Metal stoichiometry of isolated and arsenic substituted metallothionein: PIXE and ESI-MS study.

The stoichiometric analysis of the metal induced Metallothionein (MT) is pertinent for understanding the metal-MT interactions. Despite innumerable publications on MT, the literature addressing these aspects is limited. To bridge this gap, PIXE and ESI-MS analysis of the commercial rabbit liver MT1 (an isoform of MT), zinc induced isolated rat liver MT1, apo and Arsenic substituted rabbit liver MT1 have been carried out. These techniques in combination provide information about number and the signature of all the metal ions bound to MT. By using ESI-MS in the rabbit MT1, ions of Zn n MT1 (n = 0, 1, 4, 5, 6, 7) whereas, in rat MT1, the Zn1MT1 and Zn5MT1 ions are observed. PIXE analysis shows that some copper along with zinc is also present in the rabbit as well as rat MT1 which could not be assessed with ESI-MS. During As metallation reaction with rabbit MT1, with increase in arsenic concentration, the amount of arsenic bound to MT1 also increases, though not proportionally. The presence of both Zn and Cu in MT1 on Zn supplementation can be related to the role of MT in Zn and Cu homeostasis. Further, the presence of partially metallated MT1 suggests that MT1 may donate fractional amount of metal from it's fully metallated form to other proteins where Zn acts as a cofactor.

The relevance of arsenic speciation analysis in health & medicine.

Long term exposure to arsenic through consumption of contaminated groundwater has been a global issue since the last five decades; while from an alternate standpoint, arsenic compounds have emerged as unparallel chemotherapeutic drugs. This review highlights the contribution from arsenic speciation studies that have played a pivotal role in the progression of our understanding of the biological behaviour of arsenic in humans. We also discuss the limitations of the speciation studies and their association with the interpretation of arsenic metabolism. Chromatographic separation followed by spectroscopic detection as well as the utilization of biotinylated pull-down assays, protein microarray and radiolabelled arsenic have been instrumental in identifying hundreds of metabolic arsenic conjugates, while, computational modelling has predicted thousands of them. However, these species exhibit a variegated pattern, which supports more than one hypothesis for the metabolic pathway of arsenic. Thus, the arsenic species are yet to be integrated into a coherent mechanistic pathway depicting its chemicobiological fate. Novel biorelevant arsenic species have been identified due to significant evolution in experimental methodologies. However, these methods are specific for the identification of only a group of arsenicals sharing similar physiochemical properties; and may not be applicable to other constituents of the vast spectrum of arsenic species. Consequently, the identity of arsenic binding partners in vivo and the sequence of events in arsenic metabolism are still elusive. This resonates the need for additional focus on the extraction and characterization of both low and high molecular weight arsenicals in a combinative manner.

Studies on HDL associated enzymes under experimental hypercholesterolemia: possible modulation on selenium supplementation.

BACKGROUND: Atherosclerosis is a chronic disorder of the arterial wall that starts by formation of fatty streaks and gradually evolves into atherosclerotic plaques. High-density lipoproteins (HDL) blood levels are inversely correlated with atherosclerosis. This beneficial effect of HDL has been partly attributed to its antioxidant properties mediated by paraoxonase1 (PON1) or platelet-activating factor acetylhydrolase (PAF-AH). The present study was aimed to study HDL associated enzymes i.e. PON1 and PAF-AH under experimental hypercholesterolemia and their possible modulation on selenium (Se; an antioxidant) supplementation. Male Sprague Dawley rats were divided into three groups and fed on the control diet, high fat diet (HFD) and HFD + Se respectively for the period of 4 months. RESULTS: Cholesterol, triglycerides, HDL and LDL levels were significantly increased by HFD feeding. Selenium supplementation lowered the triglyceride level, whereas the other lipid values remained unchanged. Serum selenium levels were reduced by 31% and ROS levels in the liver were 2-fold increased by HFD. Se supplementation, however, diminished the HFD-induced ROS levels by 29%. Furthermore, Se also improved the HFD-mediated reduction of serum PON1 enzyme activity by 34% and PON1 protein levels by 21%. However, no significant effect of Se was detected on the reduced PAF-AH proteins levels in HFD fed rats. mRNA expression of PON1 and PAF-AH in the liver was not affected in the Se treated groups. CONCLUSION: Se supplementation appears to be protective in hypercholesterolemia by restoring the antioxidant properties of the HDL associated enzyme i.e. PON1 whereas biological system aims towards maintaining the same PAF-AH levels even on selenium supplementation indicating its probable role in both anti and pro-atherogenic activities. Therefore, Se supplementation might be a valuable approach to limit the adverse effects of hypercholesterolemia and may need further investigations.

Dietary selenium variation-induced oxidative stress modulates CDC2/cyclin B1 expression and apoptosis of germ cells in mice testis.

Oxidative stress has been linked with apoptosis in germ cells and with male infertility. However, the molecular mechanism of oxidative-stress-mediated apoptosis in germ cells has not been clearly defined so far. Because of the involvement of CDC2 and cyclin B1 in cell cycle regulation and their plausible role in apoptosis, the present study aimed to investigate the possibility that selenium (Se)-induced oxidative-stress-mediated modulations of these cell cycle regulators cause DNA damage and apoptosis in germ cells. To create different Se status (deficient, adequate and excess), male Balb/c mice were fed yeast-based Se-deficient diet (Group I) and a deficient diet supplemented with Se as sodium selenite (0.2 and 1 ppm Se in Groups II and III, respectively) for a period of 8 weeks. After the completion of the diet feeding schedule, a significant decrease in Se levels and glutathione peroxidase activity was observed in the Se-deficient group (Group I), whereas the Se-excess group (Group III) demonstrated an increase in Se levels. Increased levels of lipid peroxidation were seen in both Groups I and III when compared to Group II, indicating oxidative stress. The mRNA and protein expressions of both CDC2 and cyclin B1 were found to be significantly decreased in Groups I and III. A decrease in the immunohistochemical localization of these proteins was also observed in spermatogenic cells. The mRNA expressions of apoptotic factors such as Bcl-2, Bax, caspase-3 and caspase-9 were found to be increased in Groups I and III. A decrease in CDC2 kinase activity was also seen in these groups. Increased apoptosis was observed in Group I and Group III animals by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay indicating oxidative-stress-mediated DNA damage. These findings suggest the effect of Se-induced oxidative stress on the cell cycle regulators and apoptotic activity of germ cells, thus providing new dimensions to molecular mechanisms underlying male infertility.

Modulation of hypercholesterolemia-induced alterations in apolipoprotein B and HMG-CoA reductase expression by selenium supplementation.

Various studies demonstrated a significant association between the trace element selenium (Se), hypercholesterolemia and the risk of cardiovascular disorders. Present study was aimed to reveal the role of Se supplementation in modulation of hypercholesterolemia-induced changes in apolipoprotein B (apoB) and 3-hydroxy 3-methylglutaryl co-enzyme A (HMG-CoA) reductase expression during experimental hypercholesterolemia in Sprague-Dawley male rats. Animals were fed 0.2 and 1 ppm Se-supplemented control diet as well as 2% cholesterol-supplemented diet for 3 months. Apolipoprotein B levels were measured by ELISA and Western blot. HMG-CoA reductase mRNA expression was studied by RT-PCR. ApoB levels increased significantly on 2% cholesterol-supplemented diet feeding. On 1 ppm Se supplementation apoB levels decreased significantly. HMG-CoA reductase mRNA expression decreased significantly on cholesterol-supplemented diet feeding and on 1 ppm Se supplementation the mRNA expression further decreased. So the present results demonstrate that 1 ppm Se supplementation is responsible for down regulation of apoB and HMG-CoA reductase expression during hypercholesterolemia. These findings highlight the therapeutic potential of selenium supplementation in lipid metabolism.

Tertiary-butyl hydroperoxide induced oxidative stress and male reproductive activity in mice: role of transcription factor NF-kappaB and testicular antioxidant enzymes.

Reactive oxygen species (ROS) have been proposed as a major factor affecting male reproductive capacity. The present study has evaluated the possible role of oxidative stress during testicular pathogenesis in male mice exposed to tertiary-butyl hydroperoxide (TBHP). TBHP was administered by daily intraperitoneal injection for 2 weeks. Treatment greatly increased lipid peroxidation in the testis and led to a significant decrease in sperm concentration and motility and a reduction in litter size relative to controls. An increase in testicular mRNA abundance of redox-regulated p50 and p65 subunits of NF-kappaB was observed after TBHP treatment. Evaluation of NF-kappaB regulated antioxidant enzymes in the testis revealed an increase in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) activities and corresponding mRNA abundance. These results suggest a potential role of NF-kappaB in oxidative stress mediated changes in the physiology of male reproductive system.

Metallothionein does not sequester arsenic(III) ions in condition of acute arsenic toxicity.

The major cause of toxicity of trivalent arsenicals is due to their interaction with the sulfhydryl groups in proteins. Because of its high content, Metallothionein (MT) provides one of the most favorable conditions for the binding of As(III) ions to it. MT has long been anticipated for providing resistance in case of arsenic (As) toxicity with similar mechanism as in case of cadmium toxicity. The present study investigates whether the sequestration of As ions by MT is one of the mechanisms in providing protection against acute arsenic toxicity. A rat model study on the metal stoichiometric analysis of MT1 isoform isolated from the liver of arsenic treated, untreated and zinc treated animals has been carried out using the combination of particle induced X-ray emission (PIXE) and electrospray ionisation mass spectrometry (ESI-MS). The results revealed the absence of arsenic bound MT1 in the samples isolated from arsenic treated animals. Although, both Cu and Zn ions were present in MT1 samples isolated from all the treatment groups. Moreover, only partially metallated MT1 with varying number of Zn ions were observed in all the groups. These results suggest that the role of MT during acute arsenic toxicity is different from its already established role in case of cadmium toxicity.

Effect of selenium-induced oxidative stress on the cell kinetics in testis and reproductive ability of male mice.

OBJECTIVE: The present study evaluated the role of experimental oxidative stress (induced by feeding diets with different concentrations of selenium [Se], a trace nutrient and potent antioxidant) on male reproductive activity in mice. METHODS: To create different levels of oxidative stress in male mice, three diets with different levels of Se were fed to different groups for 8 wk. Mice in group 1 were fed a yeast-based diet, which is considered a Se-deficient diet (0.02 ppm). Mice in groups 2 and 3 were fed with an Se-deficient diet supplemented with 0.2 and 1 ppm Se as sodium selenite, respectively. RESULTS: After completion of the feeding schedule, a significant decrease in Se levels were observed in Se-deficient mice (group 1), whereas Se levels greatly increased in the Se-excess mice (group 3). Glutathione peroxidase activity was greatly decreased in the liver and testis in group 1, whereas glutathione-S-transferase activity was significantly increased in the testis. No significant change was found in activities of glutathione peroxidase and glutathione-S-transferase in group 3 compared with group 2. Cell kinetics showed a significant decrease in the number of pachytene spermatocytes and young and mature spermatids in group 1 compared with group 2. No appreciable change was observed in the germinal cell population in group 3. A significant decrease in sperm number was observed in group 1 compared with group 2. No change in these parameters was observed in group 3. The fertility status of mice in terms of percent fertility and litter size also exhibited a significant decrease in the reproductive ability of group 1. No change in these parameters was observed in group 3 compared with group 2. CONCLUSION: The present results clearly demonstrate the effect of oxidative stress generated by feeding different concentrations of Se on cell kinetics in the testis and, hence, its effect on the reproductive ability of male mice.

Hypercholesterolemia and apolipoprotein B expression: regulation by selenium status.

BACKGROUND: Apolipoprotein B (apoB) contains ligand-binding domain for the binding of LDL to LDL-R site, which enables the removal of LDL from circulation. Our recent data showed that selenium (Se) is involved in the lipid metabolism. The present study was aimed to understand the effect of Se deficiency (0.02 ppm) and selenium supplementation (1 ppm) on apoB expression in liver during hypercholesterolemia in male Sprague Dawley rats. Animals were fed with control and high cholesterol diet (2%) for 1 and 2 months. ApoB levels by ELISA and protein expression by western blot was done. Hepatic LDL receptor (LDL-R) activity (in vivo) and mRNA expression by RT-PCR was monitored. RESULTS: In selenium deficiency and on high cholesterol diet (HCD) feeding apoB levels increased and LDL-R expression decreased significantly after 2 months. On 1 ppm selenium supplementation apoB expression significantly decreased and LDL-R expression increased after 2 months. But after one month of treatment there was no significant change observed in apoB and LDL-R expression. CONCLUSION: So the present study demonstrates that Se deficiency leads to up regulation of apoB expression during experimental hypercholesterolemia. Selenium supplementation upto 1 ppm leads to downregulation of apoB expression. Further, this study will highlight the nutritional value of Se supplementation in lipid metabolism.

Studies on scavenger receptors under experimental hypercholesterolemia: modulation on selenium supplementation.

Scavenger receptors (SR) are the cell surface proteins that can bind and internalize modified lipoproteins. Because ox-LDL seems to play a key role in foam cell formation during atherogenesis, SR may be critical for pathogenesis of atherosclerosis. The present study was aimed to study the effect of selenium (Se) supplementation on SR, i.e., SRB1 and CD36 under experimental hypercholesterolemia. Male Sprague Dawley rats were divided into three groups and fed on the control diet, high cholesterol diet (HCD), and HCD + Se, respectively, for a period of 4 months. Selenium and reactive oxygen species (ROS) levels were estimated in serum and liver respectively. mRNA expression using RT-PCR and protein expression using ELISA were analyzed for SRB1 and CD36 receptors. Selenium levels decreased whereas ROS levels increased under experimental hypercholesterolemic state. Selenium supplementation (1 ppm), however, diminished the HCD-induced ROS levels. Furthermore, the protein expression of SRB1 was significantly reduced in HCD group in comparison to the control group. On the other hand, HCD-induced increase in CD36 mRNA and protein expression decreased significantly on Se supplementation. In conclusion, CD36 receptors seem to play a pro-atherogenic role under hypercholesterolemic state. Selenium supplementation, in addition, might prove to be a therapeutically valuable approach in near future to limit the adverse effect of hypercholesterolemia.

Upregulation of AP1 by tertiary butyl hydroperoxide induced oxidative stress and subsequent effect on spermatogenesis in mice testis.

Oxidative stress is detrimental to sperm function and a significant factor in the etiology of male infertility. Present study evaluates the effect of ter butyl hydroperoxide (TBHP)-induced oxidative stress on the spermatogenic process and cell number in the seminiferous tubules. Intraperitoneal injection of TBHP (84 mumol TBHP/100 g body weight) for 2 weeks to male Balb/c mice resulted in enhanced lipid peroxidation (P < 0.0001) decrease in reduced glutathione (P < 0.0001) and increase in the oxidized glutathione levels (P = 0.007) in the testis. Status of spermatogenesis after the treatment was assessed by the quantitative methods of germ cell evaluation in the seminiferous tubules. A significant decrease in the number of young spermatids (P = 0.0003) and pachytene cells (P = 0.022) was observed. A marked reduction was also seen in the mature spermatid number (P < 0.0001). An increase in testicular mRNA levels of redox-regulated cjun (P = 0.008) and cfos (P = 0.0006) subunits of activator protein 1 (AP1) was observed after TBHP treatment. Evaluation of AP1 regulated antioxidant enzymes in the testis revealed an increase in gamma-glutamyl cysteine synthetase (GCS) mRNA expression (P = 0.001). These results suggest a potential role of AP1 in oxidative stress-mediated meiotic and post meiotic changes in the spermatogenic process and regulation of cell number in male reproductive system.

Co-operative effect of glutathione depletion and selenium induced oxidative stress on API and NFkB expression in testicular cells in vitro: insights to regulation of spermatogenesis.

Previous studies have shown that transcription factors, API and NFkB exert important roles in the process by which selenium regulates spermatogenesis. Glutathione, an intracellular thiol, acts as a source of reducing power and aids in maintenance of the cellular redox status. The activities of selenium are closely related to the availability of glutathione. Presently, mouse testicular cells were cultured in the presence of BSO, a known glutathione depletor, to generate oxidative stress. Selenium (Se) was added as sodium selenite to these cells at concentrations of 0.5 microM and 1.5 microM. It was observed that at 1.5 microM, Se acted as a pro-oxidant and significantly decreased the redox ratio. RT PCR analysis revealed that cjun, cfos expression increased in testicular cells cultured with Se compared to control. However, the major outcome was that the combined effect of Se supplementation and GSH depletion resulted in reduced expression of cjun and cfos while p65 expression increased. This suggests that selenium affects both these transcription factors differently. Our study indicates that though low levels of oxidative stress generated by moderate doses of selenium augments the expression of cjun and cfos, a robust increase in the ROS generation caused by the dual effect high levels of selenium and glutathione depletion leads to decrease in the expression of these genes. The present work substantiates our in vivo experiments and indicates the detrimental effect of excess selenium supplementation on male fertility.

Role of selenium in spermatogenesis: differential expression of cjun and cfos in tubular cells of mice testis.

Selenium (Se) is an essential dietary trace element, involved in the process of male reproduction. Best known as an antioxidant, it acts through various selenoproteins viz. glutathione peroxidase, thioredoxin reductase and selenoprotein P. The aim of the present study was to identify the underlying molecular mechanism of Se in regulating spermatogenesis. Different Se status: deficient, adequate and excess Se, were generated in male Balb/c mice by feeding yeast based Se deficient diet, and deficient diet supplemented with Se as sodium selenite (0.2 and 1 ppm Se) respectively for a period of 4 and 8 weeks. Se levels and glutathione peroxidase (GSH-Px) activity were significantly reduced in the Se deficient mice and enhanced in Se supplemented group. Reduction in the number of post-meiotic germ cells viz. spermatids and spermatozoa, were observed in the deficient groups indicating loss in fertility and reproductive ability. cjun and cfos (components of transcription factor AP1) regulate cellular growth and differentiation and also exert a regulatory role in steroidogenesis and spermatogenesis. Changes in the mRNA expression of cjun and cfos were observed. Concomitant with this, western blot revealed that the protein expression profile for both these genes was significantly altered in the Se deficient and Se excess groups. Further immunohistochemical analysis showed that, both these genes had identical cellular localization indicating that they do not work alone but act synergistically as AP1. cjun and cfos expression was greater in the early mitotic stages-spermatogonia and spermatocytes in the Se adequate controls. It decreased in the meiotic stages and then again peaked around the later stages-elongating spermatids and spermatozoa. However in the Se deficient mice, weaker expression was observed in the spermatogonia with a complete absence of expression near the lumen. No visible changes in cjun/cfos expression and immunohistochemical localization were observed in the excess group compared to the Se adequate controls. In conclusion, the present study clearly demonstrates that alteration in Se supply leads to decreased expression pattern for both cJun and cFos in the testicular germ cells which might be responsible for decreased germ cell number, differentiation and reduced fertility and accounts for the mechanism of Se action in regulating spermatogenesis.

Hypercholesterolemia and tissue-specific differential mRNA expression of type-1 5'-iodothyronine deiodinase under different selenium status in rats.

Type-1 5'-iodothyronine deiodinase (5'-DI) is responsible for conversion of T4 to T3. Selenium (Se) is an integral part of this enzyme. Keeping in view the strong association between atherosclerosis and hypothyroidism, the present study examined the behavior of 5'-DI in liver, aorta and thyroid during hypercholesterolemia following different Se status, i.e., Se deficiency (0.02 ppm), adequate (0.2 ppm) and excess dose (1 ppm) in SD male rats. Animals were fed a control or high-cholesterol diet (2%) for 1 and 2 months. 5'-DI activity and mRNA expression was measured by RIA and RT-PCR respectively. In liver and aorta, 5'-DI expression significantly decreased with the Se-deficient and the high-cholesterol diet. The trend was opposite in thyroid, i.e., mRNA expression increased significantly during selenium deficiency and with a high-cholesterol feeding. But with 1 ppm Se supplementation, the 5'-DI expression increased in all the three tissues. The present study indicates that hypercholesterolemia along with selenium deficiency is co-responsible for differential regulation of 5'-DI enzyme in thyroidal vs. extrathyroidal tissues. Distinct regulation of 5'-DI in the thyroid reflects the clinical importance of this selenoprotein during hypercholesterolemia as this enzyme is essential for T3 production, which further has a vital role in the maintenance of lipid metabolism.

Hypercholesterolemia and LDL receptor mRNA expression: modulation by selenium supplementation.

Selenium (Se) status has been associated with cardiovascular disorders. Present study was aimed to elucidate the protective role of Se supplementation on LDL receptor (LDL-R) activity as well as mRNA expression during experimental hypercholesterolemia in SD male rats. Animals were fed 0.2 and 1 ppm Se supplemented control diet as well as 2% cholesterol supplemented diet for 3 months. LDL-R activity was measured in-vivo by injecting radiolabeled LDL to rats and decrease in counts per minute with time was taken as a measure of LDL clearance and in turn LDL-R activity. LDL-R mRNA expression was studied by RT-PCR. LDL-R activity and mRNA expression decreased significantly on 2% cholesterol supplemented diet feeding. On 1 ppm Se supplementation LDL-R activity as well as mRNA expression increased significantly. Present results demonstrate that Se supplementation upto 1 ppm is responsible for up regulation of LDL-R activity as well as mRNA expression, during hypercholesterolemia. These findings highlight the therapeutic potential of Se supplementation in lipid metabolism.

Attenuation of LDL receptor gene expression by selenium deficiency during hypercholesterolemia.

Selenium deficiency has been associated with hypercholesterolemia. Present study was aimed to determine the effect of selenium (Se) deficiency on LDL receptor (LDL-R) activity as well as mRNA expression during experimental hypercholesterolemia in SD male rats. Animals were fed Se adequate (0.2 ppm) and deficient (0.02 ppm) control diet as well as high cholesterol (2%) diet (HCD) for 1 and 2 months. LDL-R activity was measured in vivo by injecting radiolabeled LDL to rats and percent decrease in cpm with time was taken as a measure of LDL clearance and in turn LDL-R activity. LDL-R mRNA expression was studied by RT-PCR. LDL-R activity and mRNA expression decreased significantly on HCD feeding in both Se deficient and adequate diet fed rats after 2 months. In Se deficiency receptor activity and mRNA expression decreased significantly. After 2 months LDL-R activity and expression decreased in both the Se deficient groups and in Se adequate HCD fed group in comparison to 1 month data. But after 4 month there was no significant difference observed in LDL-R activity and mRNA expression in selenium deficiency as well as on HCD feeding. So the present results demonstrate that Se deficiency act synergistically with hypercholesterolemia to downregulate LDL-R activity as well as mRNA expression.

Effect of diethyl maleate induced oxidative stress on male reproductive activity in mice: redox active enzymes and transcription factors expression.

Free radicals and reactive oxygen species (ROS) associated with oxidative stress are likely to play a number of significant roles in male reproduction. Present study was carried out to evaluate the effect of diethyl maleate (DEM) induced oxidative stress on male fertility in mice. Intraperitoneal injection of DEM for two weeks resulted in decrease in reduced glutathione and increase in the oxidized glutathione levels in the testis. Effect on the reproductive ability in term of sperm concentration, motility and fertility status was studied. Sperm concentration and motility were found to be significantly reduced along with a significant reduction in the litter size. Expression of redox sensitive transcription factor, cjun and cfos genes, along with gamma-glutamyl cysteine synthetase (GCS) and manganese superoxide dismutase (MnSOD) expression were also studied using RT-PCR after DEM treatment. RT-PCR analysis revealed decrease in the testicular mRNA expression for cjun and cfos whereas the expression for GCS and MnSOD increased. Enzyme activity of SOD also increased. The study reflects the effect of DEM induced oxidative stress on the reproductive ability of male mice and interplay of the various components of the antioxidant defense system and redox regulated transcription factors at the transcriptional level.

Effect of experimental oxidative stress on steroidogenesis and DNA damage in mouse testis.

The objective of the present study was to evaluate the effect of oxidative stress induced by feeding various levels of selenium on steroidogenesis and DNA damage in mouse testes. To create various levels of oxidative stress in mice, diets with three different Se levels were fed to separate groups for 8 weeks. Group 1 animals were fed a yeast-based diet, which was considered a Se-deficient diet (0.02 ppm). Group 2 and 3 animals were fed a Se-deficient diet supplemented with 0.2 and 1 ppm Se as sodium selenite, respectively. After completion of the diet feeding, estimations were carried out, and results were compared with those of group 2. A significant decrease in Se levels was observed in group 1 animals, whereas they were greatly enhanced in group 3. Glutathione peroxidase (GSH-Px) activity was greatly reduced in both the liver and testes in group 1, whereas no significant changes were found in GSH-Px activity in group 3. Serum luteinizing hormone, follicle-stimulating hormone (FSH), and testosterone levels were reduced in group 1. Significant decreases of sperm number and motility were observed in group 1 when compared to group 2 male mice. No changes in these parameters were observed in group 3. DNA fragmentation was observed in both groups 1 and 3; however, the damage was more prevalent in group 1. The results clearly demonstrate the effect of oxidative stress generated by feeding various Se levels on the steroidogenesis and DNA fragmentation in mice testes.

Co-operative effect of glutathione depletion and selenium induced oxidative stress on API and NFkB expression in testicular cells in vitro: insights to regulation of spermatogenesis

Previous studies have shown that transcription factors, API and NFkB exert important roles in the process by which selenium regulates spermatogenesis. Glutathione, an intracellular thiol, acts as a source of reducing power and aids in maintenance of the cellular redox status. The activities of selenium are closely related to the availability of glutathione. Presently, mouse testicular cells were cultured in the presence of BSO, a known glutathione depletor, to generate oxidative stress. Selenium (Se) was added as sodium selenite to these cells at concentrations of 0.5 µM and 1.5 µM. It was observed that at 1.5 µM, Se acted as a pro-oxidant and significantly decreased the redox ratio. RT PCR analysis revealed that cjun, cfos expression increased in testicular cells cultured with Se compared to control. However, the major outcome was that the combined effect of Se supplementation and GSH depletion resulted in reduced expression of cjun and cfos while p65 expression increased. This suggests that selenium affects both these transcription factors differently. Our study indicates that though low levels of oxidative stress generated by moderate doses of selenium augments the expression of cjun and cfos, a robust increase in the ROS generation caused by the dual effect high levels of selenium and glutathione depletion leads to decrease in the expression of these genes. The present work substantiates our in vivo experiments and indicates the detrimental effect of excess selenium supplementation on male fertility.

Hypercholesterolemia and tissue-specific differential mRNA expression of type-1 5'-iodothyronine deiodinase under different selenium status in rats

Type-1 5'-iodothyronine deiodinase (5'-DI) is responsible for conversion of T4 to T3. Selenium (Se) is an integral part of this enzyme. Keeping in view the strong association between atherosclerosis and hypothyroidism, the present study examined the behavior of 5'-DI in liver, aorta and thyroid during hypercholesterolemia following different Se status, i.e., Se deficiency (0.02ppm), adequate (0.2ppm) and excess dose (1ppm) in SD male rats. Animals were fed a control or high-cholesterol diet (2%) for 1 and 2 months. 5'-DI activity and mRNA expression was measured by RIA and RT-PCR respectively. In liver and aorta, 5'-DI expression significantly decreased with the Se-deficient and the high-cholesterol diet. The trend was opposite in thyroid, i.e., mRNA expression increased significantly during selenium deficiency and with a high-cholesterol feeding. But with 1ppm Se supplementation, the 5'-DI expression increased in all the three tissues. The present study indicates that hypercholesterolemia along with selenium deficiency is co-responsible for differential regulation of 5'-DI enzyme in thyroidal vs. extrathyroidal tissues. Distinct regulation of 5'-DI in the thyroid reflects the clinical importance of this selenoprotein during hypercholesterolemia as this enzyme is essential for T3 production, which further has a vital role in the maintenance of lipid metabolism.