C-Phycocyanin Modulates Selenite-Induced Cataractogenesis in Rats

The present investigation is aimed to evaluate the anticataractogenic potential of C-phycocyanin (C-PC), extracted and purified from Spirulina platensis. Enucleated rat lenses were maintained in vitro in Dulbecco’s modified Eagle medium (DMEM). Group I contained DMEM, Group II and Group III contained 100 μM of sodium selenite, Group III was subdivided into three viz IIIa, IIIb, IIIc supplemented with 100, 150, 200 μg of C-PC respectively. In the in vivo study, on tenth day post partum: Group I rat pups received an intraperitoneal injection of saline, Group II, IIIa, IIIb, and IIIc rat pups received a subcutaneous injection of sodium selenite (19 μmol/kg bodyweight) Group IIIa, IIIb, IIIc also received an intraperitoneal injection of 100, 150, 200 mg/kg body weight of C-PC, respectively, from postpartum days?9–14. On termination of the experiment, the lenses from both in vitro and in vivo studies were subjected to morphological examination and subsequently processed to estimate the activities of antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, levels of reduced glutathione and lipid peroxidation products. Sodium selenite-exposed, C-PC-treated rat lenses (Group IIIc), showed significant restoration of antioxidant enzyme activity (p?<?0.05) when compared to their counterpart Group II. Group IIIc conserved the levels of GSH and lipid peroxidation products at near to normal levels as compared with Group II. Results conclude the possible role of C-PC in modulating the antioxidant enzyme status, thereby retarding sodium selenite-induced cataract incidence both in vitro and in vivo.     

Resveratrol delay the cataract formation against naphthalene‐induced experimental cataract in the albino rats

Oxidative stress‐induced toxicity plays a major role in ocular diseases such as retinal degeneration, age‐related cataract (ARC) formation and macular dystrophy. In this study, we explored the possible role of resveratrol (RSV) at the different dose levels (10, 20 and 40 mg/kg/day, ip) in an experimental model of naphthalene (1 g/kg/day, po)‐induced age‐related cataracts. Morphological changes in the eyes of the rats in two groups, the RSV and the ARC groups, were monitored weekly, and biochemical parameters in the lenses were assessed after completion of the experimental work. A comparison between the rats in the two groups showed that treatments at RSV doses of 20 and 40 mg/kg/day significantly retarded lenticular opacity, restored antioxidants (CAT, SOD, GPX, GSH), Ca²⁺ ATPase function, and protein contents, and reduced lipid peroxidation in the lenses of the animals in the RSV group. The treatment with resveratrol at a dose of 10 mg/kg/day did not show any anti‐cataractogenic effects. Based on the results of our investigation, we conclude that supplemental doses of resveratrol at 40 mg/kg/day effectively prevent cataract formation associated with the aging via increased soluble protein contents and Ca²⁺ homeostasis, apart from the antioxidant restoration. The results demonstrate that RSV treatment may be considered as a promising preventive or supplemental measure for delaying and/or preventing the formation of ARCs.     

Enhancement of murine phenytoin teratogenicity by the gamma-glutamylcysteine synthetase inhibitor L-buthionine-(S,R)-sulfoximine and by the glutathione depletor diethyl maleate

The teratogenicity of phenytoin may result from its enzymatic bioactivation to a reactive intermediate, which, if not detoxified, can interact with embryonic tissues and alter development. Glutathione (GSH) is an important cofactor/substrate for many physiological processes and for the detoxification of xenobiotic reactive intermediates. This study examined the effects of the GSH depletor diethyl maleate (DEM) and the GSH synthesis inhibitor L-buthionine-(S,R)-sulfoximine (BSO) on phenytoin embryopathy. Phenytoin, 55 mg/kg, was administered intraperitoneally (ip) to pregnant CD-1 mice at 0900 hr on gestational days 12 and 13. Pretreatment with DEM, 150 or 300 mg/kg ip, enhanced the incidence of phenytoin-induced cleft palates by 3.3-fold and 2.3-fold, respectively (P less than 0.05), without affecting the incidence of resorptions, postpartum death, or mean fetal weight. BSO, 1,800 mg/kg ip, given 0.5 hr prior to phenytoin, resulted in a 2.4-fold increase in postpartum lethality and a 5-fold increase in fetal weight loss (P less than 0.05), without altering the incidence of resorptions or cleft palates. In two subsequent studies, BSO, 680-1,018 mg/kg/day, was given in the drinking water on gestational days 9 to 13 in the first study and on days 10 to 14 in the second study. Phenytoin, 55 mg/kg ip, was given on days 11 and 12 and on days 11 to 13 in the respective studies. In the first drinking water study, BSO enhanced the incidence of phenytoin-induced fetal resorptions 3.8-fold and cleft palates 3.3-fold (P less than 0.05) but did not affect postpartum death. In the second study, BSO enhanced the incidence of resorptions, cleft palates, and postpartum death by 2-fold, 2.6-fold, and 1.7-fold, respectively (P less than 0.05). In both of the latter two studies, phenytoin-induced fetal weight loss was altered by BSO treatment (P less than 0.05). BSO alone had no embryopathic effects. These results suggest that GSH may be involved in the detoxification of a reactive intermediate of phenytoin and/or in fetal cytoprotection.     

Prevention of Selenite-Induced Cataractogenesis by an Ethanolic Extract of Cineraria maritima: An Experimental Evaluation of the Traditional Eye Medication

In the present study, the antioxidant potential of an ethanolic extract of Cineraria maritima and its efficacy in preventing selenite-induced cataractogenesis were assessed in vitro and in vivo. In the in vitro phase of the study, lenses dissected out from the eyes of Wistar rats were incubated for 24 h at 37°C in Dulbecco’s modified Eagle medium (DMEM) alone (group I), in DMEM containing 100 μM of selenite only (group II), or in DMEM containing 100 μM of selenite and 300 μg/ml C. maritima extract added at the same time (group III). Gross morphological examination of the lenses revealed dense opacification in group II, minimal opacification in group III, and no opacification in group I lenses. The mean activities of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly lower in group II than in group I or group III lenses, while malondialdehyde concentration was significantly higher in group II lenses than in group I and group III lenses. In the in vivo phase of the study, dense opacification of lenses was noted in all rat pups (100%) that had received a single subcutaneous injection of sodium selenite alone (19 μM/kg body weight) on postpartum day 10, whereas cataract formation occurred in only 33.3% of rat pups that had received selenite as well as an intraperitoneal injection of the extract of C. maritima (350 mg/kg body weight) for five consecutive days. These observations suggest that the ethanolic extract of C. maritima may prevent experimental selenite-induced cataractogenesis.     

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy

The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (gamma-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.     

The effect of buthionine sulfoximine on the glutathione level in goldfish tissues

This study describes the effect of DL-buthionine-[S,R]-sulfoximine (BSO) on the glutathione equivalents (GSH-eq = GSH + 2 GSSG) of goldfish. BSO causes depletion of cellular GSH by inhibiting gamma-glutamylcysteine synthetase, a key enzyme of the GSH biosynthesis pathway. BSO at 1,000 and 1,500 mg/kg was effective in promoting 50 and 80% depletion of GSH-eq from brain and liver, respectively, within 3 days. Lower doses of BSO failed to effectively promote hepatic GSH-eq depletion. Moreover, no evident toxic side-effects were observed (including hepatic lipid peroxidation and free radical-mediated oxidation of proteins) in goldfish in response to BSO intraperitoneal injections. We conclude that BSO can be used to deplete GSH-eq in goldfish liver and brain, but attention should be paid to species-specific variations in BSO effects.     

硒性白内障大鼠模型晶状体中GR和GSH-Px的表达

 为探讨硒性白内障大鼠晶状体中谷胱甘肽过氧化物酶 (GSH Px)和谷胱甘肽还原酶 (GR)的活性调节在硒性白内障形成中的作用及调节方式 ,采用半定量RT PCR方法 ,比较正常晶状体、核中心混浊晶状体 (核白 )和完全混浊晶状体 (全白 )中GSH Px和GR的mRNA水平及酶活性的变化 .研究发现 ,核白晶状体中 2种酶的活性和mRNA水平均升高 ,其中酶活性的升高幅度小于mRNA水平 .随着白内障的发展 ,2种酶的活性和mRNA水平均逐渐下降 .至晶状体全白时 ,2种酶的活性均显著低于正常 ;全白时GR的mRNA水平降至正常 ,GSH Px的mRNA水平则仍高于正常 .结果表明 ,硒性白内障形成与细胞内GSH Px和GR的活性调节密切相关 ,GSH Px和GR的活性调节可能主要发生在转录水平     

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.     

比较不同类型白内障形成过程中非蛋白质巯基与蛋白质巯基的动态变化及关系

本文报道了在亚硒酸钠、平阳霉素及半乳糖诱发大鼠产生白内障过程中晶状体中非蛋白质巯基及蛋白质巯基的动态变化,并探讨了其变化机理及相互关系。在亚硒酸钠诱发白内障过程中,给药24h后晶状体中非蛋白质巯基减少到正常的二分之一,以后又逐渐回升,但始终未达到正常水平,至第7天,非蛋白质巯基又再度减少。在平阳霉素及半乳糖诱发白内障过程中,晶状体中非蛋白质巯基分别在给药后的第7天及第3天开始大量减少,以后继续减少,至第15天时,其含量分别为正常的十分之一及五分之一。在体外,亚硒酸钠有促进还原型谷胱甘肽自氧化的作用,半乳糖对此作用无影响,而平阳霉素可阻止其进行,但能加强亚硒酸钠的促进作用。在三种白内障晶状体中,蛋白质巯基开始减少的时间均较非蛋白质巯基为晚,这表明只有非蛋白质巯基减少到一定程度后蛋白质巯基才会被大量氧化,同时也说明非蛋白质巯基具有保护蛋白质巯基免受氧化的作用。只有这种保护作用减弱后,才会使蛋白质巯基遭受氧化而导致白内障。     

Influence of buthionine sulfoximine and misonidazole on glutathione level and radiosensitivity of human tumor xenografts

We have studied the effect of buthionine sulfoximine (BSO; a gamma-glutamylcysteine synthetase inhibitor) administration, either alone or combined with misonidazole (MISO), on five human tumor xenografts (three melanomas: Bell, Mall, and Nall; and two rectocolic adenocarcinomas: HT29 and HRT18) transplanted into mice. Two criteria were used, the nonprotein bound sulfhydryl (NPSH) level (glutathione (GSH) and cysteine (CYS] and the fraction of surviving tumor cells after gamma irradiation. GSH and CYS were estimated by HPLC and cell survival by in vivo-in vitro clonogenic assay. Administration of BSO alone (three injections of 10 mumol/g) prior to irradiation always produced a significant reduction in the GSH level while MISO administration (1 mg/g) did not consistently influence the NPSH level. While BSO had little or no radiosensitizing effect, MISO always induced radiosensitization (enhancement ratio between 1.6 and 1.8). This effect did not depend on the fraction of surviving hypoxic cells. An increase in MISO-induced radiosensitization produced by BSO was cell-line dependent. Results do not seem to support the hypothesis of a relationship between the GSH level at the time of irradiation and the radiosensitization induced by BSO or BSO + MISO. However, BSO treatment may not have been able to reduce endogenous thiols to a low enough level to test the hypothesis.     

Susceptibility of Human Head and Neck Cancer Cells to Combined Inhibition of Glutathione and Thioredoxin Metabolism

Increased glutathione (GSH) and thioredoxin (Trx) metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC) cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO) and auranofin (AUR), respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO) and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.     

Aging reduces responsiveness to BSO- and heat stress-induced perturbations of glutathione and antioxidant enzymes

Aging alters cellular responses to both heat and oxidative stress. Thiol-mediated metabolism of reactive oxygen species (ROS) is believed to be important in aging. To begin to determine the role of thiols in aging and heat stress, we depleted liver glutathione (GSH) by administering l-buthionine sulfoximine (BSO) in young (6 mo) and old (24 mo) Fisher 344 rats before heat stress. Animals were given BSO (4 mmol/kg ip) or saline (1 ml ip) 2 h before heat stress and subsequently heated to a core temperature of 41 degrees C over a 90-min period. Liver tissue was collected before and 0, 30, and 60 min after heat stress. BSO inhibited glutamate cysteine ligase (GCL, the rate-limiting enzyme in GSH synthesis) catalytic activity and resulted in a decline in liver GSH and GSSG that was more pronounced in young compared with old animals. Catalase activity did not change between groups until 60 min after heat stress in young BSO-treated rats. Young animals experienced a substantial and persistent reduction in Cu,Zn-SOD activity with BSO treatment. Mn-SOD activity increased with BSO but declined after heat stress. The differences in thiol depletion observed between young and old animals with BSO treatment may be indicative of age-related differences in GSH compartmentalization that could have an impact on maintenance of redox homeostasis and antioxidant balance immediately after a physiologically relevant stress. The significant changes in antioxidant enzyme activity after GSH depletion suggest that thiol status can influence the regulation of other antioxidant enzymes.     

SR-2508 plus buthionine sulfoximine or SR-2508 alone: effects on the radiation response and the glutathione content of a human tumor xenograft

This study determined the radiosensitivity of the human tumor xenograft HT29 and its glutathione (GSH) and cysteine (CYS) contents after treatment with both buthionine sulfoximine (BSO) and SR-2508 or SR-2508 alone. Tumor radiosensitivity was assessed by the in vitro colony assay and thiol content was measured by high-performance liquid chromatography. The radiosensitizing effect of SR-2508 is dose dependent and increases when higher doses of radiation are given. SR-2508 given alone does not modify GSH and CYS content; however, when given with BSO, the GSH level is significantly reduced, yet radiosensitivity of the HT29 tumor is only slightly increased. These results have been compared to our previously observed results of HT29 treatment with misonidazole (MISO), BSO, or MISO + BSO.     

Hepatic glutathione and nitric oxide are critical for hepatic insulin-sensitizing substance action

We tested the hypothesis that hepatic nitric oxide (NO) and glutathione (GSH) are involved in the synthesis of a putative hormone referred to as hepatic insulin-sensitizing substance HISS. Insulin action was assessed in Wistar rats using the rapid insulin sensitivity test (RIST). Blockade of hepatic NO synthesis with N(G)-nitro-l-arginine methyl ester (l-NAME, 1.0 mg/kg intraportal) decreased insulin sensitivity by 45.1 +/- 2.1% compared with control (from 287.3 +/- 18.1 to 155.3 +/- 10.1 mg glucose/kg, P < 0.05). Insulin sensitivity was restored to 321.7 +/- 44.7 mg glucose/kg after administration of an NO donor, intraportal SIN-1 (5 mg/kg), which promotes GSH nitrosation, but not after intraportal sodium nitroprusside (20 nmol x kg(-1) x min(-1)), which does not nitrosate GSH. We depleted hepatic GSH using the GSH synthesis inhibitor l-buthionine-[S,R]-sulfoximine (BSO, 2 mmol/kg body wt ip for 20 days), which reduced insulin sensitivity by 39.1%. Insulin sensitivity after l-NAME was not significantly different between BSO- and sham-treated animals. SIN-1 did not reverse the insulin resistance induced by l-NAME in the BSO-treated group. These results support our hypothesis that NO and GSH are essential for insulin action.     

Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of Tatar buckwheat calli

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10–20 times higher and the glutathione reductase activity was 2–2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6–8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation of proembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.     

Combination of glycemic and oxidative stress in lens: implications in augmentation of cataract formation in diabetes

It is well known that the incidence of cataract is higher in diabetics as compared to non-diabetics. Its rate of maturation is also faster in the diabetics. The precise mechanism of this acceleration is not clearly understood. It is hypothesized that this could be a result of the combination of the metabolic and oxidative stress induced by glycemia itself with the age-associated increase in ambient generation of oxyradical species. In the current studies, we have investigated this possibility using the galactose cataract model. Galactosemia was induced by feeding rats a 50% galactose diet. The increased susceptibility of the glycemic lenses to physiological damage by reactive oxygen species (ROS) was studied by incubating them in Tyrode in the absence and presence of menadione. The resulting physiological damage to the lens was assessed initially in terms of its ability to maintain Na⁺-K⁺ ATPase dependent active transport of potassium ions, as represented by the uptake of rubidium ions. Subsequently, the level of ATP, indexing the general metabolic status, and the level of glutathione (GSH), indexing the status of antioxidant reserve, were also determined. The uptake of rubidium in the normal lenses incubated in the presence of the quinone was depressed to more than 50% of the controls run in the basal medium. A similar depression existed in the galactosemic lenses in comparison to the normal lenses. However, in the presence of menadione, the inhibition of the uptake was accentuated further in the case of galactosemic lenses, the uptake here being only 20% of the normal controls. Similarly, the galactosemic lenses were also more susceptible to menadione dependent decrease in ATP and GSH.     

晶状体生化的研究:正常和亚硒酸钠诱发白内障大鼠晶状体中与谷胱甘肽代谢有关的三种酶活性的测定

测定了用亚硒酸钠诱发的大鼠白内障晶状体中谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GSSG-R)和谷胱甘肽硫转移酶(GSH-S)的活性,并与正常晶休中这三种酶的活性作了比较。结果表明,核浊浑期晶状体中GSH-Px的活性比正常晶状体的高一倍,但在整个晶状体浑浊时降低,GSSG-R的活性变化与GSH-PX相似,这两种酶在代谢上是相关的。GSH-S的活性在核浑浊期不改变,但在完全浑浊后降低。     

Role of glutathione on renal mitochondrial status in hyperoxaluria

Role of glutathione on kidney mitochondrial integrity and function during stone forming process in hyperoxaluric state was investigated in male albino rats of Wistar strain. Hyperoxaluria was induced by feeding ethylene glycol (EG) in drinking water. Glutathione was depleted by administering buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis. Glutathione monoester (GME) was administered for supplementing glutathione. BSO treatment alone or along with EG, depleted mitochondrial GSH by 40% and 51% respectively. Concomitantly, there was remarkable elevation in lipid peroxidation and oxidation of protein thiols. Mitochondrial oxalate binding was enhanced by 74% and 129% in BSO and BSO + EG treatment. Comparatively, EG treatment produced only a 33% increase in mitochondrial oxalate binding. Significant alteration in calcium homeostasis was seen following BSO and BSO + EG treatment. This may be due to altered mitochondrial integrity and function as evidenced from decreased activities of mitochondrial inner membrane marker enzymes, succinate dehydrogenase and cytochrome-c-oxidase and respiratory control ratio and enhanced NADH oxidation by mitochondria in these two groups. NADH oxidation (r = -0.74) and oxalate deposition in the kidney (r = -0.70) correlated negatively with mitochondrial glutathione depletion. GME supplementation restored normal level of GSH and maintained mitochondrial integrity and function, as a result of which oxalate deposition was prevented despite hyperoxaluria. These results suggest that mitochondrial dysfunction resulting from GSH depletion could be a contributing factor in the development of calcium oxalate stones.     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

The modulatory influence of p-methoxycinnamic acid, an active rice bran phenolic acid, against 1,2-dimethylhydrazine-induced lipid peroxidation, antioxidant status and aberrant crypt foci in rat colon carcinogenesis

We investigated the chemopreventive effect of p-methoxycinnamic acid (p-MCA), an active phenolic acid of rice bran, turmeric, and Kaemperfia galanga against 1,2-dimethylhydrazine-induced rat colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 consisted of control rats that received a modified pellet diet and 0.1% carboxymethyl cellulose. The rats in Group 2 received a modified pellet diet supplemented with p-MCA [80 mg/kg body weight (b.wt.) post-orally (p.o.)] everyday. The rats in Groups 3-6 received 1,2-dimethylhydrazine (DMH) (20 mg/kg b.wt.) via subcutaneous injections once a week for the first 4 weeks; additionally, the rats in Groups 4, 5 and 6 received p-MCA at doses of 20, 40 and 80 mg/kg b.wt./day p.o., respectively, everyday for 16 weeks. The rats were sacrificed at the end of the experimental period of 16 weeks. The DMH-treated rats exhibited an increased incidence of aberrant crypt foci (ACF) development; an increased crypt multiplicity; decreased concentrations of tissue lipid peroxidation markers such as thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD) and lipid hydroperoxides (LOOH); decreased levels of tissue enzymic antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR); and decreased levels of non-enzymic antioxidants such as reduced glutathione (GSH) and vitamins C, E and A in the colon. Supplementation with p-MCA significantly reversed these changes and significantly inhibited the formation of ACF and its multiplicity. Thus, our findings demonstrate that p-MCA exerts a strong chemopreventive activity against 1,2-dimethylhydrazine-induced colon carcinogenesis by virtue of its ability to prevent the alterations in DMH-induced circulatory and tissue oxidative stress and preneoplastic changes. p-MCA was more effective when administered at a dose of 40 mg/kg b.wt. than at the other two doses tested.