Ravindra M. Satpute

Oxidative stress mediated cytotoxicity of cyanide in LLC-MK2 cells and its attenuation by alpha-ketoglutarate and N-acetyl cysteine

Cyanide is a rapidly acting mitochondrial poison that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia followed by cell death. Cyanide is predominantly a neurotoxin but its toxic manifestations in non-neuronal cells are also documented. This study addresses the oxidative stress mediated cytotoxicity of cyanide in Rhesus monkey kidney epithelial cells (LLC-MK2). Cells were treated with various concentrations of potassium cyanide (KCN) for different time intervals and cytotoxicity was evidenced by increased leakage of intracellular lactate dehydrogenase, mitochondrial dysfunction (MTT assay) and depleted energy status of cells (ATP assay). Cytotoxicity was accompanied by lipid peroxidation indicated by elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) (DCF-DA staining), diminished cellular antioxidant status (reduced glutathione (GSH), glutathione peroxidase, superoxide dismutase and catalase). These cascading events triggered an apoptotic kind of cell death characterized by oligonucleosomal DNA fragmentation and nuclear fragmentation (Hoechst 33342 staining). Apoptosis was further confirmed by increased caspase-3 activity. Cyanide-induced cytotoxicity, oxidative stress, and DNA fragmentation were prevented by alpha-ketoglutarate (A-KG) and N-acetyl cysteine (NAC). A-KG is a potential cyanide antidote that confers protection by interacting with cyanide to form cyanohydrin complex while NAC is a free radical scavenger and enhances the cellular GSH levels. The study reveals cytotoxicity of cyanide in cells of renal origin and the protective efficacy of A-KG and NAC.

Protection of PC12 cells from chemical ischemia induced oxidative stress by Fagonia arabica

Fagonia arabica (Zygophyllaceae) is an important Ayurvedic herb, grows throughout arid regions of India, has been widely used as a folk remedy by the indigenous people for its anti-inflammatory, analgesic and antipyretic effects. In the present study, antioxidant potential of F. arabica and the associated mechanism of antioxidant defence in rat pheochromocytoma (PC12) cells subjected to chemical ischemia was studied. Effect of total extract of F. arabica was studied for its antioxidant potential on the chemical ischemia induced PC12 cells. Alterations in the activities of cellular antioxidant enzymes (SOD, CAT, GSH-Px and GSH-R) were measured. Antioxidant potential of herb (ABTS), extent of lipid peroxidation (MDA and 4-HAE), total antioxidant status (TAS) and total glutathione (reduced, oxidized and their ratio) were evaluated. F. arabica scavenges the free radicals (ABTS(.)+), and showed a concentration dependent antioxidant activity, highest being at 1000 microg/ml. Its treatment with ischemic cells ameliorates the GSH and TAS levels and also helps the cells to restore the activities of the cellular antioxidative enzymes and also reduced the degree of lipid peroxidation. F. arabica scavenges the free radicals and attenuates oxidative stress mediated cell injury during ischemia.

Acute toxicity of some synthetic cyanogens in rats and their response to oral treatment with alpha-ketoglutarate

Oral toxicity of several cyanogens and their reversal by alpha-ketoglutarate (A-KG; oral) were studied in rats. LD(50) of acetonitrile (ATCN), acrylonitrile (ACN), malononitrile (MCN), propionitrile (PCN), sodium nitroprusside (SNP), and succinonitrile (SCN) was 4891, 143.3, 69.8, 122.9, 69.8 and 488.7 mg/kg, respectively while the protection index of A-KG (ratio of LD(50) of cyanogens in the presence or absence of A-KG) was>2.0 against MCN (7.6), PCN (2.7) and SNP (3.6) only. We further studied the efficacy of A-KG against acute toxicity of these three cyanogens (0.75 LD(50)) on various hematological and biochemical variables in blood and soft tissues 24h post-exposure. We observed increase in white blood cells (SNP), plasma alanine (PCN, SNP) and aspartate (PCN) aminotransferase, lactate dehydrogenase (MCN, PCN, SNP), Na(+) (MCN, PCN) and cyanide (PCN), and decrease in K(+) (MCN, SNP) accompanied by an increase in brain, kidney and liver malondialdehyde (PCN), decrease in brain glutathione peroxidase, glutathione reductase (PCN, SNP), reduced glutathione (MCN, PCN, SNP) and cytochrome oxidase (PCN), liver rhodanese (PCN, SNP), and kidney cytochrome oxidase (PCN). The study indicates that (i) PCN was most toxic among all the cyanogens and (ii) beside cyanide, A-KG could be considered as an effective antidote for cyanogens.

Differential diagnosis of tuberculous meningitis from partially-treated pyogenic meningitis by cell ELISA

BackgroundTuberculous meningitis (TBM) is a major global health problem, and it is sometimes difficult to perform a differential diagnosis of this disease from other diseases, particularly partially-treated pyogenic meningitis (PTPM). In an earlier study, we demonstrated the presence of a 30-kD protein antigen in cerebrospinal fluid (CSF) of TBM patients. We have also shown that lymphocytes from CSF of TBM patients respond differently to this antigen than do those from PTPM patients. The purpose of this study was to develop an assay that can discriminate between TBM and PTPM.MethodsWe developed a cell enzyme-linked immunosorbant assay (Cell ELISA) to quantitatively measure production of antibodies against the 30-kD protein in B cells from CSF of TBM and PTPM patients.ResultsThe cell ELISA yielded 92% (11/12) sensitivity and 92% (11/12) specificity for the differential diagnosis of TBM from PTPM.ConclusionWhen induced with the 30-kD protein antigen, B cells derived from CSF of TBM patients respond to IgG production within 24 h while those derived from PTPM patients do not respond.

Activity and gene expression profile of certain antioxidant enzymes in different organs of rats after subacute cyanide exposure: Effect of alpha-ketoglutarate

Oxidative stress plays a crucial role in mediating cyanide toxicity. The present study addresses the effect of cyanide on activity and gene-expression profile of certain antioxidant enzymes and the expression of heat shock protein (HSP-70) in different organs of rats. Rats were treated with 0.50 LD50 (7.0 mg/kg) of potassium cyanide (KCN; oral) and/or alpha-ketoglutarate (A-KG; 1.0 g/kg; oral) daily for 14 days, and various biochemical variables were measured in brain, liver, and kidney after 7 and 14 days of treatments and a 7-day recovery period. Cyanide significantly reduced the activity of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CA) in all the organs after 7 days, while the activity of GPx in brain, liver, and kidney, GR in liver, and CA in brain remained diminished up to 14 days. The gene-expression profile of corresponding enzymes did not show any difference between the control and treatment groups. Elevated levels of malondialdehyde were observed in brain and kidney 7 and 14 days after cyanide. Cyanide also increased the expression of HSP-70 activity in brain after 7 days alone. Regression of toxicity was observed after the withdrawal of KCN. Treatment of A-KG was found to prevent all the biochemical alterations caused by cyanide. This study reveals that oxidative stress caused by cyanide was independent of the expression of antioxidant enzyme activity at the gene level, and all changes responded favorably to A-KG, indicating its therapeutic potential.

Oxidative stress and tissue pathology caused by subacute exposure to ammonium acetate in rats and their response to treatments with alpha-ketoglutarate and N-acetyl cysteine

Ammonia is a widely used industrial chemical that is recognized as a potent neurotoxin and environmental pollutant. The present study addresses the oxidative stress and tissue pathology caused by 4 weeks of exposure to ammonium acetate (AMA; 100 mg/kg daily; orally) in rats, and their response to oral treatments with alpha-ketoglutarate (A-KG; 1.0 g/kg), a potential cyanide antidote, and/or N-acetyl cysteine (NAC; 10 mg/kg), an antioxidant. The organ–body weight index of brain and liver was significantly increased by AMA but kidney was unaffected. Also, plasma ammonia levels were significantly elevated without any concomitant change in blood gas status and hematology but levels of catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and reduced glutathione (GSH) in the brain and liver were diminished, accompanied by elevated levels of malondialdehyde. Levels of glutathione disulfide (GSSG) were unaffected, but the ratio of GSH:GSSG was reduced. Plasma alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase and total bilirubin were raised but urea, uric acid and creatinine levels were not altered. AMA also caused temporal, hepatic and renal pathology. However, the renal pathology was not supported by any biochemical alterations. A-KG or NAC alone afforded less protection against AMA as compared to both given together. The protective efficacy of A-KG can be ascribed to its ability to detoxify ammonia and additionally both A-KG and NAC have antioxidant properties as well. The study suggests a new therapeutic regimen for ammonia poisoning.

Time- and Temperature-Dependent Changes in Cytochrome c Oxidase Activity and Cyanide Concentration in Excised Mice Organs and Mice Cadavers

Postmortem stability of cyanide biomarkers is often disputed. We assessed the time and temperature‐dependent changes in cytochrome c oxidase (CCO) activity and cyanide concentration in various organs of mice succumbing to cyanide. Immediately after death, excised mice organs and mice cadavers were stored at room temperature (35°C ± 5°C) or in frozen storage (−20°C ± 2°C). At various times after death, CCO activity and cyanide concentrations were measured in excised mice organs or organs removed from mice cadavers. The study revealed that (i) measuring both the biomarkers in mice cadavers was more reliable compared to excised mice organs, (ii) measuring temporal CCO activity and cyanide concentration in vital organs from mice cadavers (room temperature) was reliable up to 24 h, and (iii) CCO activity in the brain and lungs and cyanide concentration in organs from mice cadavers (frozen) were measurable beyond 21 days. This study will be helpful in postmortem determination of cyanide poisoning.

Cytotoxicity of cyanide in primary culture of rat hepatocytes and its interaction with alpha-ketoglutarate

Cyanide is primarily a neurotoxin but its hepatotoxic and nephrotoxic potentials are also known. The present study reports the effect of alpha-ketoglutarate A-KG (2.5–20 mM; 0 min), a potential cyanide antidote on potassium cyanide (KCN; 1.25–20 mM) induced cytotoxicity in primary culture of rat hepatocytes. Cytotoxicity measured at various time points (0.5–24 h), was characterized by increased leakage of intracellular lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, accompanied by diminished mitochondrial function (MTT assay), mitochondrial membrane potential (Rhodamine 123 assay), and ATP levels. However, lipid peroxidation (malondialdehyde assay) and DNA damage were not observed. In a separate study, levels of cyanide, AKG and thiocyanate were measured in the culture medium of hepatocytes, treated with KCN (5 mM) and/or A-KG (5 or 10 mM; 0 min), and in the serum of rats given oral treatment of KCN (10 mg/kg) and/or A-KG(0.5, 1 or 2 g/kg; 0 min). Cyanide and A-KG interaction was best exhibited when both were added in equimolar dose in vitro. In rats, cyanide levels were significantly reduced by 1 and 2 g/kg A-KG. It can be concluded from the results that, (i) a very high dose of cyanide is required to produce cytotoxicity and other cellular perturbations in rat hepatocytes, (ii) cytotoxicity is independent of lipid peroxidation and DNA damage, (iii) A-KG provides significant protection against cyanide, particularly at equimolar dose in vitro, and (iv) a very high dose of A-KG is required for cyanide detoxification in vivo, suggesting that the dose of A-KG could be reduced by improving its bioavailability.

Comparative safety evaluation of riot control agents of synthetic and natural origin

Abstract Riot control agents (RCA) are lachrymatory, irritating compounds which temporarily incapacitate the uncontainable crowd. Ortho-Chlorobenzylidene-malononitrile (CS), 2-chloroacetophenone (CN), dibenz[b,f]1:4-oxazepine (CR), and nonivamide (PAVA) are synthetic RCAs, while oleoresin extract of chili known as oleoresin capsicum (OC) a natural irritant has been in use by various law enforcement agencies. Though efficacy of these agents is beyond doubt, they suffer from certain drawbacks including toxicity, production cost, and ecological compatibility. Presently, we have evaluated the safety of CR, OC, and PAVA on inhalation variables along with oral lethality. Additionally, the liver function test (LFT) in serum and lungs function was evaluated in broncho-alveolar-lavage fluid (BALF), both collected on the 14th day after RCA exposure. Animals then sacrificed and histopathology of liver and lungs was carried out. Results showed OC and PAVA to be more toxic than CR with an oral LD50 of 150 and 200 mg/kg body weight, respectively, while CR was safe at >3 g/kg body weight. All three agents caused severe impairment of respiratory variables bringing down normal respiration by >80% with rise in sensory irritation. Recovery from the irritating effect of CR was more rapid than OC and PAVA. LFT and BALF variables were not significantly different from that of control. There were no remarkable histopathological changes in liver and lungs. Hence, as per results, CR is safest among all synthetic and natural origin RCAs and can be safely used for effective dispersion of disobedient mob.