Antonios Kyriakopoulos

Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-linking during sperm maturation

A 34 kD selenoprotein purified from rat testis was identified as a specific sperm nuclei glutathione peroxidase (snGPx) with similar properties to phospholipid hydroperoxide glutathione peroxidase (PHGPx). The determination of its primary structure by analysis of its first N‐terminal amino acids, database search, polymerase chain reaction, and sequencing of the cDNA showed that it differs from PHGPx in its N‐terminal sequence. This sequence, which is encoded for by an alternative exon in the first intron of the PHGPx gene, shows more than 50% homology to the protamine sequences and contains a nuclear localization signal. In rats, snGPx is highly expressed in the nuclei of the late spermatids where it is the only selenoprotein present. Its appearance coincides with the reorganization of DNA, which leads to highly condensed chromatin stabilized by cross‐linked protamine thiols. In selenium‐depleted rats where the concentration of snGPx had decreased to one‐third of the normal level, chromatin condensation was severely disturbed. We provided evidence that snGPx acts as a protamine thiol peroxidase responsible for disulfide cross‐linking by reduction of reactive oxygen species. Its dual function in chromatin condensation and the protection of sperm DNA against oxidation is necessary to ensure male fertility and sperm quality.

Selenium deficiency increases susceptibility to glutamate-induced excitotoxicity.

Excitotoxic brain lesions, such as stroke and epilepsy, lead to increasing destruction of neurons hours after the insult. The deadly cascade of events involves detrimental actions by free radicals and the activation of proapoptotic transcription factors, which finally result in neuronal destruction. Here, we provide direct evidence that the nutritionally essential trace element selenium has a pivotal role in neuronal susceptibility to excitotoxic lesions. First, we observed in neuronal cell cultures that addition of selenium in the form of selenite within the physiological range protects against excitotoxic insults and even attenuates primary damage. The neuroprotective effect of selenium is not directly mediated via antioxidative effects of selenite but requires de novo protein synthesis. Gel shift analysis demonstrates that this effect is connected to the inhibition of glutamate‐induced NF‐κB and AP‐1 activation. Furthermore, we provide evidence that selenium deficiency in vivo results in a massive increase in susceptibility to kainate‐induced seizures and cell loss. These findings indicate the importance of selenium for prevention and therapy of excitotoxic brain damage.

Studies on the distribution and characteristics of new mammalian selenium-containing proteins

It has now been established that the essential effects of selenium in mammals are owing to the presence of several biologically active selenium compounds. Seleno-enzymes identified so far include several glutathione peroxidases and the type 1 iodothyronine de-iodinase. Some other selenoproteins have been sequenced and characterized. After in vivo labelling of rats with 75Se and protein separation using gel electrophoretic methods, more than 25 selenium-containing proteins or protein sub-units were detected. Some of the results of the investigations on these compounds are summarized and discussed here. By determining the pattern in a large number of tissues information on the distribution of the selenium-containing proteins was obtained. Their biological significance is not yet known but several findings indicate that some of these proteins may have important functions, especially in the brain and the endocrine and reproductive organs. More detailed information is already available on a 34 kDa-protein found in the testis and spermatozoa. Studies on the effects of dosage and chemical form of dietary selenium indicated that the tissue levels of the seleno-enzymes are homeostatically controlled and cannot be increased by additional supply. The increase in the tissue selenium observed with high selenium intake was found to be mainly caused by the non-specific incorporation of the element into a large number or proteins. The formation of most of the other selenium-containing proteins has priority over that of the cytosolic and plasma glutathione peroxidases. Thus the selenium requirement, which was calculated for optimum plasma glutathione peroxidase activity, also covers the amounts needed for normal levels of the other biologically important selenium compounds.

Subcellular distribution of selenoproteins in the liver of the rat.

After in vivo labeling with [75Se]selenite, the intracellular distribution of selenoproteins in the liver was investigated in selenium-adequate and selenium-deficient rats. In the subcellular fractions, which were obtained by differential centrifugation, the proteins were separated by means of SDS-PAGE and the selenium compounds were identified via their 75Se activity. In this way twelve selenium-containing proteins or protein subunits with molecular weights between 12,100 and 75,400 were found. Glutathione peroxidase was concentrated in the cytosol and in the mitochondria. With the newly detected selenoproteins, some were enriched in the cytosol, one was mainly found in the nuclear fraction and some, which were present mainly in the mitochondrial and microsomal fractions, are most probably membrane-bound. In the liver of selenium-depleted rats the selenium administered was used predominantly to restore the levels of some of the newly found selenoproteins, while in the liver of selenium-adequate animals most of the selenium retained was incorporated into the glutathione peroxidase. The differences in the distribution among the subcellular fractions and the specific incorporation of the element in selenium deficiency into certain compounds suggest that there are several metabolic pathways for selenium and that the selenoproteins are involved in several different processes of intracellular metabolism.

The brain selenoproteome: priorities in the hierarchy and different levels of selenium homeostasis in the brain of selenium-deficient rats.

The application of radionuclides for the localization of essential trace elements in vivo and the characterization of their binding proteins is a story of intermittently made improvements of the techniques used for their detection. In this study we present the use of neutron activation analysis and different autoradiographic imaging methods including real‐time digital autoradiography to reveal new insights in the hierarchy of selenium homeostasis. Selenoproteins containing the essential trace element selenium play important roles in the CNS. Although the CNS does not show the highest selenium concentration in the case of selenium‐sufficient supply in comparison with other organs, it shows a high priority for selenium uptake and retention in the case of dietary selenium deficiency. To characterize the hierarchy of selenium supply in the brain, in vivo radiotracer labeling with 75Se in rats with different selenium status was combined with autoradiographic detection of 75Se in brain tissue sections and 75Se‐labeled selenoproteins after protein separation by two‐dimensional gel electrophoresis. This study demonstrates significant differences in the uptake of 75Se into the brain of rats with different selenium status. A brain region‐specific uptake pattern of the radiotracer 75Se in selenium‐deficient rats could be revealed and the CSF was identified as a key part of the brain selenium homeostasis.

Identification of Selenocysteine and Selenomethionine in Protein Hydrolysates by High-performance Liquid Chromatography of Their o-Phthaldialdehyde Derivatives

A method for the identification of selenocysteine and selenomethionine in protein hydrolysates was developed. The proteins were subjected to acid hydrolysis after they had been carboxymethylated to prevent decomposition of selenocysteine during this process. After precolumn derivatization of the amino acids with o-phthaldialdehyde, the hydrolysate was chromatographed on C18 columns. The selenoamino acids were detected either by the fluorescence of their o-phthaldialdehyde derivatives (detection limit 30 pmol for selenomethionine and 170 pmol for selenocysteine) or by selenium determination in the eluate using atomic absorption spectrometry (detection limit 0.3 pmol) or, with 75Se-labelled compounds, the measurement of the tracer activity. With the latter procedure the detection limit, which depends on the specific activity of the Se tracer, could be decreased to the femtomole range. The method was successfully applied to the identification of selenocysteine in several newly found mammalian selenium-containing proteins.

Cellular and Subcellular Distribution of Selenium and Selenium-Containing Proteins in the Rat

Although it has been known for over more than forty years that selenium is essential for the mammalian organism, our knowledge of the metabolism and the functions of the element is still incomplete. In order to investigate some of the still unanswered questions, several studies have been carried out on rats. The animals were either supplied with adequate amounts of selenium or were depleted by feeding with a seleniumdeficient diet over longer periods of time, sometimes for several generations. By combining methods for trace element analysis, tracer techniques and various biochemical procedures, the distribution of selenium was determined among tissues, tissue fractions, certain types of cells, subcellular compartments and proteins. In this way information was obtained on the regulation of selenium and on new selenium-containing proteins and their sites of action. Our main findings in these two areas of research will be presented and discussed.

Selenium Deficiency Abrogates Inflammation-Dependent Plasma Cell Tumors in Mice

The role of the micronutrient, selenium, in human cancers associated with chronic inflammations and persistent infections is poorly understood. Peritoneal plasmacytomas (PCTs) in strain BALB/c (C), the premier experimental model of inflammation-dependent plasma cell transformation in mice, may afford an opportunity to gain additional insights into the significance of selenium in neoplastic development. Here, we report that selenium-depleted C mice (n = 32) maintained on a torula-based low-selenium diet (5-8 micro g of selenium/kg) were totally refractory to pristane induction of PCT. In contrast, 11 of 26 (42.3%) control mice maintained on a selenium adequate torula diet (300 micro g of selenium/kg) and 15 of 40 (37.5%) control mice fed standard Purina chow (440 micro g of selenium/kg) developed PCT by 275 days postpristane. Abrogation of PCT was caused in part by the striking inhibition of the formation of the inflammatory tissue in which PCT develop (pristane granuloma). This was associated with the reduced responsiveness of selenium-deficient inflammatory cells (monocytes and neutrophils) to chemoattractants, such as thioredoxin and chemokines. Selenium-deficient C mice exhibited little evidence of disturbed redox homeostasis and increased mutant frequency of a transgenic lacZ reporter gene in vivo. These findings implicate selenium, via the selenoproteins, in the promotion of inflammation-induced PCT and suggest that small drug inhibitors of selenoproteins might be useful for preventing human cancers linked with chronic inflammations and persistent infections.

Effects of long-term selenium yeast supplementation on selenium status studied in the rat.

To investigate the selenium status during long-term dietary supply of selenium yeast, 30-day-old male rats were fed for 379 days a methionine-adequate low-selenium diet supplemented with 0.2mgSe/kg (selenium-adequate diet) or 1.5mgSe/kg (high-selenium diet) in the form of selenium yeast that contained 60% of the element as l-selenomethionine. Their selenium load was determined at several intervals by neutron activation analysis of the selenium concentrations in the main selenium body pools, skeletal muscle and liver. After 64 days the tissue selenium concentrations plateaued in both groups and then stayed at that level. Compared with the selenium-adequate group, elevated tissue selenium concentrations were found in the high-selenium group, but the increase by a factor of 3.5 in the muscle and by a factor of 2.3 in the liver was smaller than the 7.5-fold increase in the selenium intake. In the selenium-adequate group about 50% of the muscle selenium and 30% of the liver selenium and in the high-selenium group about 85% of the muscle selenium and 70% of the liver selenium were estimated to be present in non-selenoprotein forms. During selenium depletion the liver glutathione peroxidase activity in the high-selenium group remained unaffected for 4 weeks and then decreased more slowly than that in the selenium-adequate group. From these results it can be concluded that selenium incorporated from the selenium yeast diet into non-selenoprotein forms can serve as an endogenous selenium source to maintain selenoprotein levels in periods of insufficient selenium supply.

Long-term selenium supplementation of humans: selenium status and relationships between selenium concentrations in skeletal muscle and indicator materials.

Supplementation with elevated doses of l-selenomethionine (SeM) or selenium-enriched yeast that contains SeM as the main selenium species is frequently used as a protective or therapeutic measure. Information on the effects of long-term selenium supplementation on the body selenium status is, however, rather scarce. We therefore investigated fifteen male test persons who had taken selenium yeast and/or SeM supplements in medium doses of 62.5-125 microg Se/day or high doses of 200-262.5 microg Se/day for periods ranging from 1 year to 24 years. Seven non-supplemented men served as controls. As skeletal muscle is the main selenium pool, thigh muscle biopsy samples were taken. The selenium concentrations in these biopsies and in samples of the indicator materials blood, blood plasma, blood cells, head hair and toenails were determined by neutron activation analysis. Compared with the controls, the muscle selenium level was raised with additional selenium supplementation, but the relative increase in the mean muscle selenium concentration (by factors of about 1.6 and 2 for the medium and high doses, respectively) was lower than that in the selenium intake. Highly significant correlations found between the selenium concentrations in muscle and whole blood (R=0.90), red blood cells (R=0.91), blood plasma (R=0.87), head hair (R=0.89) and toenails (R=0.85) show that in humans supplemented in this way the selenium status can be assessed in a relatively easy way by analysis of the selenium retention in these indicator materials.