gastric mucosal DNA is also protected by the extract in vitro. Bark extract is more effective than melatonin, vitamin E, desferrioxamine and alpha-phenyl N-tert butylnitrone, the known antioxidants having antiulcer effect. Standardisation of the bioactive extract by high pressure liquid chromatography indicates that peak 1 of the chromatogram coincides with the major bioactive compound, a phenolic glycoside, isolated from the extract. The pharmacological effects of the bark extract are attributed to a phenolic glycoside which is apparently homogeneous by HPLC and which represents 10% of the raw bark extract. A single dose of 1g of raw extract per kg b.w. (mice) given in one day and application of 0.6g raw extract per kg b.w. per day by oral route over 15 days to a cumulative dose of 9g per kg was well tolerated and was below the LD(50). It is also well tolerated by rats with no significant adverse effect. It is concluded that Neem bark extract has therapeutic potential for the control of gastric hyperacidity and ulcer.

Melatonin protects against stress-induced gastric lesions by scavenging the hydroxyl radical.

The antiulcer effect of melatonin on gastric lesions caused by restraint‐cold stress or by indomethacin (IMN) was studied with the intent of determining the mechanism of action of the indole. Melatonin dose‐dependently prevents both stress and IMN‐induced gastric damage with around 90% inhibition at a dose of 60 mg per kg BW. When compared with already‐marketed antiulcer drugs, such as ranitidine and omeprazole, melatonin was found to be more effective than ranitidine but less effective than omeprazole in preventing stress ulcer. When compared with other antioxidants, melatonin was more potent than glutathione and essentially equipotent to α‐tocopherol in blocking stress‐induced ulcer. As stress‐induced gastric lesions are mainly caused by oxidative damage due to hydroxyl radicals (OH), the effect of melatonin in scavenging the OH generated during stress conditions, as well as in an in vitro model system, was studied. The results indicate that melatonin at the dose of 60 mg per kg BW caused an 88% reduction of endogenous OH during stress. Melatonin was also highly effective in scavenging OH generated in vitro by a Cu2+‐ascorbate system. In this case, melatonin at 100 μM reduced OH by 80%. Melatonin was also found to be a more potent radical scavenger than benzoate, a known OH scavenger. The results indicate that melatonin prevents stress‐induced gastric lesions by scavenging the endogenous OH. As it also protects against IMN‐induced gastric damage, it probably also offers gastroprotection by maintaining endogenous prostaglandin levels.

Melatonin inhibits free radical‐mediated mitochondrial‐dependent hepatocyte apoptosis and liver damage induced during malarial infection

Abstract:  We showed earlier that malarial infection significantly induces liver apoptosis mediated by oxidative stress mechanisms. Thus, a nontoxic antioxidant–antiapoptotic molecule may be beneficial for hepatoprotection. Melatonin remarkably prevents hepatocyte apoptosis in mice induced during malaria as indicated by caspase 3 and TUNEL assays as well as transmission electron microscopy (TEM) of the liver tissue. The mitochondrial apoptotic pathway, which plays a critical role in liver cell death during malarial infection, was almost completely suppressed by melatonin as it corrects both the overexpression of Bax and down‐regulation of bcl‐2 as revealed by semiquantitative RT‐PCR. Fluorometric studies using JC‐1 documented that melatonin also restores mitochondrial transmembrane potential (ΔΨm) in malaria‐infected mice liver. The antiapoptotic effect of melatonin is associated with its antioxidant role because melatonin protects liver from oxidative stress induced during malaria by scavenging the hydroxyl radicals, preventing the depletion of reduced glutathione, inhibiting lipid peroxidation and protein carbonyl formation. The effective antioxidant dose of melatonin to protect liver from oxidative stress during malaria is 20 times lower than that of known antioxidants, vitamin C and vitamin E. Apoptosis of hepatocytes during malarial infection is well correlated with dysfunction of the liver while melatonin offers hepatoprotective effects as indicated by different liver function tests. Thus, melatonin may well be effective in combating oxidative stress‐induced apoptosis and liver damage during malaria infection.

Smoking and the pathogenesis of gastroduodenal ulcer--recent mechanistic update.

Peptic ulcer is a common disorder of gastrointestinal system and its pathogenesis is multifactorial, where smoking and nicotine have significant adverse effects. Smoking and chronic nicotine treatment stimulate basal acid output which is more pronounced in the smokers having duodenal ulcer. This increased gastric acid secretion is mediated through the stimulation of H2-receptor by histamine released after mast cell degranulation and due to the increase of the functional parietal cell volume or secretory capacity in smokers. Smoking and nicotine stimulate pepsinogen secretion also by increasing chief cell number or with an enhancement of their secretory capacity. Long-term nicotine treatment in rats also significantly decreases total mucus neck cell population and neck-cell mucus volume. Smoking also increases bile salt reflux rate and gastric bile salt concentration thereby increasing duodenogastric reflux that raises the risk of gastric ulcer in smokers. Smoking and nicotine not only induce ulceration, but they also potentiate ulceration caused by H. pylori, alcohol, nonsteroidal anti-inflammatory drugs or cold restrain stress. Polymorphonuclear neutrophils (PMN) play an important role in ulcerogenesis through oxidative damage of the mucosa by increasing the generation of reactive oxygen intermediates (ROI), which is potentiated by nicotine and smoking. Nicotine by a cAMP-protein kinase A signaling system elevates the endogenous vasopressin level, which plays an aggressive role in the development of gastroduodenal lesions. Smoking increases production of platelet activating factor (PAF) and endothelin, which are potent gastric ulcerogens. Cigarette smoking and nicotine reduce the level of circulating epidermal growth factor (EGF) and decrease the secretion of EGF from the salivary gland, which are necessary for gastric mucosal cell renewal. Nicotine also decreases prostaglandin generation in the gastric mucosa of smokers, thereby making the mucosa susceptible to ulceration. ROI generation and ROI-mediated gastric mucosal cell apoptosis are also considered to be important mechanism for aggravation of ulcer by cigarette smoke or nicotine. Both smoking and nicotine reduce angiogenesis in the gastric mucosa through inhibition of nitric oxide synthesis thereby arresting cell renewal process. Smoking or smoke extract impairs both spontaneous and drug-induced healing of ulcer. Smoke extract also inhibits gastric mucosal cell proliferation by reducing ornithine decarboxylase activity, which synthesises growth-promoting polyamines. It is concluded that gastric mucosal integrity is maintained by an interplay of some aggressive and defensive factors controlling apoptotic cell death and cell proliferation and smoking potentiates ulcer by disturbing this balance.

Indomethacin, a Non-steroidal Anti-inflammatory Drug, Develops Gastropathy by Inducing Reactive Oxygen Species-mediated Mitochondrial Pathology and Associated Apoptosis in Gastric Mucosa A NOVEL ROLE OF MITOCHONDRIAL ACONITASE OXIDATION

We have investigated the role of mitochondria on the development of indomethacin (a non-steroidal anti-inflammatory drug)-induced gastric mucosal apoptosis and associated gastropathy in rat. Transmission electron microscopic studies indicate that indomethacin damages mitochondrial ultrastructure and causes mitochondrial dysfunction as evident from decreased stage-3 respiration, dehydrogenase activity, and transmembrane potential (ΔΨm). Mitochondrial pathology is associated with increased generation of intra-mitochondrial-reactive oxygen species, such as \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}, H2O2 and ·OH, leading to oxidative stress. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} is the most effective to damage mitochondrial aconitase, leading to the release of iron from its iron-sulfur cluster. The released iron, by interacting with intra-mitochondrial H2O2, forms ·OH. Immunoprecipitation of mitochondrial aconitase and subsequent Western immunoblotting indicate carbonylation of aconitase along with the loss of activity in vivo after indomethacin treatment. The release of iron has been documented by fluorescence imaging of mucosal cells by using Phen Green SK, a specific probe for chelatable iron. Interestingly, intra-mitochondrial ·OH generation is crucial for the development of mitochondrial pathology and activation of mitochondrial death pathway by indomethacin. Scavenging of ·OH by dimethyl sulfoxide or α-phenyl-n-tert-butylnitrone, a spin-trap, prevents indomethacin-induced mitochondrial ultrastructural changes, oxidative stress, collapse of ΔΨm, and mitochondrial dysfunction. The scavengers also restore indomethacin-induced activation of caspase-9 and caspase-3 to block mitochondrial pathway of apoptosis and gastric mucosal damage. This study, thus, reveals the critical role of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}-mediated mitochondrial aconitase inactivation to release intra-mitochondrial iron, which by generating ·OH promotes gastric mucosal cell apoptosis and gastropathy during indomethacin treatment.

Gallic Acid Prevents Nonsteroidal Anti-Inflammatory Drug-Induced Gastropathy in Rat by Blocking Oxidative Stress and Apoptosis

Nonsteroidal anti-inflammatory drug (NSAID)-induced oxidative stress plays a critical role in gastric mucosal cell apoptosis and gastropathy. NSAIDs induce the generation of hydroxyl radical ((*)OH) through the release of free iron, which plays an important role in developing gastropathy. Thus, molecules having both iron-chelating and antiapoptotic properties will be beneficial in preventing NSAID-induced gastropathy. Gallic acid (GA), a polyphenolic natural product, has the capacity to chelate free iron. Here, we report that GA significantly prevents, as well as heals, NSAID-induced gastropathy. In vivo, GA blocks NSAID-mediated mitochondrial oxidative stress by preventing mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. In vitro, GA scavenges free radicals and blocks (*)OH-mediated oxidative damage. GA also attenuates gastric mucosal cell apoptosis in vivo as well as in vitro in cultured gastric mucosal cells as evident from the TUNEL assay. GA prevents NSAID-induced activation of caspase-9, a marker for the mitochondrial pathway of apoptosis, and restores NSAID-mediated collapse of the mitochondrial transmembrane potential and dehydrogenase activity. Thus, the inhibition of mitochondrial oxidative stress by GA is associated with the inhibition of NSAID-induced mitochondrial dysfunction and activation of apoptosis in gastric mucosal cells, which are responsible for gastric injury or gastropathy.

Translocation of Heme Oxygenase-1 to Mitochondria Is a Novel Cytoprotective Mechanism against Non-steroidal Anti-inflammatory Drug-induced Mitochondrial Oxidative Stress, Apoptosis, and Gastric Mucosal Injury

Background: The inherent cytoprotective mechanism involved in repair of injured gastric mucosa is not clear. Results: HO-1 is induced and translocated to mitochondria to favor repair of gastric mucosal injury induced by non-steroidal anti-inflammatory drug-mediated mitochondrial oxidative stress (MOS). Conclusion: Mitochondrial localization of HO-1 is a novel cytoprotective mechanism against MOS-mediated gastric mucosal injury. Significance: Induction of HO-1 in gastric mucosa is beneficial for gastroprotection. The mechanism of action of heme oxygenase-1 (HO-1) in mitochondrial oxidative stress (MOS)-mediated apoptotic tissue injury was investigated. MOS-mediated gastric mucosal apoptosis and injury were introduced in rat by indomethacin, a non-steroidal anti-inflammatory drug. Here, we report that HO-1 was not only induced but also translocated to mitochondria during gastric mucosal injury to favor repair mechanisms. Furthermore, mitochondrial translocation of HO-1 resulted in the prevention of MOS and mitochondrial pathology as evident from the restoration of the complex I-driven mitochondrial respiratory control ratio and transmembrane potential. Mitochondrial translocation of HO-1 also resulted in time-dependent inhibition of apoptosis. We searched for the plausible mechanisms responsible for HO-1 induction and mitochondrial localization. Free heme, the substrate for HO-1, was increased inside mitochondria during gastric injury, and mitochondrial entry of HO-1 decreased intramitochondrial free heme content, suggesting that a purpose of mitochondrial translocation of HO-1 is to detoxify accumulated heme. Heme may activate nuclear translocation of NF-E2-related factor 2 to induce HO-1 through reactive oxygen species generation. Electrophoretic mobility shift assay and chromatin immunoprecipitation studies indicated nuclear translocation of NF-E2-related factor 2 and its binding to HO-1 promoter to induce HO-1 expression during gastric injury. Inhibition of HO-1 by zinc protoporphyrin aggravated the mucosal injury and delayed healing. Zinc protoporphyrin further reduced the respiratory control ratio and transmembrane potential and enhanced MOS and apoptosis. In contrast, induction of HO-1 by cobalt protoporphyrin reduced MOS, corrected mitochondrial dysfunctions, and prevented apoptosis and gastric injury. Thus, induction and mitochondrial localization of HO-1 are a novel cytoprotective mechanism against MOS-mediated apoptotic tissue injury.

Extrathyroidal actions of antithyroid thionamides

Some compounds having thionamide structure inhibit thyroid functions. Such antithyroid thionamides include mercaptomethylimidazole (methimazole), thiourea and propylthiouracil, of which mercaptomethylimidazole is widely used to treat hyperthyroidism. Undesirable side effects develop from these drugs due to extrathyroidal actions. Antithyroid thionamides inhibit lactoperoxidase which contributes to the antibacterial activities of a number of mammalian exocrine gland secretions that protect a variety of mucosal surfaces. These drugs stimulate both gastric acid and pepsinogen secretions, thereby augmenting the severity of gastric ulcers and preventing wound healing. Increased gastric acid secretion is partially due to the H2 receptor activation, and also through the stimulation of the parietal cell by intracellular generation of H2O2 following inactivation of the gastric peroxidase-catalase system. Severe abnormalities may develop in blood cells and the immune system after thionamide therapy. It causes agranulocytosis, aplastic anemia, and purpura along with immune suppression. Olfactory and auditory systems are also affected by these drugs. Thionamide affects the sense of smell and taste and also causes loss of hearing. It binds to the Bowmans glands in the olfactory mucosa and causes extensive lesion in the olfactory mucosa. Thionamides also affect gene expression and modulate the functions of some cell types. A brief account of the chemistry and metabolism of antithyroid thionamides, along with their biological actions are presented.