Oxidative stress associated hepatic and renal toxicity induced by ricin in mice.

Ricin a glycoprotein from the Ricinus communis seeds, is known to have diverse toxic effects on cells of different visceral organs. We have studied the hepatotoxicity, nephrotoxicity, and oxidative stress following i.p. administration of ricin (25 microg/kg) in Swiss albino male mice. The results of this study revealed that activities of various enzymes like glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (gamma-GT), and lactate dehydrogenase (LDH) were increased in plasma, liver, and kidney tissues indicating damage in liver and kidney. Blood urea level was also increased. However, blood creatinine and bilirubin were not altered. Lipid peroxidation increased to 49 and 25% in hepatic and renal tissue. Total non-protein sulfhydryl content decreased in plasma (12%), hepatic (29%), and renal (16%) tissues. Superoxide dismutase activity decreased significantly in liver (43%) and kidney (37%). The activity of glutatione peroxidase was also decreased. The decrease was more prominent in kidney than liver. A significant increase, 20 to 27% in the activity of catalase was observed in plasma, liver, and kidney. These results indicate that ricin produces hepatoxicity, nephrotoxicity, and oxidative damage at 24 h of post treatment. The hepatotoxicity was more prominent than nephrotoxicity.

Acute toxicity studies of alpha-ketoglutarate: a promising antidote for cyanide poisoning.

Recently we have shown that cyanide poisoning by the oral (p.o.) route could be antagonized significantly by pretreatment or simultaneous treatment of alpha-ketoglutarate (alpha-KG), administered p.o. in rodents. The protective effect of alpha-KG was dose dependent (0.125-2.0 g kg(-1)) and the effect was significant at a dose above 1.0 g kg(-1). In order to establish the safety of alpha-KG, various haematological, biochemical and histological parameters were studied following p.o. administration of 2.0 g kg(-1)alpha-KG in female rats, and various physiological parameters were studied following p.o. administration of 2.0 or 4.0 g kg(-1)alpha-KG in anaesthetized male rats. The p.o. LD(50) of alpha-KG in male and female rats was >5.0 g kg(-1) and no toxic signs were observed in the surviving animals. Except for an increase in plasma alkaline phosphatase and urea levels after 1 h and a decrease in inorganic phosphorus levels after 7 days of treatment, no significant change in haematology, biochemistry or histology of the vital organs were observed. Mean arterial pressure and neuromuscular transmission were decreased at 4.0 g kg(-1)alpha-KG but other physiological variables such as heart rate, respiratory rate, rectal temperature, left ventricular pressure (systolic), arterial pressure (systolic) and arterial pressure (diastolic) were not altered. The changes observed at 4.0 g kg(-1)alpha-KG are unlikely to be of toxicological concern. The results indicate that alpha-KG at 2.0 g kg(-1) (p.o.)-a dose offering maximum antidotal efficacy-is non-toxic and therefore can be considered suitable for cyanide poisoning.

Protective effect of various antioxidants on the toxicity of sulphur mustard administered to mice by inhalation or percutaneous routes.

Protective effect of various antioxidants, trolox (water soluble analogue of vitamin E), quercetin (bioflavonoid) and glutathione reduced (GSH), was studied following sulphur mustard (SM) intoxication. SM, a blistering agent was administered to Swiss albino female mice through inhalation (1 LC50=42.3 mg/m3 for 1 h duration; 14 days observation for mortality) and percutaneous (1 LD50=154.7 mg/kg; 7 days observation for mortality) routes. The antioxidants were administered three times at the dose of trolox, 500 microg/kg; quercetin, 5 mg/kg and GSH, 400 mg/kg body weight by intraperitoneal injection, one immediately following SM exposure, then once each day for 2 days after SM treatment. The effect of antioxidants on survival, markers of oxidative damage and purine metabolites was investigated. Survival study animals were observed for 14 days. Oxidative markers (in blood, liver and lung) and purine metabolites (in blood and urine) were investigated 72 h after SM treatment. Survival time increased significantly following trolox and quercetin treatments through the inhalation route. Significant decrease in GSH and increase in the level of malondialdehyde (MDA) indicated oxidative damage to liver and lung tissues following SM inhalation and percutaneous exposure. Blood and urinary uric acid, end product of purine metabolism showed an increased following both routes of exposures. The antioxidants, trolox and quercetin protected the liver and lung tissues from oxidative damage caused by SM exposure through inhalation and percutaneous routes. This study showed that antioxidants could enhance survival time, protect liver and lung from oxidative damage and reduce accumulation of purine metabolites in blood following SM intoxication.

Acute inhalation toxicity study of 2-fluoroacetamide in rats.

One of the most potent rodenticides is 2-fluoroacetamide (2-FA). Toxicity of this chemical is well documented. However, its inhalation toxicity data is not available in the literature. Hence, acute inhalation toxicity study was carried out by exposing male and female rats to aerosols of 2-FA at different concentrations for 4 h in a dynamically operated whole body inhalation exposure chamber. During and after the inhalation exposure the rats were less active, and showed mild tremors and convulsions. At higher concentrations the rats died after 2-3 days. The estimated 4-h LC50 for male and female rats was 136.6 and 144.5 mg.m-3 respectively. Exposure to 0.7 LC50 for 4 h duration showed an increase in the liver weight of male and female rats 7 days after exposure. Various haematological and biochemical variables determined were within the normal limits. However, histological findings showed injured lung as indicated by desquamation and necrosis of the epithelium of the respiratory tract. Marked hypertrophy of hepatocytes displaying strong acidophilic granulated cytoplasm was observed. Focal dilatation of renal proximal tubules in kidney with cytoplasmic vacuolation, and irregularly placed pyknotic nuclei were seen. The present study shows that 2-FA is a highly toxic chemical through the inhalation route based on the LC50 value. Consequently necessary precautions should be taken during its handling.

Subacute (90 days) oral toxicity studies of Kombucha tea.

Kombucha tea (KT) is a popular health beverage and is used as an alternative therapy. KT is prepared by placing the kombucha culture in solution of tea and sugar and allowing to ferment. The inoculum is a fungus consisting of symbiotic colony of yeast and bacteria. KT is consumed in several countries and is believed to have prophylactic and therapeutic benefits in a wide variety of ailments, viz., intestinal disorders, arthritis, ageing and stimulation of immunological system. Though KT is used in several parts of the world its beneficial effects and adverse effects have not been scientifically evaluated. Since there are no animal toxicological data on KT, subacute oral toxicity study was carried out. Five groups of rats were maintained: (a) control group given tap water orally, (b) KT given 2 ml/kg orally, (c) plain tea (PT) given 2 ml/kg orally, (d) KT given in drinking water, 1% (v/v) and (e) PT given in drinking water, 1% (v/v). The rats were given this treatment daily for a period of 90 days. Weekly records of weight, feed intake, water intake and general behaviour were monitored. There was no significant difference in the growth of the animals as evidenced by the progressive body weight change. The organ to body weight ratio and histological evaluation did not show any toxic signs. The haematological and biochemical variables were within the clinical limits. The study indicates that rats fed KT for 90 days showed no toxic effects.

Dose response of sulphur mustard: behavioral and toxic signs in rats.

The present study elucidates the behavioral and toxic signs in rats following dermal application of sulphur mustard (SM). Graded doses of SM (0.10, 0.25, 0.50, 0.75 and 1.0 LD50) were topically applied to male Wister rats. The body weight as well as behavioral/toxic signs and symptoms were recorded at 1, 2, 3, and 4th day after application of SM. Sulphur mustard consistently decreased body weights of rats in a dose and time dependent manner with maximum decrease on 3rd day post treatment. Sedation and diarrhea were significant in response to doses of SM intoxication in rats. It is concluded that the body weight, sedation and diarrhea may be used as a reliable parameter in evaluating SM intoxication. It is also suggested that hydration and hypertonic saline must be used as a rescue agent within 1-3 days after exposure to SM.

Protective efficacy of calcium channel blockers in sulphur mustard poisoning.

The present study was designed to ascertain the in vivo protective efficacy of Ca(2+)-channel blockers against dermally applied sulphur mustard (SM). Male albino mice were exposed to 1.5 LD50 of SM (232 mg/kg) percutaneously and the control group received an equal volume of vehicle (polyethylene glycol 300). Prior to SM application, the animals were administered nifedipine and dextrose saline containing antibiotic by intraperitoneal route. The protection assessed by the mean survival time (MST) was determined by Dunnett's method. The MST was significantly increased in nifedipine treated group. The characteristic biochemical indices of SM intoxication, i.e. lipid peroxidation and reduced glutathione (GSH) were determined in liver from animals sacrificed at 24, 48 and 72 h after exposure. SM application (1 LD50) caused a reduction in GSH level which was restored in nifedipine treated group. SM-induced lipid peroxidation was also prevented by nifedipine administration. The protective effect of nifedipine may be related to its capacity of attenuating SM-induced lipid peroxidation and glutathione depletion.

Toxicity evaluation of freshwater cyanobacterium Microcystis aeruginosa PCC 7806: II. Nephrotoxicity in rats.

Nephrotoxic potential of laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 (Pasteur Institute) was assessed in male rats. The animals were injected intraperitoneally with 0.5, 1.0 and 2.0 LD50 doses of lyophilized cell extract. Elevated plasma urea and creatinine levels were accompanied by decrease in protein and albumin levels, followed by hematuria, proteinuria and bilirubinuria. Also decrease in kidney lactate dehydrogenase and glutamic oxaloacetic transaminase indicated possible nephrotoxic potential of the cyanobacteria. The extract also produced various hematological changes associated with stagnant type of hypoxia. High performance liquid chromatography of the culture identified the active principle (toxin) as Microcystin-LR.

Effect of topically applied sulphur mustard on antioxidant enzymes in blood cells and body tissues of rats.

The effect of sulphur mustard (0.5 LD50, percutaneous) on antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)) in blood cells (erythrocytes (RBC), leucocytes (WBC) and platelets) and body tissues (liver, kidney, spleen and brain) of rats has been investigated 24 h post exposure. The SOD activity was significantly decreased in WBC, platelets, spleen and brain as compared to control. The CAT activity was significantly inhibited in RBC, WBC and spleen as compared to control. The GSH-Px activity was signficantly depressed in WBC, spleen and liver as compared to control. It is concluded that sulphur mustard at a sublethal dose inhibited antioxidant enzyme activities in WBC and spleen. Thus, antioxidant enzymes in lymphatic tissues may be used as suitable models for assessing mustard toxicity. The study suggests the formation of reactive oxygen species in sulphur mustard intoxication.

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood.

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C18 cartridges pre-coated with Tween-20. A Polygosil C18 column is used with acetonitrile-water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.

Dose dependent effects on lymphoid organs following a single dermal application of sulphur mustard in mice.

The effects of a single dermal application of sublethal doses [15.5, 7.75 and 3.88 mg kg-1] of bis(2-chloroethyl)sulphide [sulphur mustard, SM] on body weight, organ/body weight ratio, haematology, histology and cellularity of spleen and thymus were studied after 7 days, in Balb/c mice. A progressive fall in body weight was noticed from the fifth day onwards after SM treatment. A dose-dependent decrease in the relative weights of spleen, liver and peripheral lymph nodes, and an increase in adrenal weight were also seen. An increase in red blood cell count, packed cell volume and haemoglobin concentration following SM intoxication were also dose dependent. These changes, together with a significant reduction in the cellularity of the spleen and thymus and degenerative histological changes, show that a single sublethal dermal dose of SM can cause considerable dose dependent systemic effects in Balb/c mice.

Do the hydrolysis products, methylamine and N,N'-dimethylurea, play any role in the methyl isocyanate-induced haematological and biochemical changes in rabbits?

The subcutaneous administration of methyl isocyanate (MIC) to female rabbits, resulted in significant increases in haemoglobin concentration, erythrocyte volume fraction and leucocyte number in blood, as well as plasma total proteins, and urea. The present study was designed to investigate whether the hydrolytic products of MIC, methylamine (MA) and N,N'-dimethylurea (DMU) play any role in eliciting these changes. Both MA and DMU administered subcutaneously in an equimolar dose to that of 1.0 LD50 MIC, 2.2 mmol kg-1, had no influence on these parameters, although there was a marginal increase in the plasma urea level shortly after the administration of DMU. This study establishes that the observed haematological and biochemical changes induced by MIC intoxication in rabbits are mostly due to MIC.

Influence of methylamine and N,N'-dimethylurea, the hydrolysis products of methyl isocyanate, on its systemic toxicity.

Subcutaneous administration of the LD50 dose of methyl isocyanate (MIC) to rats induced severe hyperglycaemia, lactic acidosis and uraemia in rats. Neither methylamine (MA) nor N,N'-dimethylurea (DMU), the hydrolysis products of MIC, administered in equimolar doses had any influence on these parameters except for a marginal transient increase in plasma urea by DMU. Methyl isocyanate administration led to haemoconcentration, resulting in an increase in the plasma concentration of total proteins and a decrease in both the plasma concentration of albumin and the plasma cholinesterase activity. The hydrolysis products of MIC had no influence on any of these parameters. Thus, it seems reasonable to suggest that the systemic effects of MIC are caused by MIC per se, in spite of its high hydrolytic instability.

Effects of topically applied sulphur mustard on tissue glycogen, blood glucose, lactate and pyruvate in mice.

Bis-2-Chloroethyl sulphide, commonly known as sulphur mustard (SM) or mustard gas, an alkylating agent, is frequently used as a chemical warfare agent. Inhibition of glycolysis has been related to skin injury and cell death. The effects of SM on tissue glycogen, blood glucose, lactate/pyruvate ratio were investigated in the present study. After a single dermal application of 1.0 LD50 SM in mice, a significant hyperglycemia was observed at 24 hr post exposure. There was a corresponding decrease in liver glycogen content, with no alteration in glycogen content of brain, muscles and kidney. Blood pyruvate and lactate levels were not appreciably altered.

Biochemical and pathological changes in response to hyperoxia and protection by antioxidants in rats.

A significant decrease in blood haemoglobin, reduced glutathione and protein in lung and liver, without any change in blood reduced glutathione, was observed in rats exposed to 80% oxygen. Hydrogen peroxide induced erythrocyte haemolysis was significantly increased following exposure to hyperoxia. The lungs of rats exposed to hyperoxia showed perivascular edema. Simultaneous treatment with antioxidants, vitamin A, C, or E, protected the animals against oxygen toxicity.

Biochemical changes induced by two new prophylactic regimens for cyanide antagonism.

Clinical efficacy of two pretreatment regimens, sodium nitrite (SN) + hydroxylamine (HA) and SN + 4-dimethylaminophenol (DMAP) were evaluated in rats by studying various biochemical variables at different time intervals. Animals given single subcutaneous (s.c.) co-administration of SN+HA or SN+DMAP showed significantly elevated levels of blood bilirubin indicating hemolytic anemia. Increased levels of blood creatine phosphokinase (CPK), lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (GOT) were indicative of aseptic necrosis at the injection site. On account of low methemoglobin reductase activity in human erythrocytes, a reduced sub-clinical dose of HA or DMAP is envisaged in humans.

Acute toxicity of methyl isocyanate in mammals. IV. Biochemical and hematological changes in rabbits.

Subcutaneous administration of methyl isocyanate (MIC) in 0.5 LD50 and 1.0 LD50 to female rabbits resulted in significant increases of hemoglobin concentration, hematocrit and leukocyte count in blood, as well as plasma total proteins, urea and cholesterol. A significant decrease in plasma albumin level was only observed in the 1.0 LD50 group. Urine of MIC intoxicated animals showed presence of protein, bilirubin, elevated urea and urobilinogen, while urine volume was reduced. The hematological and biochemical changes induced by MIC are perhaps the result of fluid loss from the vascular compartment as evidenced by the histopathological observations. This study further substantiates the view that acute toxicity of MIC is mediated in vivo by its effects on vascular beds.

Dermal intoxication of mice with bis(2-chloroethyl)sulphide and the protective effect of flavonoids.

The influence of dermal application of sulphur mustard (SM) on hepatic lipid peroxidation and the protective effect of flavonoids in SM toxicity was investigated. SM applied on the skin of mice (0.25 or 0.5 LD50) depleted glutathione (GSH) in blood and liver. Malondialdehyde (MDA) levels in the liver showed an increase indicating lipid peroxidation. Administration of vitamin E or two flavonoids, gossypin (GN) and hydroxyethyl rutosides (HR) after dermal application of SM did not alter depletion of GSH but did reduce the MDA level significantly. Survival time of mice with 1 LD50 SM applied dermally was increased by GN and HR to a greater extent than by vitamin E or sodium thiosulphate probably due to one or more of the analgesic, anti-inflammatory, antihepatotoxic, antihistaminic, mast cell stabilization, lipid peroxidation inhibitory and free radical scavenging actions of the flavonoids. The present study indicates that dermally applied SM can induce lipid peroxidation and GSH depletion, and flavonoids may be beneficial in reducing the toxicity.