Lipid peroxidation of erythrocyte membrane induced by lipoamide dehydrogenase in the presence of ADP-Fe3+.

Lipid peroxidation of rat erythrocyte membranes was induced by lipoamide dehydrogenase (LADH) (EC 1.8.1.4) in the presence of ADP-Fe3+. Superoxide dismutase (SOD) (EC 1.15.1.1) strongly inhibited the peroxidation reaction but catalase did not. Hydroxyl radical scavengers, mannitol and dimethylsulfoxide did not inhibit the lipid peroxidation. These results indicated that the lipid peroxidation was a superoxide (O2-)-dependent reaction, but the hydroxyl radical was not involved. ADP-Fe3+, in the presence of LADH, was reduced more rapidly under aerobic than anaerobic conditions and SOD under aerobic conditions strongly inhibited the iron reduction, indicating that O2- plays a predominant role in iron reduction. Hydrogen peroxide enhanced O2- generation by LADH, but the peroxidation reaction was not affected. In the presence of lipoamide, lipid peroxidation was also induced but the reactions were not inhibited by SOD. Evidently, the lipid peroxidation induced in the presence of lipoamide was O2(-)-independent. Dihydrolipoamide may be involved in the peroxidation reaction.     

Effect of L-penicillamine hydantoin, an analogue of glutathione, on rat liver glutathione peroxidase, reductase and transferase reactions.

In soluble fractions prepared from rat liver homogenates, L-penicillamine hydantoin appeared to be, on the basis of SH consumption measurements, a substrate for glutathione peroxidase but not transferase reactions. When glutathione is incubated with rat liver soluble proteins in the presence of penicillamine hydantoin, formation of oxidized glutathione is inhibited. Calculations from Lineweaver-Burk plots point out that inhibition by L-penicillamine hydantoin of the peroxide-dependent oxidations of glutathione is mixed, since both apparent Km and Vmax values are modified. Preincubation of rat liver soluble proteins with L-penicillamine hydantoin led to a progressive inactivation of glutathione peroxidase. The kinetics of this inactivation process with respect to time and inactivator concentration were studied. Inclusion in the preincubation mixture of SH-containing molecules such as dithiothreitol, L-cysteine or glutathione protected the enzyme against inactivation. However, none of these molecules and neither hydantoin, Triton X-100, phenol, nor dialysis could reverse the enzyme from inactivated to activated form. Mitochondrial glutathione peroxidase was inhibited and inactivated by L-penicillamine hydantoin to the same extent as its cytosolic counterpart. Modifications by penicillamine hydantoin of various subcellular markers enzymes (lactate dehydrogenase, N-acetyl beta-glucosaminidase, arylsulfatase C, butyryl-CoA dehydrogenase, lauryl-CoA and glycolate oxidases) were of weak amplitude consisting of either inhibition, inactivation or stimulation.     

Lipid Peroxidation of Erythrocyte Membrane Induced by Lipoamide Dehydrogenase in the Presence of ADP-Fe3+

Abstract: Lipid peroxidation of rat erythrocyte membranes was induced by lipoamide dehydrogenase (LADH) (EC 1.8.1.4) in the presence of ADP-Fe³⁺. Superoxide dismutase (SOD) (EC 1.15.1.1) strongly inhibited the peroxidation reaction but catalase did not. Hydroxyl radical scavengers, mannitol and dimethylsulfoxide did not inhibit the lipid peroxidation. These results indicated that the lipid peroxidation was a superoxide (O₂^-)-dependent reaction, but the hydroxyl radical was not involved. ADP-Fe³⁺, in the presence of LADH, was reduced more rapidly under aerobic than anaerobic conditions and SOD under aerobic conditions strongly inhibited the iron reduction, indicating that O₂^- plays a predominant role in iron reduction. Hydrogen peroxide enhanced of generation by LADH, but the peroxidation reaction was not affected. In the presence of lipoamide, lipid peroxidation was also induced but the reactions were not inhibited by SOD. Evidently, the lipid peroxidation induced in the presence of lipoamide was O₂^- -independent. Dihydrolipoamide may be involved in the peroxidation reaction.     

慢性脑缺血大鼠脑组织线粒体蛋白质组与能量代谢相关性研究

目的探讨慢性脑缺血大鼠脑组织线粒体蛋白质组与能量代谢相关性,以期揭示慢性脑缺血疾病的线粒体机制。方法通过差速离心分离大鼠脑线粒体,检测脑线粒体能量代谢相关的氧化磷酸化指标,应用凝胶电泳技术构建大鼠脑线粒体蛋白质组表达图谱;通过结扎大鼠双侧颈总动脉建立大鼠慢性脑缺血模型,分析比较慢性脑缺血时脑线粒体蛋白质表达谱的改变,从脑线粒体蛋白质组角度阐述慢性脑缺血与能量代谢的关系。结果实验发现,与正常对照组相比,慢性脑缺血大鼠脑线粒体ADP/O及氧化磷酸化效率明显降低,蛋白质组结果显示慢性脑缺血大鼠NADH脱氢酶复合体亚基、细胞色素C氧化酶亚基、丙酮酸脱氢酶(硫辛酰胺)β、乙酰辅酶A乙酰转移酶、烯醇化酶、醛缩酶C等表达量降低,而3-含氧酸辅酶A转移酶、4-氨基丁酸转氨酶等表达量增加。结论实验表明,慢性脑缺血大鼠能量代谢功能及能量代谢相关酶发生了很大变化,慢性脑缺血疾病机制与线粒体具有密切关系。     

Histochemical localization of rhodanese activity in rat liver and skeletal muscle

A previously described histochemical technique was applied to the localization of rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) activity in rat skeletal muscle and liver. The physiological function of rhodanese is controversial, but it and other sulfurtransferases can catalyze the conversion of cyanide to the much less toxic thiocyanate. The volume of distribution of cyanide in human and dog is said to correspond roughly to the blood volume. Because of this and other observations, it was hypothesized that sulfurtransferase activity associated with the vascular endothelium on smooth muscle layers of blood vessels might play a role in cyanide detoxification. However, little enzyme activity as identified histochemically was associated with those sites in comparison with others examined. As expected, high activity was found in the liver and moderately high levels were present in skeletal muscle. In muscles sectioned longitudinally, points of rhodanese staining occurred in linear arrays along the lengths of the muscle fiber corresponding to the location of mitochondria within the fiber. The original technique called for incubation of tissue sections with both thiosulfate and cyanide. When thiosulfate was omitted, staining for rhodanese activity was still clearly identifiable in both liver and muscle sections with cyanide alone. In muscle sections the inclusion of both thiosulfate and cyanide resulted in a preferential staining of type I fibers presumably because of their higher content of mitochondria. Thus, this technique is a potential alternative to the NADH dehydrogenase stain for distinguishing between type I and type II muscle fibers. Incubation of tissue sections with only thiosulfate produced results that did not appear to differ from those obtained when both substrates were omitted. From these observations it may be inferred that the endogenous pool of sulfane-sulfur available to sulfurtransferases is larger than any alleged endogenous pool of cyanide. Although sulfurtransferase activity in muscle appeared to be lower than that in liver, the total body muscle mass is greater than the liver mass. Thus, these results support other evidence that skeletal muscle may make a significant contribution to total cyanide biotransformation in the absence of exogenously added thiosulfate.     

Histological demonstration of wheat germ lectin binding sites in the liver of normal and ANIT treated rats

Wheat-germ lectin peroxidase conjugate was used to stain the liver of normal rats and rats given -naphthylisothiocyanate (ANIT). Changes in patterns in bile duct and canalicular staining were compatible with the hypothesis that cell damage caused by ANIT is essentially restricted to bile ducts.     

Inhibitory effect of CDA-II, a urinary preparation, on aflatoxin B(1)-induced oxidative stress and DNA damage in primary cultured rat hepatocytes.

The effects of CDA-II (cell differentiation agent II; a urinary preparation) on both aflatoxin B(1) (AFB(1))-induced cell injury and DNA damage were investigated using cultured rat hepatocytes. CDA-II was able to suppress both the lipid peroxidation and lactate dehydrogenase leakage induced by AFB(1). Glutathione (GSH) depletion by AFB(1) was replenished by CDA-II treatment. Under these experimental conditions, CDA-II enhanced the activity of GSH peroxidase, but not GSH S-transferase. By evaluation of unscheduled DNA synthesis, CDA-II reduced AFB(1)-induced DNA damage in hepatocyte cultures. These findings suggest that CDA-II can inhibit cytotoxicity of AFB(1) through enhancing the activity of GSH peroxidase and preventing GSH depletion.     

不同产地、不同年限人参中3种同工酶活力比较

目的:对不同产地,不同生长年限(4年、5年)的人参中过氧化物酶(Peroxidase POD)、过氧化氢酶(Catalase CAT)、苹果酸脱氢酶(Malate Dehydrogenase MDH)活力进行比较。方法:采用中性缓冲液提取总蛋白,应用愈创木酚比色法测定过氧化物酶活力,过氧化氢比色法测定过氧化氢酶活力,草酰乙酸比色法测定苹果酸脱氢酶活力。结果:不同产地人参的同工酶活力存在一定差异,4年、5年生人参同工酶活力具有相似性,差异较小。结论:POD、CAT、MDH的活力可以作为人参品种鉴定及药材优选的评价指标。     

Studies on the mechanism of methanol poisoning: purification and comparison of rat and human liver 10-formyltetrahydrofolate dehydrogenase

Methanol poisoning in primates and humans is due to formate accumulation as a result of low rates of formate oxidation. This toxicity is not seen in rats, where formate oxidation rates are high. Formate oxidation in vivo is dependent on hepatic tetrahydrofolate levels and on the activity of the enzyme 10-formyl-tetrahydrofolate (10-formyl-H4folate) dehydrogenase (EC 1.5.1.6). Because hepatic 10-formyl-H4folate dehydrogenase activity is lower in human liver than in rat liver, studies were performed investigating the properties of this enzyme in rat and human liver. 10-Formyl-H4folate dehydrogenase was purified to homogeneity from rat and human liver and was found to possess similar subunit molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (96,000). N-Terminal amino acid analysis of the pure proteins showed an identical sequence for the first 16 amino acids. Antibodies raised in rabbits against the rat liver enzyme were inhibitory toward the activity of both rat and human liver enzymes and appeared to recognize only the 10-formyl-H4folate dehydrogenase in cytosolic preparations of rat and human liver. Immunoblots of pure rat and human liver 10-formyl-H4folate dehydrogenase showed similar staining intensity. It is concluded that rat and human liver 10-formyl-H4folate dehydrogenase possess very similar properties and that the activity of the enzyme in human liver is lower than that of rat liver, due to a reduced amount of enzyme protein in human liver. This may be an important factor in regulating formate oxidation in humans and may explain, in part, the accumulation of formate and the mechanism of toxicity of methanol in humans.     

Influence of thioctic acid on the glutathione antioxidant system and the activity of some NADPH-generating enzymes in rat heart under conditions of chronic alcohol intoxication

Administration of thioctic acid in doses of 35 and 70 mg/kg into rats under conditions of chronic alcohol intoxication resulted in the concentration of reduced glutathione and the activities of glutathione peroxidase, glutathione reductase, NADP-isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase in rat heart approaching the control levels. The obtained data provided deeper insight into the molecular mechanisms of thioctic acid action and a basis for further research aimed at expanding the range of indications for use of thioctic acid.     

Immunolocalization of glutathione S-transferase in the rat uterus

Immunohistochemical localization of glutathione S-transferase (GST) and enzyme cytochemical staining for endogenous peroxidase (Px) activity were studied in rat uteri. Both enzymes were clearly detected in the endometrium of the uterus taken from proestrus to estrus during the estrous cycle. Based on our data, the biological significance of GST in endometrium was discussed.     

Catalysis of nitrofuran redox-cycling and superoxide anion production by heart lipoamide dehydrogenase

Heart lipoamide dehydrogenase (LADH) catalyzed redox-cycling and O2-. production by (5-nitro-2-furfurylidene)amino derivatives using NADH as electron donor. NADH was a much more effective electron donor than NADPH for the nitroreductase activity. O2-. production was demonstrated by cytochrome c reduction, adrenochrome formation and the effect of superoxide dismutase. Under optimum conditions, nitroreductase activity was about 1% of LADH activity. One electron oxygen reduction and NADH oxidation correlated in 2:1 stoichiometry. The nitroreductase kinetics was in accordance with an ordered bi-bi mechanism. Nitrofuran derivatives bearing unsaturated five- or six-membered nitrogen heterocycles were more effective substrates than those bearing other groups, namely nifurtimox, nitrofurazone, nitrofurantoin and 5-nitro-2-furoic acid. Other nitro compounds (chloramphenicol, benznidazole, 2-nitroimidazole and 5-nitroindole) were ineffective. With the triazole, traizine and imidazole nitrofuran derivatives, the nitroreductase pH curve showed a maximum at pH 8.8, different from the pH optimum for the lipoamide reductase and diaphorase activities. Spectroscopic observations demonstrated pH-dependent structural changes in the triazole(I) and triazine derivatives which would affect their behavior as nitroreductase substrates. The nitroreductase activity was inhibited by p-chloromercuribenzoate and enhanced by cadmium and arsenite, whereas the NADH-induced LADH inactivation failed to affect the nitroreductase activity. In the absence of oxygen. LADH catalyzed nitrofuran reduction to products more reduced than the nitroanion, which were not reoxidized by oxygen. The anaerobic nitrofuran reduction was inhibited by cadmium and arsenite. The assayed nitrofuran compounds did not inhibit LADH lipoamide reductase activity, at variance with their action on glutathione reductase (Grinblat et al., Biochem Pharmacol 38: 767-772, 1989).     

Acute toxicity, genotoxicity, and dermal carcinogenicity assessment of isooctyl acrylate

Isooctyl acrylate (IOA) monomer is a complex mixture comprised predominantly of isomeric, eight-carbon alkyl esters of acrylic acid. Limited evidence from animal studies suggests that certain acrylate esters may be carcinogenic by the dermal route of exposure. The following studies were performed with IOA monomer: acute oral toxicity limit test in rats, primary dermal and ocular irritancy in rabbits, Ames Salmonella microsome assay, Saccharomyces cerevisiae D3 recombinogenicity assay, L5178Y TK +/- mouse lymphoma cell assay, and C3H/10T1/2 mouse embryo cell transformation assay. Finally, a limited dermal carcinogenicity bioassay was performed in which aliquots (25 microliters) of IOA monomer (5% v/v in acetone), IOA polymer (19% w/v in 70:30 acetone/heptane), or acetone (vehicle control) were applied to the shaved backs of male C3H/HeJ mice three times per week for the animals' lifetimes. IOA monomer had an acute oral LD50 in rats greater than 5000 mg/kg, was slightly irritating to the eyes and skin of rabbits on single exposures, and exhibited no genotoxic or cell-transforming potential. In the dermal carcinogenicity bioassay, no significant difference in mean survival times was observed between either treatment group and the control group. Animals treated with IOA monomer exhibited moderate dermatitis, surface crusting, hyperkeratosis, epidermal hyperplasia, diffuse melanosis, and one benign melanoma at the treatment size. Animals treated with IOA polymer exhibited varying degrees of dermatitis, surface crusting, and hyperkeratosis. Neither IOA monomer nor IOA polymer was carcinogenic under the conditions of the study.     

Delayed protection of tetramethylpyrazine on neonatal rat cardiomyocytes subjected to anoxia-reoxygenation injury

The aim of this study was to investigate the cardioprotective effects and the possible mechanisms of delayed preconditioning induced by tetramethylpyrazine (TMP) in cultured neonatal rat cardiomyocytes subjected to anoxia-reoxygenation injury. Cultured neonatal rat cardiomyocytes were preconditioned using TMP at different concentrations (100, 200 and 500 microM). Cell viability, lactate dehydrogenase release, malondialdehyde formation, superoxide dismutase activity and glutathione peroxidase activity were measured to determine the protective effects against anoxia-reoxygenation injury. The expression of heat shock protein 70 (Hsp70) was measured 24 hr after TMP preconditioning by Western blot analysis. The results showed that TMP decreased lactate dehydrogenase release, increased cell viability, suppressed malondialdehyde formation and augmented activities of superoxide dismutase and glutathione peroxidase in a concentration-dependent manner. Moreover, the delayed protection was abolished by pre-treating with either protein kinase C inhibitor chelerythrine chloride or PD98059, a selective inhibitor of extracellular signal-regulated protein kinase 1/2, respectively, and the expression of Hsp70 was significantly increased in 24 hr after TMP preconditioning that was also suppressed by chelerythrine chloride or PD98059. These results suggest that TMP can induce delayed cardioprotective effects by activation of protein kinase C and extracellular signal-regulated protein kinase 1/2 signalling pathways and subsequent increased expression of Hsp70 in rat neonatal cardiomyocytes.     

The influence of vitamin E and methionine on the activity of enzymes and the morphological picture of liver of rats intoxicated with sodium fluoride

The aim of this study was to investigate the effects of vitamin E and methionine on the activity of enzymes regulating carbohydrate metabolism and enzymes associated with glutathione as well as to examine the morphology of the liver in rats exposed to sodium fluoride.The study was conducted in 18 male rats of Wistar strain. The rats were divided into three groups: a control group, which received distilled water and two experimental groups, which received sodium fluoride (10 mg/kg of body mass/24 h) in water solution. Animals in the second experimental group received 3 mg of vitamin E/rat/24 h and 2 mg methionine/rat/24 h. The experiment lasted 35 days. In supernatants obtained after homogenization of rat liver slices, the activity of the following enzymes was assayed: fructose-1,6-biphosphate aldolase (ALD) malate dehydrogenase (MDH), lactate dehydrogenase (LDH), sorbitol dehydrogenase (SDH) the activity of glutathione peroxidase (GPx), glutathione transferase (GST) and glutathione reductase (GR). Pathomorphological evaluation was conducted on preparations made by standard paraffin method, followed by staining with hematoxylin and eosin. The administration of antioxidants counteracted changes in the activity of the enzymes and the morphological abnormalities of the liver induced by NaF. Antioxidants may be important in preventing toxicity of fluoride compounds.     

Melatonin ameliorates bisphenol A-induced biochemical toxicity in testicular mitochondria of mouse

Bisphenol A (BPA) is a monomer of polycarbonate plastic used to manufacture plastic baby bottles and lining of food cans. It has endocrine-disrupting potential and exerts both toxic and estrogenic effects on mammalian cells. We studied BPA-induced perturbation of mitochondrial marker enzymes in testes of Swiss albino mice and its amelioration by melatonin. Mice exposed to standardized dose of BPA (10 mg/kg body weight) orally for 14 days showed decrease in activities of marker mitochondrial enzymes such as succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, monoamine oxidase and NADH dehydrogenase. Besides, it also affected activities of antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione peroxidase. BPA also caused lipid peroxidation (LPO) and decrease in reduced glutathione (GSH) content of mitochondria. Concomitant melatonin administration (10 mg/kg body weight; intraperitoneally for 14 days) lowered mitochondrial lipid peroxidation. It also restored the activity of mitochondrial marker enzymes and ameliorated decreased enzymatic and non-enzymatic antioxidants of mitochondria. These results demonstrate that melatonin has a potential role in ameliorating BPA-induced mitochondrial toxicity and the protection is due to its antioxidant property or by the direct free radical scavenging activity.     

固定时间对口腔黏膜鳞状细胞癌组织染色的影响

目的探讨固定时间对口腔黏膜鳞状细胞癌组织HE染色和免疫组织化学（immunohistochemistry，IHC）染色的影响。方法选取10例患者口腔黏膜鳞状细胞癌组织，每例各取4块，分别固定8h、24h、48h和1周，进行HE染色和高分子量细胞角蛋白（cytokeratin high molecular weight, CKH，CKH，克隆号3413E12）和广谱细胞角蛋白（cytokeratin pan，ckpan，克隆号AE1／AE3）IHC染色，观察细胞形态的优良、染色情况及IHC染色的阳性强度和阳性率。结果HE染色和IHC染色结果均显示口腔黏膜鳞状细胞癌组织形态和染色、阳性强度和阳性率最佳为固定24h，8h为最差。固定1周者染色效果较48h差。结论本研究表明对口腔黏膜鳞状细胞癌组织，24h为最佳固定时间；组织固定不充分对HE染色和IHC染色的影响要大于固定时间过长的影响；固定时间越长，对HE染色和IHC染色效果影响越大。     

Preventive effects of Laminaria japonica aqueous extract on the oxidative stress and xanthine oxidase activity in streptozotocin-induced diabetic rat liver

Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of diabetes mellitus type 1 and type 2. Xanthine oxidase (XO) has been proposed as one of the sources of free radical formation in diabetes. We therefore investigated the preventive effects of Laminaria japonica aqueous extract (LJE) on alterations in the activity of hepatic XO and oxidative stress in the streptozotocin-induced experimental diabetes. We found that lipid peroxide levels and xanthine oxidase activity were increased, whereas glutathione (GSH), GSH reductase and GSH peroxidase were decreased in the liver of streptozotocin-induced diabetic rats. Pretreatment with LJE of 100 mg/kg orally for 5 d significantly reduced blood glucose levels and hepatic lipid peroxidation in the diabetic rats. In addition, the content of glutathione was restored to the control level by LJE pretreatment. Furthermore, LJE significantly suppressed the increased activity of XO and type conversion of the xanthine dehydrogenase to XO in diabetic rat liver. The results suggest that Laminaria japonica would be of great value in preventing hyperglycemia in diabetes mellitus as a dietary supplement possibly, through its antioxidant activity.     

HEPATOPROTECTIVE EFFECTS OF PICROLIV IN ALCOHOL-FED ALBINO RATS

Alcohol abuse is thought to be a risk factor for the cause of liver damage, hyperlipidemia and insulin resistance. An alcohol-fed rat model was developed by chronic administration of ethanol to rats which caused significant alteration in liver function as revealed by elevated serum transaminases, alkaline phosphatase, acid phosphatase, sorbitol dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase and bilirubin and decline in total serum protein content over the control group after 25 days of feeding in both male and female rats. Hepatic glutathione, lipid peroxides, glutathione peroxidase, alcohol dehydrogenase, aldehyde dehydrogenase, glycogen and total protein in liver were also significantly altered. A slightly elevated fasting blood glucose profile, 1.5 fold higher serum insulin levels and impaired glucose tolerance was prevalent in ethanol treated rats. In the present study, the effect of picroliv, an irridoid glycosidic fraction of Picrorhiza kurroa, on the above said parameters of these alcoholic rats was studied. Picroliv significantly reverted most of the above said altered blood and hepatic parameters in the alcohol-fed male and female rats to almost normal levels.     

Detection of immunoglobulin depositions by immunofluorescence and/or immunoperoxidase (P-AP method) in patients with IgA nephropathy

Deposition of immunoglobulins in the glomeruli by immunoperoxidase (IP) and/or immunofluorescence (IF) using unfixed materials obtained from 25 patients with IgA nephropathy (IgAN) was evaluated. The results indicated that the deposition of IgA, IgM or IgG in the glomerular capillary walls using a peroxidase anti-peroxidase (P-AP) method was more prominently detected than by using IF. The results of double staining indicated that both P-AP and IF were able to stain mesangial and capillary depositions of IgA was more widely detected by P-AP than by IF. It is concluded that the P-AP method has the advantage of indicating the deposition of immunoglobulins in glomerular capillary walls, and evaluating the histopathological significance together with IF in patients with IgAN.