Prenatal alcohol-induced neuroapoptosis in rat brain cerebral cortex: protective effect of folic acid and betaine.

PURPOSE: Alcohol consumption in pregnancy may cause fetal alcohol syndrome (FAS) in the infant. This study aims to investigate prenatal alcohol exposure related neuroapoptosis on the cerebral cortex tissues of newborn rats and possible neuroprotective effects of betaine, folic acid, and combined therapy. METHODS: Pregnant rats were divided into five experimental groups: control, ethanol, ethanol + betaine, ethanol + folic acid, and ethanol + betaine + folic acid combined therapy groups. We measured cytochrome c release, caspase-3, calpain and cathepsin B and L. enzyme activities. In order to observe apoptotic cells in the early stages, TUNEL method was chosen together with histologic methods such as assessing the diameters of the apoptotic cells, their distribution in unit volume and volume proportion of cortical intact neuron nuclei. RESULTS: Calpain, caspase-3 activities, and cytochrome c levels were significantly increased in alcohol group while cathepsin B and L. activities were also found to be elevated albeit not statistically significant. These increases were significantly reversed by folic acid and betaine + folic acid treatments. While ethanol increased the number of apoptotic cells, this increase was prevented in ethanol + betaine and ethanol + betaine + folic acid groups. Morphometric examination showed that the mean diameter of apoptotic cells was increased with ethanol administration while this increase was reduced by betaine and betaine + folic acid treatments. CONCLUSION: We observed that ethanol is capable of triggering apoptotic cell death in the newborn rat brains. Furthermore, folic acid, betaine, and combined therapy of these supplements may reduce neuroapoptosis related to prenatal alcohol consumption, and might be effective on preventing fetal alcohol syndrome in infants.

Protective effect of calpain inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal on acute alcohol consumption related cardiomyopathy.

Excessive alcohol consumption and alcoholism cause medical problems with high mortality and morbidity rates. In this study we aimed to decrease the alcohol related tissue damage by inhibiting calpain activation which plays an important role in apoptosis and necrosis, in rats with cardiomyopathy induced by acute alcohol consumption. Male Sprague-Dawley rats were separated into four groups (control, vehicle, alcohol and alcohol + inhibitor) with 10 rats in each. Control group received isocaloric maltose while vehicle group received isocaloric maltose with DMSO, and alcohol group received 8 g/kg absolute ethanol by gavage. Inhibitor group received 20 mg/kg calpain inhibitor 1 intraperitonally prior to alcohol administration. Calpain activities, cathepsin L levels and cytochrome c release rates were significantly increased in alcohol group compared to control group (p < 0.05). Serum CK MB and BNP levels of alcohol group were excessively increased compared to control group (respectively p < 0.001 and p < 0.01). Serum BNP levels of alcohol + inhibitor group were significantly (p < 0.05) decreased compared to alcohol group. In addition to these, histological evaluation of light microscope images and the results of DNA fragmentation and immunohistochemical caspase-3 activity results showed significant improvement of these parameters in alcohol + inhibitor group compared to alcohol group. Results of our biochemical and histological evaluation results revealed that the calpain inhibitor N-acetyl-leu-leu-norleucinal may have an ameliorating effect on acute alcohol consumption related cardiac tissue damage due to its effects on cell death pathways.

Effect of kefir and low-dose aspirin on arterial blood pressure measurements and renal apoptosis in unhypertensive rats with 4 weeks salt diet.

Abstract We aim to study the effect of low-dose aspirin and kefir on arterial blood pressure measurements and renal apoptosis in unhypertensive rats with 4 weeks salt diet. Forty adult male Sprague-Dawley rats were divided into five groups: control, high-salt (HS) (8.0% NaCl), HS+aspirin (10 mg/kg), HS+kefir (10.0%w/v), HS+aspirin +kefir. We measured sistolic blood pressure (SBP), mean arterial pressure (MAP), diastolic pressure, pulse pressure in the rats. Cathepsin B, L, DNA fragmentation and caspase-3 activities were determined from rat kidney tissues and rats clearance of creatinine calculated. Although HS diet increased significantly SBP, MAP, diastolic pressure, pulse pressure parameters compared the control values. They were not as high as accepted hypertension levels. When compared to HS groups, kefir groups significantly decrease Cathepsin B and DNA fragmentation levels. Caspase levels were elevated slightly in other groups according to control group. While, we also found that creatinine clearance was higher in HS+kefir and HS+low-dose aspirin than HS group. Thus, using low-dose aspirin had been approximately decreased of renal function damage. Kefir decreased renal function damage playing as Angiotensin-converting enzyme inhibitor. But, low-dose aspirin together with kefir worsened rat renal function damage. Cathepsin B might play role both apoptosis and prorenin-processing enzyme. But not caspase pathway may be involved in the present HS diet induced apoptosis. In conclusion, kefir and low-dose aspirin used independently protect renal function and renal damage induced by HS diet in rats.

Effects of polysaccharide from fruiting bodies of Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus on alcoholic liver injury.

In the present study, the curative effects of crude polysaccharides (PSs) from mushrooms on the symptoms of alcoholic liver injury were investigated. PSs from Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus fruiting bodies were administered by gavage at levels of 100 mg per kg body weight per day for 7 d after the onset of the disease. The caspase-3 activity, mitochondrial membrane potential, mitochondrial outer membrane integrity of the liver tissues of sacrificed rats, and the serum alanine aminotransferase (ALT) levels were determined. In addition, light and transmission electron microscope (TEM) studies were performed for histopathological and cytological evaluations on liver sections. PSs from A. brasiliensis decreased ALT level and mitochondrial membrane potential and increased the outer membrane integrity; microscopic examinations also revealed normal hepatocytes and tissue. On the basis of our data, it can be argued that crude PSs from Agaricus brasiliensis have therapeutic potential for alcoholic liver injury.

Consumption of Coprinus comatus polysaccharide extract causes recovery of alcoholic liver damage in rats.

CONTEXT: Excess use of alcohol is known to be associated with liver diseases such as fatty liver, alcoholic hepatitis, and cirrhosis. Various practices may be applied to prevent or treat the damage caused by chronic alcoholism. Coprinus comatus (O.F. Müll.) Pers. (Agaricaceae) is a macrofungus that has been reported to aid the recovery of murine livers damaged by benzopyrene. OBJECTIVE: In this study, the possible therapeutic effects of three different doses (50, 100, and 150 mg/kg) of C. comatus polysaccharide (PS) extract were studied in rats subjected to an alcoholic diet. The histological and biochemical results were compared between the control and experimental groups. MATERIALS AND METHODS: Modified Lieber-Decarli's calorie-adjusted liquid alcohol diet was given orally for 60 d. In addition to histopathology, alanine transaminase (ALT), aspartate transaminase (AST), mitochondrial membrane integrity, total cytochrome-c oxidase activity (TotalStCox), total mitochondrial cytochrome-c oxidase activity (TotalMtStCox), and caspase-3 values were used as liver parameters, and liver sections from all experimental groups were examined by electron microscopy. RESULTS: Using histopathological assessment, it was observed that there was a decline in liver hepatocyte vacuolization in the treatment group fed 50 mg PS/kg. The TotalStCox and TotalMtStCox values of this group differed from the EtOH control group (p < 0.05). DISCUSSION AND CONCLUSION: Daily administration of 50 mg/kg of C. comatus PS extract considerably reduced the negative effects of alcohol on liver structure and function.

Investigation of the possible protective role of gallic acid on paraoxanase and arylesterase activities in livers of rats with acute alcohol intoxication.

Gallic acid, a polyphenyl class natural product from gallnut and green tea, is known to be antioxidant, anti-inflammatory and radical scavenger. In this study, we aimed to investigate the possible protective effects of gallic acid on paraoxonase and arylesterase activities in liver exposed to acute alcohol intoxication. Paraoxonase and arylesterase activities in liver tissue and serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels were measured. Histological investigations were also made. In our study, we observed a significant increase of serum alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities, which are indicators of liver damage after acute ethanol consumption. Gallic acid therapy has significantly reduced the increase in these biomarkers, indicating a possible hepatoprotective effect of gallic acid. Ethanol consumption caused a significant decrease in liver paraoxonase activity (P < 0.001). Gallic acid treatment partly restored this decreased paraoxonase activity, which resulted from ethanol administration. A gallic acid dose of 100 mg/kg was observed as highest restoring effect for paraoxonase activity (P < 0.05). The activity of arylesterase was decreased in the ethanol group as compared with the control group, but this was not significant. However, 50 mg/kg of gallic acid treatment restored the loss of this activity due to ethanol exposure (P < 0.001). We observed that gallic acid ameliorates the liver damage caused by excessive alcohol consumption in a dose-dependent way. Our results in this study showed that gallic acid might have a protective effect against alcoholic liver disease.

Curative effect of crude exopolysaccharides of some macrofungi on alcohol-induced liver damage.

Abstract The authors investigate the curative effects of crude exopolysaccharides (EPS) produced by four Basidomycetes strains on the symptoms of alcoholic liver injury. EPSs were administered to experimental groups at levels of 100 mg per kg body weight per day for 7 days using an oral zonde needle after the onset of the disease. Serum levels of alanine transaminase (ALT), lactate dehydrogenase (LDH), DNA fragmentation, caspase-3 activities, and mitochondrial outer membrane integrity were determined following sacrifice of the rats. Light and transmission electron microscope (TEM) studies were performed on liver sections for histopathological and cytological evaluations. EPS that was obtained from Coprinus comatus OBCC 1014 decreased serum ALT level and increased outer membrane integrity, and allowed for the regaining of histologically and cytologically normal hepatocyte and tissue views. As a result, based on the obtained data, it can be argued that among all studied mushroom strains crude exopolysaccharides from Coprinus comatus OBCC 1014 strain have therapeutic potential for alcoholic liver injury according to control groups.

Preventive role of gallic acid on alcohol dependent and cysteine protease-mediated pancreas injury.

In order to investigate an association between alcohol consumption and lysosomal cysteine protease induced pancreatic injury and preventive effect of gallic acid as dose-dependent, we determined myeloperoxidase and malondialdehyde levels, serum amylase activities and cathepsin B and L activities in the cytosolic and lysosomal fractions of pancreatic tissue in the ethanol (8 g/kg) and ethanol plus gallic acid (at different doses 50, 100 and 200 mg/kg) given rats. Absolute ethanol (8 g/kg) was given by oral gavage. Gallic acid was dissolved in the saline (2 ml/kg) and administered before 30 min the oral administration of ethanol. Pancreatic myeloperoxidase and also malondialdehyde levels and serum amylase activities were measured. Besides, histological investigations were made. Cathepsin B activities in the cytosolic fraction were decreased by gallic acid (200 mg/kg) and increased in ethanol given rats. Cytosolic/lysosomal ratio of cathepsin B and L were found to be low in the all doses of gallic acid as compared to ethanol group. Serum amylase, pancreatic myeloperoxidase activities and malondialdehyde levels in the ethanol group were higher than in the control group. These were not statistically significant for myeloperoxidase and malondialdehyde. Also, our histopathologic results indicated that ethanol administration increased pancreatic tissue injury. Gallic acid especially at 200 mg/kg improved ethanol-mediated pancreatic tissue damage.In conclusion, gallic acid treatments were decreased release of lysosomal cathepsin B and L enzymes into cytoplasmic fraction and prevented alcohol mediated pancreatic tissue injury. Preventive effect of gallic acid might be dose-dependent.

Betaine (trimethylglycine) as a nutritional agent prevents oxidative stress after chronic ethanol consumption in pancreatic tissue of rats.

In this study, we investigated the free radical-mediated cytotoxic effects of chronic ethanol consumption on the pancreatic tissue and a possible cytoprotective effect of betaine as a methyl donor and an important participant in the methionine cycle. Twenty-four male Wistar rats were divided into control, ethanol, and ethanol+betaine groups. Prior to sacrifice, all groups were fed 60 mL/diet per day for two months. Rats in the ethanol group were fed with ethanol 8 g/kg/day. The ethanol+betaine groups were fed ethanol plus betaine (0.5 % w/v). Malondialdehyde levels and adenosine deaminase, superoxide dismutase, and xanthine oxidase activities were determined in pancreatic tissues of rats. Compared to control group, MDA levels increased significantly in the ethanol group (p<0.05). MDA levels in the ethanol+betaine group were significantly decreased compared to the ethanol group (p<0.05). ADA activity in the ethanol+betaine group decreased significantly when compared to the ethanol group (p<0.05). XO activities in ethanol-fed rats were decreased significantly compared to the control group (p<0.05). XO activity in the betaine group was increased significantly (p<0.05) compared to the ethanol group. SOD activity in the ethanol group decreased significantly compared to control group (p<0.001). SOD activity in the ethanol+betaine group decreased significantly (p<0.05) compared to the control group. We think that betaine, as a nutritional methylating agent, may be effective against ethanol-mediated oxidative stress in pancreatic tissue.

The protective effect of dexanabinol (HU-211) on nitric oxide and cysteine protease-mediated neuronal death in focal cerebral ischemia.

We hypothesized that dexanabinol can prevent neuronal death by protecting neuronal lysosomes from nitric oxide (NO)-mediated toxicity, and in turn, by suppressing the release of cathepsins during cerebral ischemia. Focal cerebral ischemia was induced in two sets of animals by permanent middle cerebral artery occlusion. The first set was used to monitor NO concentration and cathepsin activity, while the second was used for histological examination with hematoxylin and eosin, and TUNEL staining. In post-ischemic brain tissue, NO content and cathepsin B and L activity increased (p < 0.05). Dexanabinol treatment reduced NO concentration and cathepsin activity to the control level (p > 0.05). The number of eosinophilic and apoptotic neurons increased in the post-ischemic cerebral cortex (p < 0.05). However, dexanabinol treatment lowered both of these (p < 0.05). We conclude that dexanabinol might be a useful agent for the treatment of stroke patients.

Chronic alcohol consumption augments loss of sialic acid residues and alters erythrocyte membrane charge in type II diabetic patients.

In this study, the effects of alcohol consumption on erythrocyte membrane properties in type 2 diabetic patients were investigated. Therefore, we measured total and lipid-bound sialic acid (LSA) levels, sialidase activities, and erythrocyte membrane negative charge. Three groups, including control group (n = 20), alcohol-consuming diabetic patients group (n = 14), and diabetic patients without alcohol consumption group (n = 42), were created. Plasma total sialic acid (TSA) levels of the alcohol-consuming diabetic group were elevated as compared to the healthy control and diabetic group (p < 0.001 and p < 0.01, respectively). TSA levels of the diabetic group were significantly elevated as compared to the healthy control group (p > 0.001). Plasma LSA levels of the alcohol-consuming diabetic group were higher than that in the healthy control and diabetic group (p < 0.05 and p < 0.05, respectively). LSA levels of the diabetic group were found to be high as compared to the healthy control group (p < 0.05). Plasma sialidase activities of the alcohol-consuming diabetic group and diabetic group were significantly elevated as compared to the healthy control group (p < 0.05 and p < 0.05, respectively). Sialidase activities of the alcohol-consuming diabetic group were elevated as compared to the diabetic group, but this was not statistically significant (p > 0.05). Erythrocyte membrane negativity levels of the alcohol-consuming diabetic group and diabetic group were significantly decreased (p < 0.001 and p < 0.001, respectively) as compared to the healthy control group. Erythrocyte membrane negativity levels of the alcohol-consuming diabetic group were decreased as compared to the diabetic group, but this was not statistically significant (p > 0.05). In conclusion, our results indicate that chronic alcohol consumption may augment membrane alterations in type 2 diabetic patients.

Nitric oxide level and adenosine deaminase activity in cerebrospinal fluid of patients with subarachnoid hemorrhage.

OBJECTIVE: Adenosine and nitric oxide (NO) are known as vasodilatators. We investigated adenosine deaminase (ADA) activity and NO concentration in the cerebrospinal fluid (CSF) of patients with subarachnoid hemorrhage (SAH). METHODS: Forty patients with SAH and 10 controls were included in the study. Nitrate level and ADA activity were measured in CSF. SAH patients were grouped according to the presence of angiographic vasospasm, Hunt and Hess grading, Glasgow Coma Scale (GCS) and Fisher Grade (FG). RESULTS: The level of NO markers in SAH patients decreased when compared to that in the control group (p < 0.05). However, NO markers in patients with vasospasm was higher than in that of patients without vasospasm (p < 0.05). ADA activity increased in patients with SAH (p < 0.01) and also patients with angiographic vasospasm (p < 0.05). ADA activity in the poor-grade SAH group was higher than that in the good-grade SAH group. The group with the lower GCS showed increased ADA activity compared to those with a higher GCS score (p < 0.01). Furthermore, patients with FG 4 had a higher level of ADA activity compared to FG 1 and 2 and FG 3 (p < 0.001 and p < 0.01, respectively). CONCLUSION: Decreased NO level may participate in the early development of vasospasm. However, the increased level of ADA activity in the SAH patients with the poor clinical and consciousness level may have resulted from the ischemic cerebral insult.

Effects of Black Hoof Medicinal Mushroom, Phellinus linteus (Agaricomycetes), Polysaccharide Extract in Streptozotocin-Induced Diabetic Rats.

In this article we report the healing effects of a Phellinus linteus fruiting body hot water extract (PLE) in streptozotocin (STZ)-induced diabetic rats. PLE was given before and after STZ. The preprotective, protective, and postprotective effects of PLE on STZ-induced oxidative stress were studied using biochemical (caspase 3 activity, cytosolic-to-lysosomal ratio of cathepsin B and L, DNA fragmentation levels), ordinary histological and immuno-histochemical investigation parameters. Following oral administration of PLE after STZ application, the serum glucose concentration significantly decreased up to 41.13% compared with the control group (P < 0.05). The hypoglycemic potential of the PLE was further supported by an increase of insulin secretion in the islets of Langerhans. In addition, the number of cells in Langerhans islets increased by 45.89% when PLE was given after STZ application. On the other hand, the use of PLE before oxidative stress could not prevent the onset of diabetes. This is, to our knowledge, the first study of the effect of application time of orally administered Ph. Linteus hot water extract on STZ-induced diabetes.

Effect of Different Psychoactive Substances on Serum Biochemical Parameters.

BACKGROUND: Psychoactive substances affect mainly central nervous system and brain function causing changes in behavior. OBJECTIVES: The purpose of this study was to determine the effects of different psychoactive substances on serum biochemical parameters. PATIENTS AND METHODS: The study included 324 drug dependents, and 69 controls. The patient group was determined according to DSM-IV (The diagnostic and statistical manual of mental disorders, fourth edition) criteria. All patients and control subjects were tested for routine biochemical parameters and urine toxicology parameters for psychoactive substance use. Cases and controls with accompanying diseases like diabetes, cancer, metabolic disorders etc. are excluded from the study. Moreover, an association between urine toxicology results and changes in biochemical parameters was evaluated for statistical significance. RESULTS: There was a statistically significant difference in the Gamma-Glutamyl Transferase (GGT), uric acid, creatinine, urea, albumin, Aspartate Aminotransferase (AST) medians between the dependent and control groups (P < 0.05). We found a statistically significant difference in sodium and albumin levels between the opium-dependent and control groups (P < 0.05). In the benzodiazepin dependent group, we found a significant difference in GGT, urea, glucose, sodium, T protein, and AST levels (P < 0.05). Moreover, a statistically significant difference was observed in triglyceride and GGT levels between the ethyl glucuronide and control groups (P < 0.05). CONCLUSIONS: In psychoactive substance dependents, serum routine biochemistry parameters can be used to predict the need for intensive monitoring and treatment programs.

Effect of boric acid on oxidative stress in rats with fetal alcohol syndrome.

To the best of our knowledge, this is the first study concerning the effect of boric acid (BA) administration on fetal alcohol syndrome (FAS). In this study, the aim was to investigate prenatal alcohol-induced oxidative stress on the cerebral cortex of newborn rat pups and assess the protective and beneficial effects of BA supplementation on rats with FAS. Pregnant rats were divided into three groups, namely the control, alcohol and alcohol + boric acid groups. As markers of alcohol-induced oxidative stress in the cerebral cortex of the newborn pups, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels were measured. Although the MDA levels in the alcohol group were significantly increased compared with those in the control group (P<0.05), the MDA level in the alcohol + boric acid group was shown to be significantly decreased compared with that in the alcohol group (P<0.01). The CAT activity of the alcohol + boric acid group was significantly higher than that in the alcohol group (P<0.05). The GPx activity in the alcohol group was decreased compared with that in the control group (P<0.05). These results demonstrate that alcohol is capable of triggering damage to membranes of the cerebral cortex of rat pups and BA could be influential in antioxidant mechanisms against oxidative stress resulting from prenatal alcohol exposure.

The neuroprotective effect of acute moderate alcohol consumption on caspase-3 mediated neuroapoptosis in traumatic brain injury: the role of lysosomal cathepsin L and nitric oxide.

Our aim in this study was to investigate the effect of moderate acute alcohol administration on cysteine protease mediated neuronal apoptosis and nitric oxide production in the traumatic brain injury. A total of 29 adult Sprague-Dawley male rats weighing 250-300 g were used. The rats were allocated into four groups. The first group was the control (sham-operated) group in which only a craniotomy was performed, the others were alcohol, trauma and trauma+alcohol groups. Caspase-3 enzyme activity in the trauma group increased significantly in comparison with the control group. The alcohol given group showed a decreased caspase-3 enzyme activity compared to the trauma group. The level of caspase-3 enzyme activity in the alcohol+trauma group decreased in comparison to the trauma group. SF/FEL ratio of cathepsin-L enzyme activity in the trauma group was significantly higher than in the control group. Our results indicate that moderate alcohol consumption may have protective effects on apoptotic cell death after traumatic brain injury. Protective effects of moderate ethanol consumption might be related to inhibition of lysosomal protease release and nitric oxide production.

Erythropoietin prevents nitric oxide and cathepsin-mediated neuronal death in focal brain ischemia.

We examined the preventive effect of human recombinant erythropoietin (HrEPO) on nitric oxide (NO)-mediated toxicity to neurons and cysteine protease release into cytoplasm, which is attributed to neuronal death in brain ischemia. Focal cerebral ischemia was induced by permanent occlusion of middle cerebral artery in two sets of rat. The first set was used to monitor NO concentration and cathepsin activity, while the second was used for histological examination with hematoxylin and eosin, and TUNEL staining. A group in both set was administered human recombinant erythropoietin (HrEPO). NO content, cathepsins B and L activity increased significantly in the post-ischemic cerebral tissue (p<0.05). HrEPO treatment reduced NO concentration and cathepsin activity to control level (p>0.05). A significant increase in the number of necrotic and apoptotic neurons was observed in the post-ischemic cerebral cortex (p<0.05). HrEPO treatment was markedly lowered both of these (p<0.05). It is concluded that HrEPO prevents neuronal death by protecting neuronal liposomes from NO-mediated toxicity and suppressing the release of cathepsins.

Effects of chronic ethanol consumption on brain synaptosomes and protective role of betaine.

To evaluate the cytotoxic effects of chronic ethanol consumption on brain cerebral synaptosomes and preventive role of betaine as a methyl donor and S-adenosylmethionine precursor, 24 male Wistar rats were divided into three groups: control, ethanol (8 g/kg/day) and ethanol plus betaine(0.5% w/v) group. Animals were fed 60 ml/diet per day for two months, then sacrificed. Malondialdehyde (MDA), protein carbonyl contents and adenosine deaminase (ADA) activities were determined in synaptosomal/mitochondrial enriched fraction isolated from rat cerebral cortexes. When compared to controls, ethanol containing diet significantly increased MDA levels (P < 0.05), also increased protein carbonyl levels and adenosine deaminase activities. But these were not statistically significant (P > 0.05). However, adding betaine to ethanol containing diet caused a significant decrease in MDA, protein carbonyl levels and adenosine deaminase activities (P < 0.05). These results indicate that betaine may appear as a protective nutritional agent against cytotoxic brain damage induced by chronic ethanol consumption.