hCG treatment raises H<Subscript>2</Subscript>O<Subscript>2</Subscript> levels and induces germ cell apoptosis in rat testis

The clinical significance of exogenous hCG treatment is to stimulate steroidogenesis and spermatogenesis in the testis. However, the pathogenesis of detrimental effects on the testis arising out of chronic hCG treatment is yet to be clearly ascertained. In the present study we have shown that hCG treatment (100 IU/day) to rats for 30 days raises testicular oxidative stress leading to germ cell apoptosis and impairment of spermatogenesis. The treatment raises testicular H₂O₂ levels along with increase in lipid peroxidation and concomitant decrease in the enzymatic antioxidant activities like superoxide dismutase, catalase and glutathione-s-transferase. The rise in the number of apoptotic germ cells was associated with up regulation of Fas protein expression and caspase-3 activity in the testis. However, serum testosterone which was elevated by 15 days of hCG treatment declined to pretreatment levels by 30 days. No significant alteration in serum gonadotropins was observed. The above findings indicate that the pathogenesis of deleterious effects following chronic hCG treatment is due to increase in testicular oxidative stress with high H₂O₂ availability leading to apoptosis among germ cells.     

Ferulic acid supplements abrogate oxidative impairments in liver and testis in the streptozotocin-diabetic rat

The primary objective of this study was to assess the efficacy of ferulic acid (FA), a phenolic antioxidant, in ameliorating oxidative stress in the testis and liver of diabetic pubertal rats. Male (6 wk old) rats were rendered diabetic by an acute dose (60 mg/kg body weight, intraperitoneal) of streptozotocin (STZ) and were given oral supplementation of FA (50 mg/kg body weight/d on alternate days) for 4 weeks. The protective efficacy of FA was assessed by measuring markers of oxidative stress in the testis and liver along with the effect of stress on lipid profile in serum/testis. Terminally, the testis (cytosol and mitochondria) of STZ-administered rats exhibited a marked elevation in the status of lipid peroxidation and enhanced reactive oxygen species (ROS) production compared to the non-diabetic controls. FA treatment completely normalized the oxidative impairments in the testis. Further, STZ-induced depletion of reduced glutathione (GSH) and elevated protein carbonyl content in the testis were restored to normalcy by FA treatment. The protective effects of FA were also discernible in the testis in terms of restoration of activities of various antioxidant enzymes in the diabetic rats. Furthermore, STZ-induced oxidative impairments in the liver were also abrogated significantly by FA treatment. STZ-induced perturbations in serum and testicular lipid profiles in the diabetic rats were also significantly attenuated by FA treatment. Collectively, these results indicate that oral supplementation of FA can significantly mitigate diabetes-associated oxidative impairments in the testis as well as in the liver and suggests the efficacy of FA as a complementary therapeutic agent in the management of diabetes-associated oxidative stress-mediated complications.     

d-Aspartic acid induced oxidative stress and mitochondrial dysfunctions in testis of prepubertal rats

Previously we demonstrated the potential of d-aspartic acid (d-Asp), an acidic amino acid to induce oxidative response in prepubertal rat testis in vitro. In the present study, we determined the extent of oxidative stress in the testis of prepubertal rats that were administered d-Asp (100 and 500 mg/kg bw/d, i.p. 7 days). d-Asp treatment significantly elevated the levels of reactive oxygen species, malondialdehyde and hydroperoxide in cytosol and mitochondria of testis, which were accompanied by enhanced glutathione levels, elevated activities of glutathione-dependent enzymes and catalase suggesting a state of oxidative stress. Further, the activities of d-aspartate oxidase and 3β-hydroxy steroid dehydrogenase were elevated in the testis. The testis mitochondria of d-Asp-treated rats showed altered citric acid and complex enzyme activities, reduction in membrane potential, increased permeability and intracellular Ca²⁺ levels. Collectively, these findings suggest the potential of d-Asp to induce oxidative perturbations in the testis of prepubertal rats and this mechanism may in part be responsible for the observed physiological effects.     

Time-dependent oxidative stress caused by benznidazole

Benznidazole (BZN) is a nitroimidazole derivative which has a notable trypanocide activity, and it is the only drug used in Brazil and Argentina for the treatment of Chagas' disease. The drug in current use is thought to act, at least in part, by inducing oxidative stress within the parasite. Imidazolic compounds are involved in the production of reactive oxygen species (ROS). In order to evaluate the effect of BZN on ROS production and on the antioxidant status of the host, male rats were treated for different periods of time (2, 4, 6, 10 and 30 days) with 40 mg BZN/kg body weight. After treatment, biomarkers of oxidative stress such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) and glutathione reductase (GR), and also thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), total glutathione (TG) and oxidized glutathione (GSSG) concentrations, were measured in crude hepatic homogenates. Our results revealed that BZN is able to cause tissue damage as shown by increased TBARS content, inhibition of some antioxidants and induction of other antioxidants in a concentration- and time-dependent manner. The tissue damage measured as TBARS increased up to the 10th day of treatment. GST activity was inhibited during the BZN treatment. On the other hand, CAT and GR showed similar increased activities at the beginning, followed by decreased activities at the end of the treatment. After 30 days of treatment, GR activity remained low while CAT activity was high, compared to controls. The SOD activities remained unchanged throughout the experimental period. GSH showed lower values at the beginning of BZN treatment but the hepatic concentrations were enhanced at the end of the experimental period. Total glutathione showed a similar profile, and oxidized glutathione showed higher values in rats treated with BZN. In conclusion, these results indicate that, at therapeutic doses, BZN treatment elicits an oxidative stress in rat hepatocytes.     

A male contraceptive targeting germ cell adhesion

Throughout spermatogenesis, developing germ cells remain attached to Sertoli cells via testis-specific anchoring junctions. If adhesion between these cell types is compromised, germ cells detach from the seminiferous epithelium and infertility often results. Previously, we reported that Adjudin is capable of inducing germ cell loss from the epithelium. In a small subset of animals, however, oral administration of Adjudin (50 mg per kg body weight (b.w.) for 29 d) resulted in adverse effects such as liver inflammation and muscle atrophy. Here, we report a novel approach in which Adjudin is specifically targeted to the testis by conjugating Adjudin to a recombinant follicle-stimulating hormone (FSH) mutant, which serves as its 'carrier'. Using this approach, infertility was induced in adult rats when 0.5 microg Adjudin per kg b.w. was administered intraperitoneally, which was similar to results when 50 mg per kg b.w. was given orally. This represents a substantial increase in Adjudin's selectivity and efficacy as a male contraceptive.     

Piperine activates testicular apoptosis in adult rats

Piperine, an alkaloid present in the fruits of commonly used spice pepper, is known to impair reproductive functions. In the present study, piperine was administered to adult male rats at the dose levels of 1, 10, and 100 mg/kg body weight for 30 days to evaluate its effects on the testis. A significant decrease in the activities of antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in the testis was observed at 10 and 100 mg of piperine administration when compared with the controls. A dose‐dependent increase in lipid peroxidation and hydrogen peroxide generation was also observed. Sialic acid levels in the testis were also found to be decreased when piperine was administered at 10 and 100 mg dose levels. Immunofluorescence studies demonstrated a dose‐dependent increase in caspase 3 and Fas protein in testicular germ cells after piperine treatment. These observations indicate that piperine induces oxidative stress and thereby triggers apoptosis in the testis, contributing to hampered reproductive functions. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:382–388, 2008; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20251     

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress,BAX, and caspase 2

Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function. The work was supported by grants P50 DK45179 and DK53072.     

Oxidative stress,spermatogenesis and fertility

Reactive oxygen species production and glutathione depletion in mammalian male germ cells are physiological events that are requisite to the functional maturation and capacitation of spermatozoa. In relation to this oxidative stress, an oxidation of the bulk of protein sulfydryl groups takes place during the final phases of male germ cell maturation. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase catalyzes this reaction, and accounts for both the assembly of the mid-piece of spermatozoa and chromatin condensation. This process highlights the role of H2O2 and selenium in spermatogenesis and provides a mechanism for coupling a 'physiologically controlled' oxidative stress to a specialized phenotypic function.     

Proteasome inhibition induces glutathione synthesis and protects cells from oxidative stress: relevance to Parkinson disease

The cause of selective dopaminergic neuronal degeneration in Parkinson disease has still not been resolved, but it has been hypothesized that oxidative stress and the ubiquitin-proteasome system are important in the pathogenesis. In this report, we investigated the effect of proteasome inhibition on oxidative stress-induced cytotoxicity in PC12 cells, an in vitro model of Parkinson disease. Treatment with proteasome inhibitors provided significant protection against toxicity by 6-hydroxydopamine and H(2)O(2) in a concentration-dependent manner. The measurement of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate demonstrated that lactacystin, a proteasome inhibitor, significantly reduced 6-hydroxydopamineand H(2)O(2)-induced reactive oxygen species production. Proteasome inhibitors elevated the amount of glutathione and phosphorylated p38 mitogen-activated protein kinase (MAPK) prior to glutathione elevation. The treatment with lactacystin induced the nuclear translocation of NF-E2-related factor 2 (Nrf2) and increased the level of mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme in glutathione synthesis. Furthermore, SB203580, an inhibitor of p38 MAPK, abolished glutathione elevation and cytoprotection by lactacystin. These data suggest that proteasome inhibition afforded cytoprotection against oxidative stress by the elevation of glutathione content, and its elevation was mediated by p38 MAPK phosphorylation.     

Time-dependent oxidative stress caused by benznidazole

Abstract

Benznidazole (BZN) is a nitroimidazole derivative which has a notable trypanocide activity, and it is the only drug used in Brazil and Argentina for the treatment of Chagas' disease. The drug in current use is thought to act, at least in part, by inducing oxidative stress within the parasite. Imidazolic compounds are involved in the production of reactive oxygen species (ROS). In order to evaluate the effect of BZN on ROS production and on the antioxidant status of the host, male rats were treated for different periods of time (2, 4, 6, 10 and 30 days) with 40 mg BZN/kg body weight. After treatment, biomarkers of oxidative stress such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) and glutathione reductase (GR), and also thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), total glutathione (TG) and oxidized glutathione (GSSG) concentrations, were measured in crude hepatic homogenates. Our results revealed that BZN is able to cause tissue damage as shown by increased TBARS content, inhibition of some antioxidants and induction of other antioxidants in a concentration- and time-dependent manner. The tissue damage measured as TBARS increased up to the 10th day of treatment. GST activity was inhibited during the BZN treatment. On the other hand, CAT and GR showed similar increased activities at the beginning, followed by decreased activities at the end of the treatment. After 30 days of treatment, GR activity remained low while CAT activity was high, compared to controls. The SOD activities remained unchanged throughout the experimental period. GSH showed lower values at the beginning of BZN treatment but the hepatic concentrations were enhanced at the end of the experimental period. Total glutathione showed a similar profile, and oxidized glutathione showed higher values in rats treated with BZN. In conclusion, these results indicate that, at therapeutic doses, BZN treatment elicits an oxidative stress in rat hepatocytes.     

Germ cell death and their removal during initial stages of testicular ischemia and cryptorchidism: a comparative analysis

Germ cell death and their removal from the seminiferous epithelium are common in the affected testis in conditions of unilateral ischemia or cryptorchidism; the similarities and differences, however, have not been studied between these two conditions. The present study was designed to examine the severity of the effect on testicular germ cells during the initial stages of both ischemia and cryptorchidism, which have significant implications on the restoration of fertility following surgical repair. Complete absence of spermatids was observed following 12 hr of ischemia as compared to 7 days of cryptorchidism. Germ cell removal in either case was in the direction of lumen to basement membrane leaving only a single layer of cells by 24 hr of unilateral ischemia as compared to 15 days of cryptorchidism. Levels of intratesticular testosterone was found lower in cryptorchidism (7 days) but not in ischemia till 24 hrs. Giant cells frequently observed in cryptorchid testis were absent in the ischemic seminiferous epithelium. There was a gradual increase in the number of apoptotic and non-viable cells; the latter was more than 95% by 24 hr of ischemia. In contrast, approximately 85% testicular cells were nonviable till 15 days of cryptorchidism. The 1c peak representing the population of haploid cells was significantly reduced in cryptorchidism (7 days), while the peak was completely abolished by 24 hr of ischemia. Rise in the levels of oxidative stress in the affected testis was observed identically during the initial stages. These findings indicate that coupled with the rise in tissue oxidative stress, the number of apoptotic/nonviable germ cells was alarmingly high (> 80%) by 15 days of cryptochidism or 24 hr of ischemia. Restoration of complete spermatogenesis following surgical repair may not be possible in such cases because of these acute adverse effects.     

Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally cryptorchid rat testis.

The effect of experimental cryptorchidism on the level of oxidative stress and antioxidant functions in rat testis was studied. Adult male Sprague-Dawley rats were rendered unilaterally cryptorchid (by suturing one testis to the abdominal wall) and killed 1, 3, or 7 days after the operation. As an indicator of oxidative stress, lipid peroxidation was measured by the diene conjugation method in testis homogenates. The activities of the antioxidant enzymes were determined either in the 10,000 x g supernatant fraction (glutathione [GSH] peroxidase, GSH transferase, hexose monophosphate shunt) or in crude testis homogenates (superoxide dismutase, catalase). An expected reduction (48%) in weight of the abdominal testes was evident by postoperative Day 7. The catalytic activities per testis of superoxide dismutase (Cu/Zn form) and catalase were found to decrease in cryptorchidism. The effect was seen on the first postoperative day and was most profound on Day 7 after surgery. The principal antioxidant enzyme, superoxide dismutase, was most sensitive to cryptorchidism, the activity in the abdominal testes being 74% or 85% (per gram of tissue or per whole testis, respectively; p less than 0.01). After impairment of the reactive oxygen detoxifying capacity, lipid peroxidation was increased in the abdominal testis by 46% (p less than 0.01) on postoperative Day 7. Slight concomitant increases were detected in the activities of GSH-peroxidase (p less than 0.01), GSH-transferase (p less than 0.001), and the hexose monophosphate shunt (p less than 0.001). This effect was seen only when calculated per gram of tissue, not per whole testis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidative stress impairs the repair of oxidative DNA base modifications in human skin fibroblasts and melanoma cells

Irradiation of mammalian cells with solar light is associated with the generation of reactive oxygen species (ROS) and oxidative stress, which is mediated in part by endogenous photosensitizers absorbing in the visible range of the solar spectrum. Accordingly, oxidative DNA base modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) are the predominant types of DNA damage in cells irradiated at wavelengths >400 nm. We have analysed the repair of oxidative purine modifications in human skin fibroblasts and melanoma cells using an alkaline elution technique, both under normal conditions and after depletion of glutathione. Similar repair rates were observed in fibroblasts and melanoma cells from three different patients (t1/2 approximately 4h). In both cell types, glutathione depletion (increased oxidative stress) caused a pronounced repair retardation even under non-toxic irradiation conditions. Furthermore, the cleavage activity at 8-oxoG residues measured in protein extracts of the cells dropped transiently after irradiation. An addition of dithiothreitol restored normal repair rates. Interestingly, the repair rates of cyclobutane pyrimidine dimers (t1/2 approximately 18 h), AP sites (t1/2 approximately 1h) and single-strand breaks (t1/2 <30 min) were not affected by the light-induced oxidative stress. We conclude that the base excision repair of oxidative purine modifications is surprisingly vulnerable to oxidative stress, while the nucleotide excision repair of pyrimidine dimers is not.     

Formation of reactive oxygen species in L929 cells after exposure to 900 MHz RF radiation with and without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

The aim of this study was to investigate the induction of reactive oxygen species in murine L929 fibrosarcoma cells exposed to radiofrequency (RF) radiation at 900 MHz, with or without co-exposure to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent environmental carcinogen produced during chlorination of drinking water. Both continuous-wave and GSM mobile phone signals were applied for 10 or 30 min at specific absorption rates of 0.3 and 1 W/kg. Simultaneous sham exposures were performed for each exposure condition. MX treatment was performed at a subtoxic level of 500 microM, and the RF-field exposure was carried out during the first 10 or 30 min of the chemical treatment. The formation of reactive oxygen species was followed soon after the exposure and at different harvesting times until 1 h after RF-field treatment. The studied provided no indication that 900 MHz RF-field exposure, either alone or in combination with MX, induced formation of reactive oxygen species under any of the experimental conditions investigated. In contrast, exposure to MX resulted in a statistically significant increase in the formation of reactive oxygen species for all the treatment durations investigated, confirming that MX is an inductor of oxidative stress in L929 cells.     

Lipid Peroxidation and Antioxidant Enzyme Activity in Response to Interval Hypoxic Training

This study revealed the metabolic parameters of reactive oxygen species, including erythrocyte superoxide dismutase, catalase, and glutathione peroxidase activity, and oxidative stress markers, including total prooxidant activity and plasma concentration of thiobarbituric acid reactive substances, in response to 14-day interval hypoxic training (IHT). The study included healthy subjects and patients with essential hypertension, who had a decreased activity of the main antioxidant enzymes due to a marked oxidative stress, as revealed by previous studies. In all subjects, the oxidative stress markers decreased and the enzyme activity increased in four days after the IHT course. However, the differences in metabolism of the reactive oxygen species between the patients and the healthy subjects persisted. It is suggested that, even with a different antioxidant enzyme system baseline, IHT may contribute to adaptive activity of this system.     

Germ cells of male mice express genes for peroxisomal metabolic pathways implicated in the regulation of spermatogenesis and the protection against oxidative stress

Peroxisomes are organelles with main functions in the metabolism of lipids and of reactive oxygen species. Within the testis, they have different functional profiles depending on the cell types. A dysfunction of peroxisomes interferes with regular spermatogenesis and can lead to infertility due to spermatogenic arrest. However, so far only very little is known about the functions of peroxisomes in germ cells. We have therefore analyzed the peroxisomal compartment in germ cells and its alterations during spermatogenesis by fluorescence and electron microscopy as well as by expression profiling of peroxisome-related genes in purified cell populations isolated from mouse testis. We could show that peroxisomes are present in all germ cells of the germinal epithelium. During late spermiogenesis, the peroxisomes form large clusters that are segregated from the spermatozoa into the residual bodies upon release from the germinal epithelium. Germ cells express genes for proteins involved in numerous metabolic pathways of peroxisomes. Based on the expression profile, we conclude that newly identified functions of germ cell peroxisomes are the synthesis of plasmalogens as well as the metabolism of retinoids, polyunsaturated fatty acids and polyamines. Thus, germ cell peroxisomes are involved in the regulation of the homeostasis of signaling molecules regulating spermatogenesis and they contribute to the protection of germ cells against oxidative stress.     

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide

Summary. The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.     

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15–44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-l-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H₂O₂ which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H₂O₂ significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H₂O₂ significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H₂O₂. NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H₂O₂.     

Dietary Antioxidant,Quercetin, Protects Sertoli‐Germ Cell Coculture from Atrazine‐Induced Oxidative Damage

Quercetin (QT), a dietary‐derived flavonoid, is ubiquitous in fruits and vegetables and plays an important role in human health by virtue of its antioxidant function. The present study was designed to examine the effects of QT on oxidative damage that was induced by the herbicide, atrazine (ATZ), in mixed cultures of Sertoli‐germ cells. Results showed that treatment with QT increased cell viability and decreased catalase activity, malondialdehyde, and reactive oxygen species (ROS) levels. QT treatment also increased the mRNA expression of glutathione peroxidase (GSH‐Px), glutathione reductase (GR), glutathione‐S‐transferase, and superoxide dismutase‐1 and could not reversed to the control levels ATZ‐induced steady‐state mRNA levels of these antioxidant genes as well as the level of glutathione and activities of GSH‐Px and GR. QT has protective effect against ATZ‐induced oxidative stress through a reduction in ROS levels and lipid peroxidation. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:477‐485, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21449     

Increased efflux of glutathione conjugate in acutely diabetic cardiomyocytes

In diabetes, cell death and resultant cardiomyopathy have been linked to oxidative stress and depletion of antioxidants like glutathione (GSH). Although the de novo synthesis and recycling of GSH have been extensively studied in the chronically diabetic heart, their contribution in modulating cardiac oxidative stress in acute diabetes has been largely ignored. Additionally, the possible contribution of cellular efflux in regulating GSH levels during diabetes is unknown. We used streptozotocin to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac GSH. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. The effect on GSH was not associated with any change in either synthesis or recycling, as both gamma-glutamylcysteine synthetase gene expression (responsible for bio syn thesis) and glutathione reductase activity (involved with GSH recycling) remained unchanged. However, gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was elevated. GSH conjugate efflux mediated by multidrug resistance protein 1 also increased in diabetic cardiomyocytes, an effect that was blocked using MK-571, a specific inhibitor of this transporter. As MK-571 also decreased oxidative stress in diabetic cardiomyocytes, an important role can be proposed for this transporter in GSH and reactive oxygen species homeostasis in the acutely diabetic heart.