Analysis of gene mutations among South Indian patients with maple syrup urine disease: Identification of four novel mutations.

Maple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1α, E1β and E2 subunits of the branched-chain α-keto acid dehydrogenase complex, respectively. Because disease causing mutations play a major role in the development of the disease, prenatal diagnosis at gestational level may have significance in making decisions by parents. Thus, this study was aimed to screen South Indian MSUD patients for mutations and assess the genotype-phenotype correlation. Thirteen patients diagnosed with MSUD by conventional biochemical screening such as urine analysis by DNPH test, thin layer chromatography for amino acids and blood amino acid quantification by HPLC were selected for mutation analysis. The entire coding regions of the BCKDHA, BCKDHB and DBT genes were analyzed for mutations by PCR-based direct DNA sequencing. BCKDHA and BCKDHB mutations were seen in 43% of the total ten patients, while disease-causing DBT gene mutation was observed only in 14%. Three patients displayed no mutations. Novel mutations were c.130C>T in BCKDHA gene, c. 599C>T and c.121_122delAC in BCKDHB gene and c.190G>A in DBT gene. Notably, patients harbouring these mutations were non-responsive to thiamine supplementation and other treatment regimens and might have a worse prognosis as compared to the patients not having such mutations. Thus, identification of these mutations may have a crucial role in the treatment as well as understanding the molecular mechanisms in MSUD.

Dietary grapes (Vitis vinifera) feeding attenuates ethanol-induced oxidative stress in blood and modulates immune functions in mice.

Ethanol metabolism is known to induce overwhelming production of reactive oxygen species (ROS) and also to cause associated immune dysfunction. Several interventional agents of plant origin, in particular fruits and vegetables have been used to counteract these alterations induced by ethanol. In this study, we investigated the efficacy of dietary feeding of skin and flesh of grapes (Vitis vinifera L.) on the alterations in immune and vascular functions in mice with liver abnormalities induced by chronic ethanol consumption. Results revealed that feeding of both grape skin and flesh (2.5 g/kg body wt/day) effectively attenuated the oxidative stress and alterations in immune function and angiogenesis induced by chronic ethanol consumption (1.6 g/kg body wt/day for 12 weeks) in mice. The antioxidant actions of the grape skin and flesh as observed in this study might be attributed to the polyphenols present in the grapes.

Effects of long-term ethanol consumption on adhesion molecules in liver.

Adhesion molecules play an important role in the pathogenesis of several diseases. In this study, expression of adhesion molecules was examined in the setting of chronic alcohol induced liver damage of male albino Wistar strain rats (16-18 weeks-old, 200-220 g) in a time dependent manner. Decreased protein level and increased activities of liver marker enzymes in response to the chronic ethanol (1.6 g ethanol/kg body weight/day) exposure, indicated that these animals suffered from liver damage in a time-dependent manner. Flow cytometric analysis revealed that chronic ethanol treatment induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression in liver tissues of rats with duration of ethanol exposure. The results suggest that the adhesion molecules may be associated with the initiation of hepatic injury during alcohol intoxication.

Protective effect of resveratrol and vitamin E against ethanol-induced oxidative damage in mice: Biochemical and immunological basis.

The metabolism of ethanol gives rise to the generation of excess amounts of reactive oxygen species and is also associated with immune dysfunction. We examined the efficacy of resveratrol and vitamin E on the immunomodulatory activity and vascular function in mice with liver abnormalities induced by chronic ethanol consumption by measuring the protein, liver-specific transaminase enzymes, antioxidant enzymes and non-enzymes such as reduced glutathione (GSH) content, thiobarbituric acid reactive substance (TBARS) level, nitrite level, and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and cytokines such as interleukin (IL)-2, IL-4, IL-10, tumor necrosis factor (TNF)-, gamma interferon (IFN-), vascular endothelial growth factor (VEGF)-A and transforming growth factor (TGF)-1 in mice blood. Ethanol (1.6 g/kg body wt/day) exposure for 12 wks significantly increased TBARS and nitrite levels and GST activity, and significantly decreased GSH content and the activities of SOD, CAT, GR and GPx in whole blood hemolyzate of 8-10 wks-old male BALB/c mice (weighing 20-30 g). Ethanol exposure also elevated the activities of transaminase enzymes (AST and ALT), IL-10, TNF-, IFN-, VEGF-A and TGF-1, while decreasing the albumin concentration and IL-4 activity in the serum. Both resveratrol (5 mg kg-1 day-1) and vitamin E (80 mg kg-1 day-1) treatment significantly reduced AST, ALT, GST, IL-10, TNF-, IFN-, VEGF-A and TGF-1 activities and levels of TBARS and nitrite, and elevated albumin content, GSH level and activities of SOD, CAT, GR and GPx, compared to ethanol-treated group. Thus, results from the study demonstrated that both resveratrol (5 mg kg-1 day-1) and vitamin E (80 mg kg-1 day-1) can effectively ameliorate ethanol (1.6 g kg-1 day-1)-induced oxidative challenges, immunomodulatory activity and angiogenesis processes.

Time-dependent effects of ethanol on blood oxidative stress parameters and cytokines.

Alcohol consumption is implicated in the genesis of a spectrum of liver abnormalities, which are associated with a number of factors. In the present study, time-dependent effects of ethanol on cytokines (TNF-alpha, IL-2, IL-4, IL-10, IFN-gamma, VEGF-A and TGF-beta1) in serum, and blood oxidative stress parameters such as reduced glutathione content, TBARS level and activities of GPx, GR, GST, catalase and SOD in 8-10 weeks-old male BALB/c mice have been investigated. Ethanol administered @ 1.6 g/kg body wt/day significantly increased the activities of liver marker enzymes AST, ALT and ALP. Serum nitrite levels and haemolysate TBARS level also increased, while total antioxidant status in serum and GSH content in whole blood hemolysate decreased from 4th week onwards of exposure. In spite of the increased serum nitrite level and GST activity in the haemolysate, albumin level in serum, GPx and GR activities in haemolysate decreased after 12 weeks of exposure. Chronic ethanol treatment did not show any effect on IL-2, but IL-4 level was reduced and other cytokines such as IL-10, TNF-alpha, IFN-gamma, TGF-beta1 and VEGF-A levels were increased significantly after 12 weeks. The study indicates a relationship between free radical generation and immune response, and suggests that ethanol-induced liver damage is associated with oxidative stress and immunological alterations in a time-dependent manner.

Effect of lecithin with vitamin-B complex and tocopheryl acetate on long-term effect of ethanol induced immunomodulatory activities.

The alcoholic liver disease usually causes overall immunological alterations which might be attributed to hepatic disease, to ethanol action, and/or to malnourishment. In the present study, efficacy of lecithin with vitamin-B complex to treat ethanol induced immunomodulatory activity was compared with the effect of lecithin alone and tocopheryl acetate (vitamin E). Ethanol (1.6 g/kg body wt/day for 12 weeks) exposure increased thiobarbituric acid reactive substance (TBARS) level, while decreased superoxide dismutase (SOD) activity and reduced glutathione (GSH) content in whole blood hemolysate of 8-10 week-old male BALB/c mice (weighing 20-30 g). The activities of transaminase (AST and ALT) enzymes, interleukin (IL)-10 and gamma interferon (IFN-gamma) elevated, while IL-2 and IL-4 reduced in mice serum due to ethanol exposure. These suggested that oxidative stress and immunomodulatory activities were interdependent and associated with ethanol induced liver damage. Lecithin treatment significantly reduced AST (32.44%), ALT (32.09%), IL-10 (25.63%) activities and TBARS content (12.76%) compared to ethanol treated group. However, lecithin with vitamin-B complex treatment, significantly reduced AST (62.83%); ALT (61.96%); IL-10 (35.88%); IFN-gamma (22.55%) activities and TBARS content (31.58%), while significantly elevated GSH content (36.49%) and SOD activity (61.21%). Tocopheryl acetate treatment significantly reduced AST (62.83%); ALT (61.54%); IL-10 (36.35%): IFN-gamma (23.28%) activities and TBARS content (35.84%). while significantly elevated GSH content (28.76%) and SOD activity (62.42%) compared to ethanol treated group. These findings persuasively argued that lecithin with vitamin-B complex was a new promising therapeutic approach in controlling ethanol induced immunomodulatory activities involving liver damage processes. Prevention of oxidative stress with correction of nutritional deficiency caused alteration in the ethanol-induced immunomodulatory activities and associated liver diseases.

Modulation of lecithin activity by vitamin-B complex to treat long term consumption of ethanol induced oxidative stress in liver.

Alcoholic liver disease (ALD) develops as a consequence of priming and sensitizing mechanisms rendered by cross-interactions of primary mechanistic factors and secondary risk factors. Chronic alcohol abuse and its progression to ALD are associated with abnormal metabolism and low tissue or plasma levels, or both, of many micronutrients. Glutathione depletion is considered the most important sensitizing mechanism. In the present study efficacy of lecithin with vitamin-B complex to treat ethanol induced oxidative stress was compared with the effect of lecithin alone, tocopheryl acetate (vitamin E), as well as capacity of hepatic regeneration during abstention. Ethanol (1.6g / kg body weight/ day for 4 weeks) affects body weight in 16-18 week old male albino rats of Wistar strain weighing 200-220 g. Thiobarbituric acid reactive substance level, nitrite content, protein carbonyl group level, redox ratio (oxidized to reduced glutathione ratio), superoxide dismutase activity, and glutathione s-transferase activity significantly increased on ethanol exposure. Whereas reduced glutathione content, and activities of catalase, glutathione reductase and glutathione peroxidase significantly reduced due to ethanol exposure. These changes were reversed by different treatment. The results suggest that tocopheryl acetate (vitamin E) could partially reverse these changes and act as a potential therapeutic agent. However, lecithin with vitamin-B complex treatment is a promising therapeutic approach. Furthermore, preventive measures were more effective than curative treatment. Prevention of oxidative and nitrosative stress along with correction of nutritional deficiency is one of the proposed mechanisms for the therapeutic approach.

Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver.

The production of reactive oxygen species (ROS) is considered to be a major factor in oxidative cell injury. The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against such hepatic damage. Silymarin, derived from the milk thistle plant, Silybium marianum, has been used in traditional medicine as a remedy for diseases of the liver and biliary tract. In the present study, the effect of hepatoprotective drug silymarin on body weight and biochemical parameters, particularly, antioxidant status of ethanol-exposed rats was studied and its efficacy was compared with the potent antioxidant, ascorbic acid as well as capacity of hepatic regeneration during abstention. Ethanol, at a dose of 1.6 g/kg body wt/day for 4 wks affected body weight in 16-18 week-old male albino rats (Wistar strain weighing 200-220 g). Thiobarbituric acid reactive substance (TBARS) level, superoxide dismutase (SOD), and glutathione-s-transferase (GST) activities were significantly increased, whereas GSH content, and catalase, glutathione reductase (GR) and GPx (glutathione peroxidase) activities significantly reduced, on ethanol exposure. These changes were reversed by silybin and ascorbic acid treatment. It was also observed that abstinence from ethanol might help in hepatic regeneration. Silybin showed a significant hepatoprotective activity, but activity was less than that of ascorbic acid. Furthermore, preventive measures were more effective than curative treatment.

Alcohol abuse-duration dependent decrease in plasma testosterone and antioxidants in males.

Ethanol is a testicular toxin and it causes fertility abnormalities with low sperm count and impaired sperm motility in men. The present study was designed to investigate plasma testosterone level and hypothalamic pituitary gonadal (HPG) axis function in alcoholic men and also effect of ethanol on systemic oxidative stress. Forty six male alcohol abusers in the age group 20-40 years were selected. Fifty five, males in the same age group served as control. Alcohol abusers had significantly low plasma testosterone with low luteinizing hormone and follicle stimulating hormone. In addition they had significantly high thiobarbituric acid reactive substances (TBARS), superoxide dismutase and glutathione S-transferase, and low glutathione, ascorbic acid, catalase, glutathione reductase and glutathione peroxidase. Moreover, serum testosterone level in alcoholics negatively correlated with duration of alcohol abuse, and TBARS. Duration dependent decreased serum testosterone level in alcohol abusers might be due to 1) increased oxidative stress which can damage Leydig and supporting Sertoli cells and 2) impaired HPG axis.

Alcohol: its health and social impact in India.

Alcoholic beverages have been used in human societies since the beginning of recorded history. The patterns of alcohol intake around the world are constantly evolving, and alcohol is ubiquitous today. Research has contributed substantially to our understanding of the relation of drinking to specific disorders, and has shown that the relation between alcohol consumption and health outcomes is complex and multidimensional. Increases in the average volume of drinking are predicted for the most populous regions of the world in Southeast Asia including India. Cultural differences apparently influence the pattern of alcohol consumption. In addition, alcohol is linked to categories of disease whose relative impact on the global burden is predicted to increase. Therefore, it is appropriate to implement policies with targeted harm reduction strategies. The crucial need, from a public health perspective, is for regular means of coordination whereby prevention of alcohol-related problems is taken fully into account in policy decisions about alcohol control and regulation in the market for alcoholic beverages.

Effect of exogenous lecithin on ethanol-induced testicular injuries in Wistar rats.

Infertility is well-established harmful effect in chronic alcoholism and so far, there is no effective treatment for this condition. The study was conducted to determine the effects of lecithin, a known hepatoprotective on ethanol induced testicular injuries in male albino rats of Wistar strain. Five groups (n=6) of animals were used. Group I served as control. Group II received daily 1.6 g ethanol/kg body weight/day for 4 weeks orally. Group III received 1.6 g ethanol + 500 mg lecithin/kg body weight/day for four weeks orally. Group IV received 1.6 g ethanol/kg body weight for/day 4 weeks and followed by 500 mg lecithin/kg body weight/ day for four weeks orally. Group V received 1.6 g ethanol/kg body weight/ day orally for 4 weeks, followed by 4 weeks abstinence. Twenty-four hours after the last treatment the rats were sacrificed using anesthetic ether. Testes were removed and used for the estimation of extent of lipid peroxidation and tissue levels of antioxidants and steroidogenic enzymes. Lecithin protected testes from ethanol induced oxidative stress. However, the drug did not show any considerable effect on the activities of testicular delta5, 3beta-HSD and 17beta-HSD. In conclusion, ethanol induced oxidative stress can be reversed by treatment with lecithin. However the effect of lecithin on steroidogenesis was not promising.

Biochemical toxicity study on rat brain upon co-exposure to aluminium and ethanol.

Aluminium and alcohol are well known neuro toxins. Co-exposure of these neuro toxins has been studied in rats. Alcohol exposure significantly affected the aluminium content, protein content, acid phosphatase activity, alkaline phosphatase activity, alanine aminotransferase activity, glutathione-S-transferase activity, and glucose 6-phosphate dehy-drogenase activity of brain. Aluminium exposure, on the other hand, contributed significantly only in the alterations of aluminium content, acid phosphatase activity, and aspartate aminotransf erase activity of brain of rats in the present study. The interaction of both aluminium intoxication and alcohol exposure is significant only in the case of acid phosphatase and glutathione-S-transferase activities of brain. Therefore, from the observations of the present investigation, it can be suggested that the general neurotoxic-ity produced by aluminium is not modified by alcohol. However, the aluminium load and oxidative stress, caused by aluminium exposure, may be influenced by alcohol co-exposure.

Effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity in rats.

In order to find out the effect of chronic ethanol administration on testicular antioxidant system and steroidogenic enzyme activity, male rats fed with ethanol 1.6g/kg body weight per day for four weeks were studied. Besides a drastic reduction in body and testis weight, there was decrease in ascorbic acid, reduced glutathione and activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase in the testicular tissue of the treated animals. Simultaneously, there was increase in lipid peroxidation and glutathione S-transferase activity. Activities of 3 beta-hydroxy steroid dehydrogenase and 17 beta-hydroxy steroid dehydrogenase were also found decreased in the treated animals. The results indicate that chronic ethanol administration resulted in increase in oxidative stress and decrease in the activities of steroidogenic enzymes in the rat testes.

Experimental therapeutic intervention with ascorbic acid in ethanol induced testicular injuries in rats.

Ascorbic acid treatment significantly increased the activities of testicular delta5, 3beta-HSD and 17beta-HSD. Moreover, the treatment was also associated with significant decrease in oxidative stress in the testis. Ethanol induced oxidative stress and decreased steroidogenesis can be reversed by treatment with ascorbic acid.

Possible role of Granulocyte Macrophage Colony Stimulating Factor receptor (GM-CSF R) in malaria.

Malaria has been reportedly increasing in incidence on the globe. Evidence from clinical studies supports a role for cytokines in the pathogenesis of cerebral malaria. Given the stimulatory effect of the ligand GM-CSF on the synthesis and release of the pyrogenic cytokine TNF alpha, the present study has been undertaken to investigate a possible role of GMCSF receptor in the pathogenesis of both Plasmodium vivax and Plasmodium falciparum malaria. An enzyme immunoassay developed by us at our laboratory for the quantitation of GM-CSF receptor has been used. No changes in the concentration of the receptor have been indicated either at the time of diagnosis or after treatment. In addition, an intercomparison of the receptor concentration between the P. vivax and P. falciparum groups does not show any significant difference. The results suggest that GM-CSF receptor has no significant role in the pathogenesis of either type of malaria.

Activity of Ocimum sanctum (the traditional Indian medicinal plant) against the enteric pathogens.

Aqueous & alcoholic extracts of O. sanctum were prepared. Two concentrations of these extracts (30 mg & 60 mg) were tried against the enteric pathogens & candida albicans by Agar diffusion method. Wide zones of inhibition were observed at 60 mg concentration of extract. Aqeous extract showed wider zone of inhibition when compared to alcoholic extract. Aqueous extract showed wider zones of inhibition for Klebisella, E. Coil, Proteus & Staphylococcus aureus. Alcoholic extract showed wider zone for vibrio cholerae.

Assessment of immunological parameters during tumour development in a murine model.

Natural Killer activity assessed by 51Cr release assay from K-562 cells showed detectable activity from 5th day after tumour transplantation, reaching a peak on 12th day and thereafter showing a gradual decline in the activity. Antibody dependent cell mediated cytotoxicity estimated by 51Cr labelled sheep red blood cells anti SRBC system demonstrated a peak activity on 5th day. Cytotoxic T lymphocyte activity detected by 51Cr release of Dalton's lymphoma ascites target cells showed a peak on 10th day. Antibody complement mediated cytotoxicity revealed a similar pattern as natural killer cell activity.