Aflatoxin B1-induced DNA damage in Labeo rohita: protective effect of an antioxidant supplement, Amrita Bindu.

The present study was undertaken to investigate the effect of potent hepatocarcinogen aflatoxin B1 in adduct formation and DNA damage in Labeo rohita. Also, the salubrious efficacy of an antioxidant supplement Amrita Bindu (based on Indian system of Medicine) was investigated. Fish weighing 175-250 g were administered intraperitoneally a single dose of 100 microg aflatoxin B1/100 g body wt. and another group was given 20% solution of Amrita Bindu along with aflatoxin B1 at 100 microg/100 g body wt. On the 3rd and 6th day, the liver tissue was analyzed for aflatoxin concentration, aflatoxin-DNA adduct formation and DNA damage measured in terms of single strand breaks. The fishes administered with aflatoxin B1 showed elevated concentration of aflatoxin along with a parallel increase in the DNA adduct when compared with the controls. While the fish co-administered with Amrita Bindu showed 34% and 24% reduction in aflatoxin deposition (accumulation) and aflatoxin-DNA adduct formation respectively on the 3rd day, a further reduction by around 41% and 33% in aflatoxin deposition and DNA adduct formation respectively was observed on the 6th day. Furthermore, the increased single strand breaks (measured by alkaline single cell gel assay) and fragmentation observed in agarose gel electrophoresis in aflatoxin B1 administered fish were significantly reduced by Amrita Bindu co-administration. In conclusion, this is the first report to show aflatoxin B1-induced DNA adduct formation and DNA damage in one of the major Indian culturable fish, Labeo rohita. Also, our observations show that the antioxidant supplement, Amrita Bindu, has a potential role in ameliorating the aflatoxin B1-induced DNA damage thus suggesting its applicability in preventing the vital macromolecule DNA.

Excessive free radical generation in the blood of children suffering from asthma.

The aim of the present study is to evaluate the biochemical parameters in blood relevant to oxygen free radicals and antioxidant defenses in children with asthma. A total of 210 asthmatic children, aged 5-18 years, were studied at two different times, once during a severe episode of wheeze (during episode category) and the other after recovery (resting condition). A total of 180 healthy children participated in the study as age and sex matched healthy controls. Superoxide and hydroxyl radical assays were used as a measure of free radical formation. Antioxidant enzymes and free radical scavengers in blood were also assayed. Lipid peroxidation products were assayed in plasma and erythrocytes to evaluate the imbalance (if any) between oxidant (radical) formation and their inactivation. Serum IgE concentrations and peak expiratory flow rate (PEFR) were used as measures of allergic reactions and residual lung capacity, respectively. Excessive production of superoxide and hydroxyl radicals were noted in the blood cells in asthmatics and were correlated to the severity of disease measured as PEFR. Superoxide dismutase and free radical scavengers in blood were significantly lower in asthma, even during resting condition. The present observations endorse the correlation between disease severity and oxygen radical production in asthma subjects. Oxygen metabolites may play a direct or indirect role in the modulation of airway inflammation. Excessive superoxide and hydroxyl radical production may be used as a marker for susceptibility to asthma and for monitoring therapeutic measures.

Studies on some trace and minor elements in blood. A survey of the Kalpakkam (India) population. Part I: Standardization of analytical methods using ICP-MS and AAS.

Blood is one of the widely used specimens for biological trace element research because of its biological significance and ease of sampling. We have conducted a study of the blood of the Kalpakkam township population for trace and minor elements. For this purpose, analytical methods have been developed and standardized in our laboratory for the elemental analysis of blood plasma and red cells. Inductively coupled plasma-mass spectrometry (ICP-MS), a relatively new technique, has been applied for the analysis of trace elements. Details regarding spectral interference and matrix interference encountered in the analysis of blood and the methods of correcting them have been discussed. Flame atomic absorption spectrometry (AAS)/atomic emission spectrometry (AES) has been applied for the determination of minor elements. Precision and accuracy of these methods have also been discussed.

Studies on some trace and minor elements in blood. A survey of the Kalpakkam (India) population. Part III: Studies on dietary intake and its correlation to blood levels.

In our studies on elemental levels in blood of the Kalpakkam population, it was found that the reference values for many elements were normal, but some deficiency with respect to Se was noticed. As a followup study, the dietary ingredients of the local population were analyzed for trace and minor elements to assess the dietary intake of these elements. Details of the analytical methods developed using the technique of inductively coupled plasma-mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) have been described. The dietary intake of many of these trace and minor elements were found to be quite adequate according to the recommended dietary allowance (RDA) levels prescribed, except for Se and Zn. The dietary intake of Se was found to be in the range 20-50 micrograms/d (as opposed to the RDA of 50-200 micrograms/d), whereas the intake of Zn was found to be in the range 8-10 mg/d (as opposed to the RDA of 15 mg/d). Although the deficiency of Se intake was reflected in the blood, that of Zn was not, probably owing to the high level of homeostasis for this element. Fish and egg were found to be rich sources of Se, followed by cereals and pulses, which were found to be the major sources of Zn.

Studies on some trace and minor elements in blood. A survey of the Kalpakkam (India) population. Part II: Reference values for plasma and red cell, and correlation with coronary risk index.

Since data on the trace element levels in Indian population are lacking, we chose to conduct a survey of the Kalpakkam township population. People in the age group 40-55 were included in this study. Reference values for trace and minor elements of the blood of the Kalpakkam population were arrived at by carrying out the analysis of plasma and red cells of healthy subjects of the Kalpakkam population. Although the "reference values" for many elements were found to be normal and comparable to values available in the literature, slight deficiency with respect to Se was noticed. Subjects with high coronary risk index were also included in the study to assess the possible correlation of elemental and lipid profile. A study of box plots showed that the elements Se, Mg, Na, K, and Fe show significant differences between "high risk" coronary risk index (CRI > 5) and "no risk" (CRI < 4.5). In the plasma, the levels of Mg, Na, and K were found to be less in the high-risk group. In red cells, the amount of Se, Fe, and K were found to be significantly less in the "high-risk" group as compared to the "no-risk" group.

Abnormal antioxidant status in impaired glucose tolerance and non-insulin-dependent diabetes mellitus.

A total of 105 subjects with impaired glucose tolerance were classified into two groups, 51 subjects with plasma glucose > 11.1 mmol l-1 in one of the blood samplings during OGTT, but at 2 h being less than < 11.1 mmol l-1 were classified as early hyperglycaemics. Fifty-four cases were classified as true IGT, with fasting plasma glucose < 7.8 mmol l-1 and post plasma glucose level between 7.8 and 11.1 mmol l-1. Age and sex matched groups of normals (healthy adults) and NIDDM cases without symptomatic secondary complications were also included in the study. Lipid peroxidation (LPO) product in plasma, erythrocyte, and erythrocyte cell membrane were found to be significantly elevated (p < 0.001) in IGT, early hyperglycaemia and diabetes mellitus while glycosylated haemoglobin was also higher. Antioxidant enzymes superoxide dismutase and catalase were significantly lower in red blood cells obtained from IGT and early hyperglycaemic groups. They were closer to the levels showed in NIDDM confirming that antioxidant deficiency is already present in subjects classified as impaired glucose tolerant. Among the antioxidant scavengers, reduced glutathione (GSH) and ascorbic acid are reduced by 15% and 20% in IGT and NIDDM, respectively. We conclude that antioxidant status is poor in both IGT and NIDDM, suggesting an overlap of frank diabetic state in those classified as IGT. It is possible that antioxidant therapy might retard progression from IGT to NIDDM.

Antioxidant status and lipid peroxidation in type II diabetes mellitus with and without complications.

1. This study was conducted on 467 cases of non-insulin-dependent diabetes mellitus and 180 healthy controls. Lipid peroxidation products in plasma and erythrocytes were assayed as thiobarbituric acid reactive substances, along with the erythrocyte antioxidant enzymes, namely superoxide dismutase, catalase and glutathione peroxidase. In addition, scavenger vitamins A, C and E and reduced glutathione levels in blood were also measured. 2. Lipid peroxidation was significantly raised within the first 2 years of diagnosis, and superoxide dismutase, catalase, reduced glutathione and vitamins C and E were significantly lowered. 3. These changes were correlated with the duration of the disease and were of a higher magnitude with the development of complications. 4. The results suggest that the antioxidant deficiency and excessive peroxide-mediated damage may appear early on in non-insulin-dependent diabetes mellitus, before the development of secondary complications.

Lipid peroxidation, antioxidant defences and red-cell membrane changes in relation to coronary risk index and symptomatic coronary heart disease.

OBJECTIVE: To establish the normal lipoprotein profile in the population and identify the early warning signs of coronary heart disease (CHD). DESIGN: Random blood sampling of healthy adults and patients with symptomatic CHD including that complicated with acute myocardial infarction. METHODS: Plasma lipids, lipoproteins, lipid peroxidation, antioxidant enzymes and scavengers, red-cell membrane lipids and glycoproteins were assayed. RESULTS AND CONCLUSION: The normal levels of plasma lipids and lipoproteins were established. Levels of plasma free fatty acids, fibrinogen, white blood cell counts, echinocytes, red-cell membrane lipids and protein-bound carbohydrate components are significantly higher in healthy subjects with coronary risk index above 4.5 than they are in normal individuals. Antioxidant defences appear to be the distinguishing factor, remaining higher in normal individuals and thus keeping lipid peroxidation under control. In symptomatic CHD, antioxidant defences are significantly lowered.

Oxidative stress and the development of diabetic complications--antioxidants and lipid peroxidation in erythrocytes and cell membrane.

Erythrocytes isolated from 131 cases of Non-Insulin Dependent Diabetes Mellitus (NIDDM) were studied for lipid peroxidation, antioxidant defences, and the maximum peroxidisable substrate in the cell membrane. Antioxidant defences are lowered in NIDDM, followed by significant rise in lipid peroxidation products. However, in the erythrocyte membrane, the total polyunsaturated peroxidisable lipids are lower than in normal erythrocytes which may be a causative factor affecting the survival of the cells.

Does oxidant stress play a role in diabetic retinopathy?

The role of oxidant stress in the causation of chronic tissue damage is being increasingly recognized. Oxidant stress is usually countered by abundant supply of antioxidants. If concomitant antioxidant deficiency occurs, oxidant stress may produce tissue damage. We took up a study on antioxidant status in non-insulin dependent diabetes mellitus (NIDDM) patients with and without retinopathy and compared them with a control non-diabetic group. The levels of superoxide dismutase (SOD) were significantly reduced in all diabetic patients, i.e., those with and without retinopathy. However, the lowest levels were found in the diabetic patients with retinopathy. Vitamin E and vitamin C levels were also markedly lower in the diabetic patients. There was a paradoxical rise in the catalase and glutathione peroxidase (GPx) in the diabetic patients with retinopathy. This may be a compensatory mechanism by the body to prevent tissue damage by increasing the levels of the two alternative antioxidant enzymes.

Amrita Bindu--a salt-spice-herbal health food supplement for the prevention of nitrosamine induced depletion of antioxidants.

Ayurveda, practised in India, identified a large number of plant components to be used in the diet for the prevention or the delayed development of degenerative disorders. They include some of the commonly used spices, namely pepper and ginger. The Materia Medica includes both naturally occurring and artificially produced salts, as a partial substitute for common salt. Health promoting herbs and spices which are classified pharmacologically as rejuvenating, nourishing, invigorating, cleansing, wound-healing, etc., are used as food additives. Amrita Bindu is a salt-spice-herbal mixture based on these principles and was tested for its effect in maintaining antioxidant defense systems in blood and liver when exposed to a carcinogenic nitrosamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Amrita Bindu supplementation prevented MNNG induced depletion of the antioxidant enzymes and the scavenger antioxidants glutathione and vitamins A, C and E. Amrita Bindu provides protection against free radical and reactive oxygen species induced tissue lipid peroxidation and the resultant tissue degeneration.

Exercise-induced cholesterol depletion and Na+,K(+)-ATPase activities in human red cell membrane.

Red cell membranes were isolated from blood samples obtained from athletes during exhaustive exercise on a bicycle ergometer and during the subsequent recovery period of 60 min. Plasma lactate levels were also determined. During exercise, cell membranes were progressively depleted of cholesterol and, at exhaustion, membrane cholesterol was less than 80% of the initial level. A parallel decline in Na+,K(+)-ATPase was also noted, while phospholipid reduction was around 5%. During recovery, the erythrocyte membrane cholesterol and Na+,K(+)-ATPase increased, but at a slow rate and were inversely proportional to plasma lactate content.

Brahmighritham, an Ayurvedic herbal formula for the control of epilepsy.

Brahmighritham is an Ayurvedic herbal recommended for the control of epilepsy. The preparation of the drug is described. It was tested for its oral effectiveness in controlling pentylenetetrazole-induced seizures in male albino rats and was compared with benzdiazepam. Alterations in the EEG pattern and gross neurological function were measured or rated 60 min after pentylenetetrazole administration. Thirty-day pretreatment with both Brahmighritham and benzdiazepam served to make the rats more insensitive to epileptogenic events.

Double-blind cross-over study of modified Anna Pavala Sindhooram in patients with hyperlipidemia or ischemic heart disease.

The plasma lipid lowering effect of modified Anna Pavala Sindhooram (APSm) was tested in 30 patients with plasma lipid abnormalities and with symptomatic ischemic heart disease (IHD) in a double-blind cross-over design. HDL2 cholesterol and total HDL cholesterol were increased during APSm therapy leading to a reduction in the coronary risk index. Plasma cholesterol, LDL and VLDL cholesterol, triglycerides, phospholipids and free fatty acids were significantly reduced during APSm therapy while withdrawal of the drug led to a slow increase in these lipids. No adverse side effects were noticed. Some patients were able to reduce their daily doses of antihypertensive and vasodilator drugs during APSm therapy.

Effect of patulin on some enzymes of carbohydrate metabolism studied in rats.

The toxic nature of the secondary metabolite of Penicillium patulum has been studied in rats. Liver, Kidney and Intestine of the experimental animals showed derangement in carbohydrate metabolism. Changes in the concentration of a few key enzymes in carbohydrate metabolism have also been studied. Glycogen phosphorylase is found to be markedly increased while the glycolytic enzymes like hexokinase and aldolase are significantly lowered. Gluconeogenesis is stimulated and this is evidenced by increased glucose-6-phosphatase and fructose-1,6-diphosphatase activity. Our results revealed that, patulin, the secondary metabolite of Penicillium patulum showed toxicity in all the organs studied.

Anna Pavala Sindhooram, a novel hypolipidemic agent reduces phospholipid levels in atherosclerosis.

Anna Pavala Sindhooram (APS) was investigated in rabbits during atherosclerosis. Changes in the metabolism of plasma and aortic phospholipids were studied by fractionation into individual lipids by tlc and following the incorporation of radiolabel from 14C-acetate into phospholipids. In these studies APS was found to lower the levels of plasma lecithin and aortic sphingomyelin. The effect of APS on plasma lecithin could be partly attributed to its action on synthesis from acetate while synthesis does not account for the effect observed in aortic sphingomyelin levels.

Control of alcohol addiction by SKV therapy--its action on water, food intake, brain function and cell membrane composition.

Alcohol being easily permeable through cell membrane causes toxic damage to many tissues. Rats drinking aqueous ethanol (25% v/v) for 120 days and 240 days showed an initial rise in body weight. The reduced rate in weight gain in chronic alcoholism is associated with a fall in food intake. Ethanol ingesting animals showed slow response to stimuli and increase in blood ethanol and serum GGTP levels. Liver plasma membrane, kidney brush-border membrane and pancreatic plasma membrane from alcoholic rats showed significant alterations in cholesterol/phospholipid molar ratio and membrane ATPases. Water retention with the enlargement of liver and kidney associated with increased fluid consumption are also seen during alcoholism. SKV by breaking alcohol dependence reduces drinking, lowers blood ethanol level and fluid intake without developing withdrawal symptoms. Restriction of ethanol intake by SKV therapy resulted in the reversal of organ enlargement and membrane composition in alcoholics.

Effect of an isolate from gymnema sylvestre, R. Br. In the control of diabetes mellitus and the associated pathological changes.

Gymnema sylvestre, R. Br., popularly known as Meshashringi in Sanskrit and Sarkaraikolli in Tamil, was investigated for the control of type I (insulin dependent) diabetes in experimental animals. The hypoglycaemic extract was found to bring about blood glucose homeostasis, by increasing serum insulin levels. The islets of langerhans appear to be restored or regulated by the herbal extract. Increased glycoprotein, which is the major metabolic abnormality in diabetes mellitus and the resultant nephropathy, retinopathy and micro and macroangiopathy, is brought under control by the administration of the leaf extract.

Effect of high-fat diet on cholesterol distribution in plasma lipoproteins, cholesterol esterifying activity in leucocytes, and erythrocyte membrane components studied: importance of body weight.

Effects of isocaloric changes in dietary fat on plasma lipoproteins and lipids and enzymes of erythrocytes and leucocytes were assessed. Subjects with a higher Brocca index showed increase in total and LDL cholesterol, significant reduction in HDL cholesterol, and increased total cholesterol:HDL cholesterol ratio after high-fat diet consumption. Due to high-fat diet feeding, erythrocyte membrane and leucocyte cholesterol and phospholipid contents were increased, cholesterol:phospholipid molar ratio was elevated, and erythrocyte enzymes (G6PD and 6PGD) and leucocyte enzymes (CEH and CES) were elevated. Erythrocyte membrane glycoprotein components showed marked increase, indicating possible alterations of membrane surfaces. The metabolic alterations were reversed slowly after resumption of the normal (low-fat) diet. Body weight plays an important role in the alterations in major lipoprotein cholesterol contents in response to changes in dietary fat composition. Cellular changes indicate alterations in structure and function of blood cells due to high-fat diet feeding.

Studies on brain structure and neurological function in alcoholic rats controlled by an Indian medicinal formula (SKV).

An Indian herbal brew known in Ayurvedic pharmacy as asavam (SKV) was tested for its effectiveness in controlling addiction to ethanol in rats. Rats on SKV therapy with free access to 15% ethanol showed a marked reduction in voluntary ethanol intake. Their performance in simple neurological tests improved and a reversal of ethanol-induced changes in the electroencephalogram and electrocardiogram were also recorded. SKV treatment appeared to correct the fatty changes in liver and the signs of haemorrhage, demyelination and spongiosis seen in the brain of ethanol-fed rats.