María Carolina Cassará
email: carolina.cassara@yahoo.com

Viviana Andrea Menzel

Klaus-Dieter Hinsch

Christine Wrenzycki

Elvira Hinsch

Voltage-dependent anion channel 1 and 2 are expressed in porcine oocytes

Keywords: immunoblotting, indirect immunofluorescence, oocyte, RT-PCR, voltage-dependent anion channel

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Voltage-dependent anion channels (VDAC) are a widespread class of pore-forming proteins [START_REF] Reymann | Further evidence for multitopological localization of mammalian porin (VDAC) in the plasmalemma forming part of a chloride channel complex affected in cystic fibrosis and encephalomyopathy[END_REF][START_REF] Junankar | Porin-type 1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres[END_REF][START_REF] Thinnes | Studies on human porin XXII: cell membrane integrated human porin channels are involved in regulatory volume decrease (RVD) of HeLa cells[END_REF][START_REF] Okada | Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume in murine cells[END_REF][START_REF] Wang | Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development[END_REF][START_REF] Roman | Direct measurement of VDAC-actin interaction by surface plasmon resonance[END_REF] mainly found in the outer mitochondrial membranes in most eukaryotic organisms (for reviews see [START_REF] Colombini | Voltage gating in the mitochondrial channel, VDAC[END_REF][START_REF] De Pinto | New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC)[END_REF] ). In addition to mitochondrial localization, in the past few years several reports demonstrated the presence of VDAC in the plasma membrane and caveolae-like domains, sarcoplasmic/endoplasmic reticulum, nuclear envelope and endosomes (for reviews see [START_REF] Bathori | Extramitochondrial porin: facts and hypotheses[END_REF][START_REF] Shoshan-Barmatz | The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death[END_REF]). VDAC have conserved sequences, and three different VDAC genes encoding distinctly expressed isoforms are known to exist in vertebrates [START_REF] Shoshan-Barmatz | The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death[END_REF][START_REF] Sampson | The murine voltagedependent anion channel gene family. Conserved structure and function[END_REF]. Little is known in mammals about tissue-specific distribution, intracellular localization and the specific role of each isoform [START_REF] Sampson | The murine voltagedependent anion channel gene family. Conserved structure and function[END_REF][START_REF] Messina | Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered[END_REF][START_REF] Rahmani | Isolation of a novel human voltage-dependent anion channel gene[END_REF]. The existing information derives mainly from recombinant proteins expressed in model organisms like yeast or mutant mice [START_REF] Sampson | The murine voltagedependent anion channel gene family. Conserved structure and function[END_REF][START_REF] Anflous | Bacterial and eukaryotic porins[END_REF][START_REF] Blachly-Dyson | Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel[END_REF][START_REF] Messina | Characterization of the human porin isoform 1 (HVDAC1) gene by amplification on the whole human genome: A tool for porin deficiency analysis[END_REF]. This paucity of information is due to the fact that to date mainly VDAC1 has been purified and characterized from different animal tissues, in contrast to VDAC2 which was until now only obtained from bovine spermatozoa [START_REF] Menzel | Molecular and functional characterization of VDAC2 purified from mammal spermatozoa[END_REF]. The basic, known function of VDAC is to form a diffusion pore with highly conserved characteristics in the outer mitochondrial membrane (OMM). The OMM functions as a link between mitochondrial metabolism and the rest of the cell. The involvement of VDAC in metabolic pathways and interactions with other supramolecular structures has been investigated [START_REF] De Pinto | New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC)[END_REF][START_REF] Shoshan-Barmatz | The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death[END_REF]. VDAC has been implicated to play a significant role in the programmed cell death pathway (for review see [START_REF] Kroemer | Mitochondrial membrane permeabilization in cell death[END_REF]). Other recent roles attributed to VDAC were the participation in calcium trafficking in the cell and the interaction with cytoskeletal proteins [START_REF] Shoshan-Barmatz | The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death[END_REF][START_REF] Schwarzer | Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex1 and the heat-shock protein PBP74[END_REF][START_REF] Xu | Actin modulates the gating of Neurospora crassa VDAC[END_REF][START_REF] Linden | Identification of porin as a binding site for MAP2[END_REF]. Currently, there is only scant information regarding the expression and the function of VDAC proteins in gametes. In a previous study we reported the presence of VDAC in the outer dense fibers (ODF) of the bovine sperm flagellum [START_REF] Hinsch | Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum[END_REF] and their involvement in the regulation of essential sperm functions [START_REF] Triphan | Localisation and function of voltagedependent anion channels (VDAC) in bovine spermatozoa[END_REF]. Furthermore, we have obtained and characterized highly enriched VDAC2 from bovine spermatozoa [START_REF] Menzel | Molecular and functional characterization of VDAC2 purified from mammal spermatozoa[END_REF]. [START_REF] Steinacker | The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels[END_REF] reported the localization of VDAC in the plasma membrane of Xenopus laevis oocytes [START_REF] Steinacker | The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels[END_REF]. The expression and subcellular distributions of Porin 1 and 2 (VDAC1 and 2) were also reported in ovaries of Drosophila melanogaster [START_REF] Guarino | Expression and localization in spermatozoa of the mitochondrial porin isoform 2 in Drosophila melanogaster[END_REF][START_REF] Specchia | Porin isoform 2 has a different localization in Drosophila melanogaster ovaries than porin 1[END_REF]. Wang et al., also identified VDAC1 and 2 in two-dimensional electrophoresis analysis of human ovary [START_REF] Wang | A two-dimensional electrophoresis reference map of human ovary[END_REF] relating it with a possible role in the atresia of follicles. At present there is no evidence regarding the presence and the function of VDAC in mammalian oocytes. This work provides the first report of the expression and localization of endogenous VDAC in immature and mature porcine oocytes as well as the purification of VDAC2 from porcine oocytes.

Materials and methods

Collection of oocytes and in vitro maturation

Ovaries of pre-pubertal gilts were transported at 37 °C from a local abattoir in physiological saline solution containing 100 mg/ml Kanamycin. Cumulus-oocyte complexes were aspirated from non-atretic follicles (3-6 mm diameter) and only oocytes with evenly granulated cytoplasm and several layers of cumulus cells were selected. The in vitro maturation was performed following the method described by Romar [START_REF] Romar | Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa[END_REF]. For all experiments, granulosa cell-free immature oocytes (germinal vesicle, GV) and mature oocytes (meiosis II, MII) were used.

RT-PCR

The total cellular RNA extracted from 10 porcine oocytes using an RNeasy mini-kit (Qiagen, Hilden, Germany) was reversibly transcribed into the firststrand cDNA through Omniscript Reverse Transcriptase (Qiagen). The subsequent amplification of the desired genes was performed using Taq DNA polymerase (Eppendorf, Hamburg, Germany) according to the manufacturer's protocol. The primer sequences derived from porcine cDNA sequences (GenBank) were constructed as follows for: VDAC1, (5´-CAAAATCTGAGAATGGACTGGAA-3´ and 5´-TTGGTGAGAAGGATGAATCAAAG-3´) and for VDAC2, (5´-AAATCAAAGCTGACAAGGAA-3´ and 5´-GACTTTTGCAGAAATGGAAG-3´). As a control for amplification 5´and 3´primers for the GAPDH gene were used (5´-CCAGAAGACTGTGGATGGCC-3´ and 5´-CTGACGCCTGCTTCACCACC-3´). The amplification was conducted for 35 cycles at 94 °C for 45 s, annealing temperature (57 °C) for 45 s, and 72 °C for 90 s with a final step of 72 °C for 5 min.

Protein detection by immunoblotting

Oocytes were incubated at 70 °C in a minimum volume of 0.9% NaCl for 1 h, then dissolved 1:1 (v/v) in oocyte sample buffer (5% SDS; 100 mM Tris-HCl, pH 7; 25 mM EDTA; 12% glycerol; 2% bromophenol blue; 10% β-mercaptoethanol) and vortexed for 5 min followed by a last heating step at 95 °C. SDS-PAGE was performed according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during assembly of the head of bacteriophage T4[END_REF] in 12% (v/v) polyacrylamide gels loaded with an extract from 300 oocytes per lane. Proteins were subjected to immunoblotting following a published protocol [START_REF] Hinsch | Generation of antisera against mouse and human synthetic ZP3 peptides[END_REF]. The antibodies used were AS P1/6 (anti-VDAC1), monoclonal anti-human type I porin (P31HL, Calbiochem, Darmstadt, Germany) and AS P2/45 (anti-VDAC2) at appropriate dilutions. Characterization and specificity of the antibodies has been previously described [START_REF] Hinsch | Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum[END_REF].

Purification and protein identification by mass spectrometry of oocyte VDAC

Protein samples were prepared from immature oocytes (3300 cells) solubilized in Triton X-100 extraction buffer (2% v/v Triton X-100; 150 mM NaCl; 50 mM Tris-HCl, pH 7.4; 2 mM DTT; 1 mM EDTA; 10 mM benzamidine; 0.2 mM PMSF). The suspension was incubated for 90 min at 4 °C and finally centrifuged at 13800xg for 45 min. The supernatant (extract A) was stored at -80 °C until use. The remaining sediment was subjected to a second solubilization treatment as described above (extract B). The proteins from extract A and B were diluted 1:1 with 2% Triton X-100 extraction buffer. Each extract was applied to a dry hydroxyapatite-celite (2:1 w/w) column [START_REF] De Pinto | A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues[END_REF]. The first two ml of the pass-through (VDAC-containing fraction) were collected separately in 1 ml fractions and stored at -20 °C until use. Both extracts were subjected to semipreparative SDS-PAGE (12% separating polyacrylamide gels) according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during assembly of the head of bacteriophage T4[END_REF] and proteins were visualized by staining with SilverQuest (Invitrogen, Karlsruhe, Germany). Protein bands were excised and subsequently subjected to MALDI-TOF mass spectrometry analysis (ChromaTec, Greifswald, Germany). Peptide mass fingerprinting database searching was carried out using available online software (http://www.matrixscience.com/).

Immunocytochemical localization of VDAC proteins

For immunofluorescence microscopy, porcine oocytes were fixed in 3.7% (w/v) paraformaldehyde (Sigma, Munich, Germany) in phosphate-buffered-saline (PBS, 120 mM NaCl, 4 mM KH 2 PO 4, 10 mM Na 2 HPO 4, pH 7.4) for 30 min at room temperature, washed three times for 5 min in PBS drops and blocked for 1 h with PBS supplemented with 1% skimmed milk powder (EuroClone, Milano, Italy; MP-PBS). Incubation with primary antibody P31HL (anti-VDAC1, Calbiochem), AS P1/6 (anti-VDAC1) and anti-VDAC2 antibody (Santa Cruz, Heidelberg, Germany) was performed for 60 min followed by three washes in 0.1% MP-PBS. Secondary FITC-conjugated antibodies were applied for 30 min: antimouse IgG (Sigma) for P31HL antibodies; anti-rabbit IgG (Sigma) for AS P1/6; and anti-goat IgG (Santa Cruz) for anti-VDAC2 antibodies. During the last 5 min of incubation, propidium iodide (100 µg/ml, Sigma) was added to detect maturation stages. Finally, labeled oocytes were mounted onto slides, examined and recorded using a laser scanning confocal microscope (LSM 510, Zeiss Axioplan imaging, Gottingen, Germany). The microscope was equipped with an Argon and HNe1 laser.

For the assessment of intracellular localization of VDAC in immature oocytes, the paraformaldhyde-fixed cells were permeabilized for 5 min in 0.1% (v/v) Triton X-100 (Serva, Heidelberg, Germany) in PBS. The antibodies used were monoclonal antihuman type I porin, P31HL (Calbiochem), AS P1/6 (anti-VDAC1), AS P2/45 (anti-VDAC2) and anti-VDAC2 antibody (Santa Cruz) at appropriate dilutions.

Results

RT-PCR

RT-PCR revealed that VDAC1 and VDAC2 mRNAs were expressed in porcine oocytes (Figure 1). Amplification of the coding sequences was detected in GV (VDAC1 lane A, VDAC2 lane B) and MII (VDAC1 lane C, VDAC2 lane D) stage oocytes. The primer pairs generated amplicons of 219 bp (VDAC1) and 231 bp (VDAC2) and the positive control GAPDH showed an amplification product of 248 bp in GV and MII (GAPDH, lane A-D). All amplicons were sequenced on both strands (MWG Biotech, Ebersberg, Germany) and confirmed by blasting. Figure 2 shows immunoblot results using protein extracts obtained from 300 GV and 300 MII stage oocytes. VDAC proteins detected with the antiserum P1/6 (anti-VDAC1, lanes 1 and 2) and AS P2/45 (anti-VDAC2, lanes 3 and 4) demonstrate the presence of the two VDAC isoforms, VDAC1 and VDAC2, in GV and MII oocyte protein extractions. A strong immunoreaction at the approximate molecular weight of 32 kDa was visible in GV (lane 1) and MII oocytes (lane 2) using anti-VDAC1 antibodies. In contrast, anti-VDAC2 immunodetection presented a different staining pattern between immature and mature oocytes. In immature cells a polypeptide band of approximately 30 kDa was observed; however, a faint immune reaction with a 50-55 kDa peptide was also visible (lane 3). In mature oocytes a single, strong immune reaction was observed at the molecular mass range of 50-55 kDa (lane 4), while the 30 kDa immunoreactive protein band was missing. In control experiments with the respective pre-immune sera, no immunoreactions were noticed (data not shown).

Protein detection by immunoblotting

Purification and protein identification by mass spectrometry of oocyte VDAC

The purification procedure of porcine oocyte VDAC was adapted from the method described for mammalian VDAC protein [START_REF] De Pinto | A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues[END_REF]. Proteins from immature porcine oocytes (n = 3300) were isolated by a two-step sequential extraction (extract A and B) as described in Material and Methods.

M a n u s c r i p t

In extract A, no VDAC protein was detected (data not shown). Extract B contained a single faint protein band of 30-32 kDa in the first fraction (data not shown), while a higher amount was found in the second fraction (Figure 3). The protein band from fraction 2 was excised from the gel and subjected to MALDI-TOF MS followed by PMF analyses. The number of peptides matched covered 31% of the VDAC2 (Sus scrofa) protein.

Immunocytochemical localization of VDAC proteins in porcine oocytes

In Figure 4 (A-C), GV stage non-permeabilized oocytes were prepared and assayed with anti-VDAC antibodies. Anti-VDAC1 antiserum, AS P1/6, and monoclonal anti-human type 1 VDAC antibodies, P31HL (Calbiochem), yielded a dense dot-like arrangement of fluorescent labeling primarily confined to the oocyte surface (Figure 4, A andB). For the polyclonal antibody AS P1/6, additional staining of the zona pellucida was observed (Figure 4 A, arrow). No distinct labeling of the oocyte surface was observed in control oocytes, which were treated only with the second antibody and the respective preimmune serum or IgG 2a (Figure 4,C). VDAC2 antibodies (Santa Cruz) displayed no fluorescent labeling; however, some cells exhibited signs of lesions and disturbance of cell integrity (data not shown). For the assessment of intracellular VDAC polypeptides, membrane-permeabilized oocytes (GV and MII stage) were probed with anti-VDAC antibodies. Oocytes were completely scanned and photographed in three planes; however, mainly the equatorial plane data was included since it gave the most descriptive information. As shown in Figure 4 for GV stage oocytes (D), the antiserum against VDAC1, AS P1/6 (D), presented a dense immunofluorescence labeling around the cortical area of the oocyte. Identical results were observed when immature oocytes were probed with another anti-VDAC1 antibody, P31HL (Calbiochem, data not shown). The assessment of MII porcine oocytes with both anti-VDAC1 antibodies yielded the same immunofluorescence pattern that has been observed in GV stage porcine oocytes (data not shown). By using anti-VDAC2 antibodies from Santa Cruz, immunolabeling was observed in most of the GV oocytes tested (Figure 4,E). In these immunofluorescence-positive cells, variable spherical clusters, not evenly distributed throughout the cortical area, were detected (details observed in the oocyte cortical area of Figure 4,F). However, when porcine MII oocytes were tested with anti-VDAC2 antibodies from Santa Cruz, no specific reaction was observed (data not shown). The anti-VDAC2 antibody (AS P2/45) displayed no specific fluorescence labeling in immature and mature porcine oocytes (data not shown).

Discussion

In previous studies, we reported the expression, localization and functional characterization of VDAC isoforms in bovine testis and spermatozoa [START_REF] Menzel | Molecular and functional characterization of VDAC2 purified from mammal spermatozoa[END_REF][START_REF] Hinsch | Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum[END_REF][START_REF] Hinsch | VDAC2 (porin-2) expression pattern and localization in the bovine testis[END_REF]. Furthermore, we presented new results that indicate the participation of VDAC proteins in essential sperm functions [START_REF] Triphan | Localisation and function of voltagedependent anion channels (VDAC) in bovine spermatozoa[END_REF]. In relation VDAC in mammal oocytes have not yet been investigated. Using RT-PCR, we demonstrated for the first time the expression of VDAC1 and VDAC2 within immature (GV) and in vitro-matured (MII) porcine oocytes. The amplification products detected in porcine oocytes corresponded to the published coding sequence. The presence of additional sequences corresponding to an unspliced variant, Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

as has been described previously for Porin 2 (VDAC2) in Drosophila melanogaster, remains to be elucidated [START_REF] Guarino | Expression and localization in spermatozoa of the mitochondrial porin isoform 2 in Drosophila melanogaster[END_REF]. Specific antibodies generated against VDAC1 and VDAC2 allowed investigation of the expression pattern of VDAC proteins in oocytes. Immunoblot analyses demonstrated the expression of VDAC1 in GV and MII maturation stages. Testing of immature porcine oocytes with anti-VDAC2 antibodies yielded VDAC2 protein having the same molecular weight range of about 30-32 kDa as has been reported for sperm VDAC2 [START_REF] Hinsch | Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum[END_REF]. Additionally, we noticed a faint immunoreactive band with a higher molecular weight of approximately 50-55 kDa. Interestingly, in mature oocytes (MII) only a single, strong immunoreaction with anti-VDAC2 antibodies was observed with the 50-55 kDa protein band while the original immunoreactive 30-32 kDa VDAC protein disappeared. This result enhances the hypothesis of the expression of two VDAC2 subisoforms, with different polypeptide lengths, and with a pattern of similar posttranslational modifications [START_REF] Menzel | Molecular and functional characterization of VDAC2 purified from mammal spermatozoa[END_REF]. [START_REF] Guarino | Expression and localization in spermatozoa of the mitochondrial porin isoform 2 in Drosophila melanogaster[END_REF] suggested that a high molecular VDAC protein in drosophila ovaries could be the product of mRNA still containing intron sequences.

sought to further investigate the identity of the immunoreactive protein bands by purifying porcine oocyte VDAC proteins to apparent homogeneity and subsequent sequencing of the polypeptides using hydroxyapatite/celite purification [START_REF] De Pinto | A simple and rapid method for the purification of the mitochondrial porin from mammalian tissues[END_REF] and MALDI TOF MS analyses, respectively. To obtain sufficient material for sequencing, high amounts of granulosa cell-free oocytes and a two-step protein extraction were necessary. Here we report, to our knowledge for the first time, the purification of the 30-32 kDa VDAC2 protein from mammalian immature oocytes. The VDAC1 and the VDAC2 immuno-related 50-55 kDa polypeptide, identified by immunological methods in immature porcine oocytes, could not be detected in the VDAC protein-containing hydroxyapatite/celite throughput. Presumably, the amount of these proteins was too low or the proteins were retained in the hydroxyapatite/celite column [START_REF] De Pinto | Interaction of non-classical detergents with the mitochondrial porin. A new purification procedure and characterization of the pore-forming unit[END_REF]. Having identified VDAC proteins in mature and immature porcine oocytes, the subcellular distribution of these proteins was studied. The cytochemical analyses of nonpermeabilized oocytes with anti-VDAC1 antibodies could reveal the presence of VDAC1 in the plasma membrane region. Very similar staining patterns were obtained with the two different anti-VDAC1 antibodies. However, AS P1/6 (antiserum), additionally displayed immunofluorescence of the zona pellucida. Compared with the specific staining pattern of the monoclonal anti-VDAC1 antibody P31HL (Calbiochem), this observation most probably is due to nonspecific binding to this extracellular oocyte matrix. The fluorescence pattern of anti-VDAC1 antibodies is reminiscent of the cytological staining observed in Xenopus laevis oocytes [START_REF] Steinacker | The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels[END_REF]. Furthermore, the dot-wise labeling pattern at the cell surface suggests a plasma membrane localization of VDAC1. Because the oocytes assayed in our study were not permeabilized, we assume that the VDAC1 epitopes detected by the two antibodies are localized at the extracellular plasma membrane domain of the oocyte. The first evidence for an extra-mitochondrial localization of VDAC protein in the plasma membrane of human B lymphocytes was published in 1989 [START_REF] Thinnes | Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell[END_REF]. Several reports on the presence of VDAC as an integral membrane protein of various kinds of cells and organelles supported this study [START_REF] De Pinto | New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC)[END_REF][START_REF] Bathori | Extramitochondrial porin: facts and hypotheses[END_REF][START_REF] Dermietzel | Cloning and in situ localization of a brain-derived porin that constitutes a large-conductance anion channel in astrocytic plasma membranes[END_REF][START_REF] Reymann | Further evidence for multitopological localization of mammalian porin (VDAC) in the plasmalemma forming part of a chloride channel complex affected in cystic fibrosis and encephalomyopathy[END_REF][START_REF] Junankar | Porin-type 1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres[END_REF]. Our results confirm the localization of VDAC1 in the plasma membrane of porcine oocytes; however, no information on the function of plasma membrane-bound VDAC1 A c c e p t e d M a n u s c r i p t in oocytes is yet available. Several studies suggest that VDAC in the plasma membrane of somatic cell types act as anion channels involved in volume regulatory mechanisms [START_REF] Thinnes | Studies on human porin XXII: cell membrane integrated human porin channels are involved in regulatory volume decrease (RVD) of HeLa cells[END_REF][START_REF] Okada | Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume in murine cells[END_REF]. The participation of VDAC1 in the volume regulatory process of mammalian spermatozoa was reported recently for gametes [START_REF] Triphan | Localisation and function of voltagedependent anion channels (VDAC) in bovine spermatozoa[END_REF]. VDAC2 was not observed in the oocyte plasma membrane, suggesting separate localizations and functions of both isoforms in the oocyte. After having found that anti-VDAC 1 antibodies bind to the outer surface of the oocyte plasma membrane, we were interested to identify VDAC proteins in the ooplasm of immature and mature porcine oocytes. Using confocal microscopy, it was possible to detect VDAC1 and VDAC2 proteins in different localizations of the porcine ooplasm. VDAC1 was clearly observed at the cortical area of immature and mature oocytes. Thus, the distribution of VDAC1 proteins seemed to be stable throughout the meiotic

In the literature, it has been reported that in the GV stage of mammalian oocytes, actin filaments are distributed as a relatively thick, uniform area around the cell cortex and are also found near the germinal vesicle. After germinal vesicle breakdown (GVBD), microfilaments are predominantly found at the cortex of the cell and around the female chromatin [START_REF] Wang | Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development[END_REF]. The interaction of VDAC with other cellular proteins, especially those of the cytoskeleton, has been reported in recent years [START_REF] Linden | Identification of porin as a binding site for MAP2[END_REF][START_REF] Roman | Direct measurement of VDAC-actin interaction by surface plasmon resonance[END_REF]. G-actin has been demonstrated to modulate VDAC gating in Neurospora crassa [START_REF] Xu | Actin modulates the gating of Neurospora crassa VDAC[END_REF], whereas in HeLa cells the dynein light chain Tctex1 was shown to regulate VDAC [START_REF] Schwarzer | Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex1 and the heat-shock protein PBP74[END_REF]. Furthermore, VDAC were identified as binding sites for microtubule-associated protein 2 (MAP2) in the outer membrane of rat brain mitochondria [START_REF] Linden | Identification of porin as a binding site for MAP2[END_REF][START_REF] Leterrier | Interactions between brain mitochondria and cytoskeleton: evidence for specialized outer membrane domains involved in the association of cytoskeleton-associated to mitochondria in situ and in vitro[END_REF]. The localization of VDAC1 at the cortical area of the oocyte might indicate an interaction of VDAC1 with cytoskeleton proteins that are predominantly found in this area of the cell. The observed pattern of VDAC1 distribution could also suggest a mitochondrial localization. The presence of VDAC protein on the outer membrane of mitochondria from eukaryotic cells is well established [START_REF] Lemasters | Voltage-dependent anion channel (VDAC) as mitochondrial governator--thinking outside the box[END_REF][START_REF] Benz | Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells[END_REF]. During porcine oocyte maturation, the distribution of oocyte mitochondria was reported in the perinuclear area in GV stage oocytes, and large mitochondrial foci form and relocate to the inner cytoplasm in mature oocytes [START_REF] Sun | Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro[END_REF]. In mouse oocytes, mitochondria are clustered at the periphery of the large nucleus and the cortical area, but they disperse through the cytoplasm during maturation. This pattern of distribution may be related to the high energy requirement in cortex, as the oocyte requires the support of cumulus cells at this stage [START_REF] Sun | Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro[END_REF]. Staining patterns observed with anti-VDAC2 antibodies differed from that of the VDAC1 isoform. In immature porcine oocytes, VDAC2 antigen was detected as distinct but not evenly distributed spherical clusters throughout the cortical area of the ooplasm. Thus, it can be speculated that VDAC1 and VDAC2 are present in different intracellular compartments of the oocyte. Our results implicate that in oocytes VDAC subtypes might be involved in the regulation of different cell functions (e.g. cell maturation or apoptosis). However, the exact localization and role of VDAC in the regulation of particular oocyte functions remain to be investigated. In mature oocytes, the labeling pattern of VDAC2 protein completely disappeared after in vitro maturation, apparently indicating an interrelation of VDAC2 distribution or integrity with the cell's developmental stage. Nevertheless, the lack of observed in mature oocytes could be also explained with a posttranslational change of the conformation of VDAC2 protein that hinders binding of the antibody to the native protein. It has been shown previously that some of the oocyte organelles exhibit dramatic changes during meiotic progression. Although VDAC were A c c e p t e d M a n u s c r i p t originally thought to be only located in the OMM, several investigations reported the presence in other cell compartments, e.g. nuclear envelope, endosomes, sarcoplasmic/endoplasmic reticulum, caveolae and other organelles of somatic cells [START_REF] Bathori | Extramitochondrial porin: facts and hypotheses[END_REF][START_REF] Shoshan-Barmatz | The voltage-dependent anion channel (VDAC): function in intracellular signalling, cell life and cell death[END_REF][START_REF] Lisanti | Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease[END_REF][START_REF] Shoshan-Barmatz | VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle[END_REF][START_REF] Shoshan-Barmatz | The voltage-dependent anion channel in endoplasmic/sarcoplasmic reticulum: characterization, modulation and possible function[END_REF]. The distribution of caveolae in Caenorhabditis elegans oocytes [START_REF] Sato | Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans[END_REF] resembles that observed in our immunofluorescence studies with anti-VDAC2. However, preliminary double staining experiments, containing anti-VDAC2 and anti-Caveolin 1 antibodies, showed only a partial overlapping of both proteins. More research is essential to determine VDAC localization in these or other organelles in mammalian oocytes. Anti-VDAC2 antibodies clearly exhibited different labeling patterns within immature oocytes. In the future it will be of great interest to study if the difference observed in VDAC2 labeling is conditioned to individual characteristics of each oocyte (e.g. oocyte quality). The identification of quality markers for oocyte fertilization capacity would be of great help in clinical applications and in vitro production. The data obtained for VDAC2 using immunofluorescence are in line with the immunoblot experiments performed in this study, where shifts of apparent molecular masses of VDAC antigens during maturation were detected. The immunoreactive band at 30-32 kDa was no longer found after oocyte maturation, and a strong immunoreaction was observed in the 50-55 kDa molecular weight range. The 50-55kDa anti-VDAC antigen, which is evidently the only VDAC2-related protein in the ooplasm of mature oocytes, could possibly represent a homodimerization/oligomerization product of these integral membrane proteins [START_REF] Distler | A targeted proteomic approach for the analysis of rat liver outer membrane proteins with extensive sequence coverage[END_REF][START_REF] Linden | Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are[END_REF][START_REF] Babel | Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle[END_REF] or a tight complex of VDAC with other polypeptides [START_REF] Roman | Direct measurement of VDAC-actin interaction by surface plasmon resonance[END_REF]. However, it is tempting to speculate that either alterations of intracellular distribution of VDAC2 or protein-protein interactions of VDAC isoforms are causally related to the shift of apparent molecular masses during the maturation of oocytes. The identification and localization of VDAC isoforms in different compartments of maturing porcine oocytes unveiled in this study could be the basis for better understanding of the molecular mechanisms of oocyte functions as well as the basis for novel clues concerning the role of VDAC proteins in the regulation of physiological processes of cells. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 27 Jul 2009 as manuscript BSR20090088 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090088 A c c e p t e d M a n u s c r i p t

Bioscience

  Reports Immediate Publication. Published on 27 Jul 2009 as manuscript BSR20090088 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090088 A c c e p t e d M a n u s c r i p t

  Bioscience Reports Immediate Publication. Published on 27 Jul 2009 as manuscript BSR20090088 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090088 A c c e p t e d

Bioscience

  Reports Immediate Publication. Published on 27 Jul 2009 as manuscript BSR20090088 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090088 A c c e p t e d M a n u s c r i p t

Bioscience

  Reports Immediate Publication. Published on 27 Jul 2009 as manuscript BSR20090088 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090088

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Immunoblotting of VDAC1 and VDAC2 from porcine oocytes Detection of VDAC isoforms in porcine GV (immature) and MII stage (mature) oocytes. Proteins of 300 oocytes per lane were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes (lane 1 and 2) were incubated with anti-VDAC1 antibody (AS P1/6, 1:100). A second set of membranes (lane 3 and 4)immunoprobed with anti-VDAC2 antibody (AS P2/45, 1:100). Bound antibodies were detected with protein A-HRP (1:3000) followed by detection with an ECL system.

Figure 3 :

 3 Figure 3: Purification of VDAC2 protein from immature porcine oocytes Protein extraction and hydroxyapatite-celite purification of 3300 GV stage porcine oocytes. Protein fractions were separated in 12% SDS-PAGE and silver stained. The second fraction obtained from extraction B yielded a single stained band (arrow). A Mascot MS/MS ion search identified the tagged band as VDAC2 (Sus scrofa).

Figure 4 :

 4 Figure 4: Immunolocalization of VDAC1 and VDAC2 protein in porcine oocytes Upper panel Confocal laser microscopy images of immature porcine oocytes in the equatorial plane a permeabilization step (A, B and C). Oocytes were incubated with anti-VDAC1 antibodies. A: AS P1/6 (1:20) and B: P31HL (Calbiochem, 1:4), revealed by FITC-labeled anti-rabbit antibody (1:20) and anti-mouse antibody (1:8), respectively. C: Sample for negative control; oocyte exposed to IgG 2a. Zona pellucida (A, arrow). Original magnification X 600. Lower panel: Subcellular immunolocalization of VDAC protein isoforms in paraformaldehyde-fixed porcine immature stage oocytes (GV). Cells were treated with D: AS P1/6 (1:80), E and F: anti-VDAC2 (Santa Cruz; 1:5). A-E: Equatorial section of GV oocyte, F: Cortical plane of GV oocyte. Bound antibodies were detected with FITC-labeled second antibodies. Original magnifications X 600.

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  p t e d M a n u s c r i p t

  Bioscience Reports Immediate Publication. Published on 27 Jul 2009 as manuscript BSR20090088 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090088

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

The authors wish to thank the colleagues of the Institute for Animal Breeding Biotechnology at the Federal Agricultural Research Centre (FAL; Neustadt, Germany) the excellent technical assistance and support. This project was supported by funds from the Deutsche Forschungsgemeinschaft GRK 533. This publication includes parts of the thesis of M. C. Cassará.


Astrid C Bosse

Josef Pallauf

Bettina Hommel

Mariana Sturm

Susanne Fischer

Nicole M Wolf

Andreas S Mueller
email: andreas.mueller@landw.uni-halle.de

Impact of selenite and selenate on differentially expressed genes in rat liver examined by microarray analysis

Keywords: Selenium, selenite, selenate, differential gene expression, microarray rat Bioscience Reports Immediate

Sodium selenite and sodium selenate are approved inorganic Se compounds in human and animal nutrition serving as precursors for selenoprotein synthesis. In recent years numerous additional biological effects over and above their functions in selenoproteins have been reported. For greater insight into these effects our study examined the influence of selenite and selenate on the differential expression of genes encoding non-selenoproteins in the rat liver using microarray technology. 5 groups of 9 growing male rats were fed a Se deficient diet or diets supplemented with 0.20 or 1.0 mg Se/kg as sodium selenite or sodium selenate for 8 weeks. Genes which were more than 2.5-fold up-or down-regulated by selenite or selenate compared to Se deficiency were selected. Glutathione peroxidase 1 was upregulated 5.5-fold by both Se compounds, whereas glutathione peroxidase 4 was upregulated only 1.4-fold. Selenite and selenate down-regulated three phase II enzymes.

Despite the regulation of many other genes in an analogous manner, frequently only selenate changed the expression of these genes significantly. In particular, genes involved in the regulation of the cell cycle, apoptosis, intermediary metabolism, and those involved in Se deficiency disorders were more strongly influenced by selenate. The comparison of seleniteand selenate-regulated genes revealed that selenate may have additional functions in the protection of the liver, and that it may be more active in metabolic regulation. In our opinion the more pronounced influence of selenate compared to selenite on differential gene expression results from fundamental differences in the metabolism of these two Se compounds.

INTRODUCTION

Since the discovery of the essentiality of selenium (Se) five decades ago, this element has been increasingly associated with many biological processes [START_REF] Hesketh | Nutrigenomics and selenium: gene expression patterns, physiological targets, and genetics[END_REF]. In contrast to other metal trace elements which interact with proteins as cofactors, the physiological functions of Se derive mainly from the incorporation of Se into the active sites of functional selenoproteins via the cotranslational synthesis of selenocysteine [START_REF] Hesketh | Nutrigenomics and selenium: gene expression patterns, physiological targets, and genetics[END_REF]. To date the existence of not less than 33 functional selenoproteins with at least one selenocysteine residue has been reported. The best investigated characteristic of Se is its antioxidant role. Six different glutathione peroxidases (GPx1 -GPx6) and two thioredoxin reductases (TrxR 1 and TrxR2) contribute largely to the maintenance of cellular antioxidant defense. The involvement in thyroid metabolism through iodothyronin deiodinase activity represents a further important Se dependent target [2]. Under conditions of low Se availability, these selenoproteins selectively are down-regulated. Whereas GPx1, exhibiting a high and ubiquitous expression and activity in mammalian tissues, and plasma glutathione peroxidase (GPx3) undergo a rapid and severe down-regulation, gastrointestinal glutathione peroxidase (GPx2) and phospholipidhydroperoxide glutathione peroxidase (GPx4) are far more conserved [3,4].

Depending on the animal species, a long term Se deficiency causes liver necrosis and myopathies [5][6][7][8]. Besides the mechanisms of functional selenoproteins, in recent years additional information on Se biochemistry has been obtained. The down-regulation of phase II detoxification enzymes such as glutathione-S-transferases and aflatoxin b1 aldehyde reductase (Afar) due to adequate Se supply impressively shows the counter regulation of cellular stress response [9,10]. Anti-carcinogenic effects of supranutritive Se dosages, investigated in animal trials and tissue culture experiments, may result from the initiation of proapoptotic mechanisms by the generation of monomethylselenol and high concentrations of reactive oxygen species [11], or from the response of phosphorylation-and signal transduction mechanisms [12]. Other hypotheses suggest the reversion of the hypermethylated DNA of tumour suppressor genes [START_REF] Stranges | Effects of long-term selenium supplementation on the incidence of type 2 diabetes[END_REF] or the reduction of prostaglandin E2

and cyclooxygenase 2 via GPx2 activity as being the underlying mechanisms for the protective effect of Se against colon cancer [START_REF] Tonks | A brake becomes an accelerator: PTP1B--a new therapeutic target for breast cancer[END_REF]. However, the results of large placebo controlled human intervention trials did not support uniquely beneficial effects of supranutritional Se supplementation in the prevention of several cancers [15,16]. The influence of Se supplementation and selenoprotein activities on obesity, insulin resistance, diabetes and hyperlipidaemia has been the subject of recent controversial discussion.

Whereas only very high supranutritive selenate doses have antidiabetic effects [17], a high activity of GPx1 as well as a high Se status derived from the permanent intake of Se supplements have lately been reported as being one possible cause of the accelerated development of obesity, insulin resistance, diabetes and hyperlipidaemia [18][19][20][21][22]. These important results were the outcome of the above mentioned cancer intervention trials and of large cross sectional studies [15,[19][20][21]. Optimum activities of all functional selenoproteins can be obtained by a daily Se uptake of 30-70 µg in humans and by a concentration of 0.15-0.30 mg Se/kg diet in animals. The inorganic Se compounds sodium selenite (+IV) and sodium selenate (+VI) are approved Se additives both in human mineral supplements and in animal feed. Both selenite and selenate have a high absorption rate from the small intestine [23], but they exhibit some fundamental differences in their absorption mechanism as well as in their intermediary metabolism. There is no doubt that selenite and selenate represent valuable inorganic Se compounds to ensure adequate selenoprotein synthesis [24,25].

Nevertheless, information is lacking whether and in what way selenite and selenate influence the expression of genes beyond functional selenoproteins. In this context the present study using microarray analysis (MA) as the analytical tool had the following aims:

• Data mining of genes regulated differentially by selenite or selenate supplementation versus a control group with Se deficiency.

•

Exploring genes responding in particular to selenite or selenate supplementation by direct comparison of the selenite and selenate expression profile. humidity, 12h light : dark cycle) and had free access to the diet and deionized water. The protocol of the animal experiment was approved by the Regional Council of Giessen and by the animal welfare committee of Giessen University. At the end of the trial, the rats were anaesthetized in a carbon dioxide atmosphere and subsequently decapitated. The liver was removed, frozen in liquid nitrogen, and stored at -80°C until analysis.

Se analysis in diets and liver

Se concentration in the diets and the livers was determined by hydride generation atomic absorption spectrometry (AAS, Unicam PU 9400 X; PU 3960 X) as reported previously [27].

RNA extraction and microarray analysis

The analysis of differentially expressed genes was performed using "10k rat oligoarrays" from MWG (Ebersberg, Germany). For the comparison of the expression data from selenite and selenate treated rats with that of their companions on Se deficiency the following criteria were applied:

•

Only genes with average Cy3 and Cy5 values that were 2-fold above the background were used.

•

Only genes that were at least 2.5-fold up-or down-regulated by selenite or selenate treatment were considered.

•

Only genes regulated in an analogous manner by selenite and selenate were considered.

•

Only genes which fulfilled the statistical assumptions were considered (c.f. statistical analysis).

Differentially expressed genes were assigned to 5 functional areas and are shown in Table 1.

Determination of GPx1 and GPx4 activity

The activities of GPx1 and GPx4 were determined spectrophotometrically (Beckmann DU 50) recording glutathione dependent peroxide reduction coupled to glutathione reductase and NADPH oxidation at 340 nm [29]. For GPx1 activity, the diluted cytosolic supernatants of rat liver homogenates served as sample material. For GPx4 activity, crude homogenates of livers were prepared in a sucrose buffer [30]. H 2 O 2 served as the substrate for GPx1 determination, whereas freshly synthesized phosphatidylcholine hydroperoxide (PCOOH)

was used for measurement of GPx4 activity [30]. One unit of GPx1 and GPx4 activity was defined as one micromole NADPH oxidized/min and normalized to 1 mg protein.

Activity of the phase II detoxification enzymes GSTa2 and epoxide hydrolase

The activity of alpha glutathione-S-transferases to which GSTa2 activity makes a large contribution was selectively determined according to a spectrophotometric protocol using the specific substrate 7-chloro-4-nitrobenzoic-2-oxa-1,3-diazole (NBD-Cl) [31]. The increase in absorption at 419 nm caused by the conjugation of NBD-Cl to 7-glutathionyl-4-nitrobenzoic-2-oxa-1,3-diazole (NBD-GSH) was recorded for 3 min. 1 U of alpha glutathione-S-transferase activity was defined as the formation of 1 µmol of NBD-GSH/min and normalized to 1 mg protein.

Epoxide hydrolase activity in 1:5 (w/v) crude liver homogenates was measured according to a spectrophotometric protocol using styrene oxide as the substrate [42]. In the first reaction were analyzed with Student's t test. At P<0.05, expression differences were accepted as statistically significant if homogeneous variances and the normality of distribution could be ensured. In order to reduce the genes differentially expressed among treatment groups to a manageable number for publication only genes which were at least 2.5-fold up-or downregulated by selenite or selenate treatment were considered (Table 1). Only genes which were regulated in an analogous manner by selenate at both dietary concentrations or in addition by selenite at one dietary concentration are displayed.

RESULTS

Effect of Se status on feed intake and live weight

After 8 weeks no significant differences in feed intake and live weight (final average weight 285.8 ± 7.0 g) were observed between rats receiving a Se deficient diet (0) compared to their littermates with Se adequate nutrition (I, II) or supranutritive Se supply (III, IV).

Concentration of Se in diets and liver

The basal diet based on Torula yeast was readily accepted by the rats and exhibited a Se concentration below the detection limit (<0.03 mg/kg). Analyzed Se levels of the supplemented diets corresponded well with the values scheduled in the experimental design (I: 0.21±0.009 mg/kg fresh matter (~90% dry matter), II: 0.19±0.010, III 0.96±0.071, 0.85±0.025). Liver Se concentration in group 0 (21.2±2.40 µg/kg fresh matter) decreased to 2% of that of rats with adequate Se intake (I: 1044±88.6, II: 1050±78.4). Supranutritive Se (III: 2233±197, IV: 2148±149) again increased liver Se concentration significantly compared to groups I and II. At both dietary levels no significant differences existed between selenite and selenate regarding their efficacy in influencing liver Se concentration.

GPx1-and GPx4 activity and expression

As a sensitive parameter of Se deficiency, GPx1 expression and activity were significantly decreased in the Se deficient group 0. (Figure 1 A,C). Independent of the Se compound, the supplementation with 0.2 mg Se/kg diet (groups I and II) resulted in a strong increase in GPx1 activity (73-fold) which was accompanied by multiplied gene expression in the MA experiment (~7.5-fold). MA data also indicated a marked increase in GPx1 expression (~6.0fold) for groups III and IV which received diets with supranutritive Se (1.0 mg Se/kg) compared to group 0 (Figure 1 A). GPx1 activity did not quite reach the level of the adequately supplemented groups (Figure 1 C). The differences in GPx1 mRNA expression, as indicated by the MA data, could be confirmed by 2-step RT-PCR analysis (Figure 1 A). In

General results for differential gene expression with regard to the comparison of selenite-or selenate feeding versus Se deficiency

Despite no differences between selenite and selenate in the regulation of the selenoproteins GPx1 and GPx4, the most challenging and unexpected finding in our study was the fact that compared to Se deficiency (group 0) both dietary selenate levels (groups II and IV) had a distinctly stronger influence on differential gene expression than selenite (groups I and III).

Thus with a ratio of 2.5 compared to Se deficiency about 300 genes were significantly regulated by selenate feeding, whereas selenite only affected the expression of 43 and 44 genes, respectively. For both inorganic Se compounds the comparison of the nutritional level (0.2 mg/kg) and the supranutritive level (1.0 mg/kg) revealed no significant differences in the total number of up-or down-regulated genes.

Specific results for differential gene expression with regard to the comparison of selenite-or selenate feeding versus Se deficiency after mapping functional areas

After analysis of the general effects of selenite and selenate on differential gene expression we mapped genes which were up-or down-regulated in an analogous manner compared to Se deficiency at least by selenate feeding (242 genes) into 5 functional areas (c.f. Material and Methods). The results of this mapping are shown in Table 1 for 44 representative genes.

By displaying the regulation ratio as well as the significance level also for the selenite groups I and III, Table 1 clearly shows that selenate feeding (groups II and IV) compared to Se deficiency (group 0) influences the differential expression of genes in all functional areas investigated much more distinctly and significantly than selenite feeding. To confirm the results of the MA analysis we validated the expression data for 3 representative target genes (GST2a, area I; epoxide hydrolase, area I; PTP1B, area II) by 2-step RT-PCR and/or enzymatic analysis in addition to GPx1-and GPx4-expression. One very challenging result of our study was the fact that Se strongly influences the regulation of phase I-and phase IIdetoxification genes (Table 1, area I). Three mRNAs of phase II-detoxifying enzymes, namely Aflatoxin B1 aldehyde reductase (Afar), Alpha glutathione-S-transferase (GSTA2)

and Epoxide hydrolase were distinctly down-regulated by Se feeding (groups I, II, III, and IV) compared to Se deficiency (group 0). For these enzymes, selenite feeding was also effective at both dietary levels. We could confirm the validity of the MA data by measurement of GSTA2 expression and activity (Figure 2 A and B) and Epoxide hydrolase activity Figure 2 C). Contrary to the down-regulation of phase II-detoxifying enzymes, the phase Ihaemoprotein cytochrome p450c was up-regulated by selenate.

In particular by selenate feeding, numerous genes controlling intermediary nutrient metabolism, the regulation of appetite and body weight and other metabolic processes (Table 1, area II) were differentially regulated compared to Se deficiency. Although selenite feeding influenced the expression of these genes into the same direction, the strength of regulation was not significant in most cases. In the case of Heme oxygenase 3 and Transferrin receptor, two genes of iron metabolism were distinctly affected in their expression (Arginase II) and even in folate metabolism (Gamma glutamyl hydrolase precursor). In area II the validity of the MA data was confirmed by the examination of the expression and the activity of the insulin antagonistic PTP1B (Figure 3). Thus Se feeding with both selenite and selenate led to a 1.7 to 2.8-fold up-regulation of PTP1B mRNA, accompanied by a ~3.0-fold increase in enzyme activity (Figure 3).

In area III (Table 1) a number of apoptosis promoting genes such as BCL2-antagonist, Dermatan sulfate proteoglycan II, FOS related antigen, Mast cell protease 1, p58, and

Results for differential gene expression with regard to the direct comparison of selenite and selenate feeding

At a ratio of 2.5 (P<0.05) a total number of 18 genes (3 down-regulated and 15 up-regulated)

were found as being differentially expressed by selenate and selenite feeding. Table 2 shows a selection of 11 differentially expressed genes from this comparison. As can be seen from The regulation of the 5-hydroxytryptamine serotonin receptor 6 and the Growth hormonereleasing hormone receptor beta indicates a more pronounced influence of selenate in liver fatty acid-and sterol metabolism as well as in central mechanisms of body weight regulation compared to selenite. The Alphab crystalline-related protein (Heat shock protein 20) and its precursor the golgi associated protein Gcp 360 which are down-regulated during the development of hepatocellular carcinoma showed a higher expression due to selenate feeding. Both genes were also found as being more highly expressed by selenate in the expression profile versus Se deficiency (c.f. Table 1). The differential regulation of Ly-49.9

antigen and the inhibitory natural killer cell receptor Nkr-p1b point to a higher influence of selenate in modulating T cell response compared to selenite. The Aldo-keto reductase akr1b-7 which was up-regulated by selenate compared to selenite plays an important role in lipid detoxification, in particular in the small intestine. Titin which increases the elasticity of cardiomyocytes showed a significantly up-regulation due to selenate feeding. Titin was also found as being more highly expressed by selenate in the expression profile versus Se deficiency (c.f. Table 1).

Additional information on functions of the regulated genes in Table 1 and However, a very important and unexpected result of our study was the finding that selenate feeding related to Se deficiency had a distinctly stronger influence on differential gene expression than selenite.

Fundamental differences in the intestinal absorption pathways, in the blood transport to peripheral organs, and in the subsequent intermediary metabolism of selenite (Se oxidation state +IV) and selenate (Se oxidation state +VI) may provide one plausible explanation for this effect [23,34]. In peripheral organs the absorption products from dietary selenite (oxidation states 0 and -I) need far fewer reduction steps than selenate (oxidation state +VI).

In Figure 4 The regulation of other genes by selenate feeding ( and of Cyclic-Adenosine-3',5'-monophosphate response element binding protein 1 (Table 1, area V) [START_REF] Liu | Increasing alpha 7 beta 1-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis without changing gene expression[END_REF][58][START_REF] Cheng | The influence of plectin deficiency on stability of cytokeratin18 in hepatocellular carcinoma[END_REF][60].

The direct comparison of selenite-and selenate regulated genes revealed additional effects of selenate e.g. in liver fatty acid-and sterol metabolism and in central mechanisms of body weight regulation (5-hydroxytryptamine serotonin receptor 6 and growth hormone-releasing hormone receptor beta) [START_REF] Suter | Effect of two 5-HT6 receptor antagonists on the rat liver: a molecular approach[END_REF], in the protection against hepatocellular carcinoma (Alphab crystalline-related protein=heat shock protein 20), in the modulation of T cell response (Ly-49.9 antigen and the inhibitory natural killer cell receptor Nkr-p1b), and in the protection against lipid toxicity (aldo-keto reductase akr1b-7) [43,62] In summary our study on the effects of the two common inorganic Se compounds selenite and selenate on the differential expression of genes other than selenoproteins showed the following:

• Compared to Se deficiency as a control, selenite (Se oxidation state +IV) and selenate (Se oxidation state +IV) resembled each other in their effectiveness for seleoprotein synthesis. Both Se compounds could be demonstrated to be equally effective in the down-regulation of not less than 3 enzymes of phase II detoxification and of a further gene with an ARE in its DNA (Afar, epoxide hydrolase, GSTa2, heme oxygenase 3). This result demonstrates mechanisms of counter-regulation of antioxidant enzymes.

• Compared to Se deficiency selenate has a distinctly stronger influence than selenite on differential gene expression in various functional areas. This distinct difference, so far unknown, may result from fundamental differences in the absorption and the intermediary metabolism of both Se compounds. This remarkable difference for both Se compounds should be considered in future investigations into Se biochemistry.

• The direct comparison of selenite-and selenate regulated genes revealed additional functions of selenate in liver fatty acid-and sterol metabolism and in central mechanisms of body weight regulation, in the protection against hepatocellular carcinoma, in the modulation of T cell response, and in the protection against lipid toxicity (aldo-keto reductase akr1b-7).

• In view of the distinct influence of Se on differential gene expression, our data could contribute to an improved understanding of mechanisms by which Se may influence diseases of the Western world. Other genes differentially regulated by selenate provided deeper insight into the beneficial effects of Se in preventing Se deficiency symptoms such as muscular dystrophy and liver necrosis.

We conclude that future studies on the pathways of Se in the treatment of diseases should consider the particular effects of the Se compound used, and that the compilation of comparative differential gene expression profiles for further selenium compounds would be of interest. (1): Selenite reacts already during its absorption with thiol compounds, such as GSH, to form selenodiglutathione and selenotrisulfides.

(1 and 2): Se metabolites delivered to tissues from selenite absorption are selenodiglutathione, selenotrisulfides or hydrogen selenide.

(2 and 3): Pathways by which orally administered selenite can modify cysteine residues in other proteins and thereby lead to changes in differential gene expression derive from the formation of methylselenol from methylselenide, which again can be produced from hydrogen selenide, obtained from the entire reduction of selenite.

(3): Methylselenol may modify cysteine residues in proteins and change the activity of these proteins via thiol modifications such as glutathionylation by methylselenolate release in the presence of glutathione.

B) Selenate pathways:

(1): Selenate may exert a stronger influence on differential gene expression starting with its unmodified uptake through the small intestine into the organism. During its reduction in peripheral organs the following reactions with cysteine containing proteins are feasible:

(1): In the first reaction step the unreactive selenate (oxidation state +VI) is reduced in a GR or TrxR dependent reaction to the highly thiol reactive selenite oxidation state +IV.

(2): In the further course selenite (selenious acid) can modify protein thiol groups to selenodiglutathione intermediates.

(3): Excess selenite (selenious acid) reacts with GSH to form hydrogen selenide. In the further course of the reaction in the presence of oxygen, superoxide anion radicals (O 2 -•) and elemental Se can be formed.

(4): The superoxide radical again can attack cysteine residues in proteins and oxidize them to sulfenic acid derivatives.

(5): In the presence of glutathione the sulfenic acid can react to a reversibly inactivated (glutathionylated) protein-thiol compound.

The pathways (6), ( 7

Bioscience

  Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042with healthy growing rats served as the basis for the examination of differential gene expression by selenite and selenate. rats (HK 51, strain of the Institute of Animal Nutrition and Nutritional Physiology) were randomly assigned to 5 groups of 9. The average initial body weight (62.8 ± 4.0 g) did not vary among groups. The rats were fed a Torula yeast based diet as described earlier [30] for 8 weeks. The diet was composed according to the current recommendations of the National Research Council (NRC) [26] with the exception of Se. The analyzed Se concentration of the basal diet (group 0) was below the detection limit (<0.03 mg Se/kg diet

  Certified samples from the National Institute of Standard and Technology (soft winter wheat flour, NIST No. 8438 and bovine liver, NIST No. 1577 b) served as reference material for Se determination in the different matrices.

Bioscience

  Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042phase I and phase II detoxification Area II: Genes involved in the regulation of intermediary metabolism and in the regulation of appetite, body weight or other metabolic processes Area III: Genes related to the cell cycle, cell development, cell death (apoptosis) or tumour metabolism Area IV: Genes related to inflammatory or immunomodulatory response Area V: Genes with other functions (e.g., hormonal function, receptor function, signal transduction)In addition to the examination of the influence of selenite and selenate on differential gene expression versus Se deficiency a direct comparison of selenite-and selenate-regulated gene expression was carried out.

  contrast only a non significant weak influence of varying Se supplementation on gene expression of GPx4 was measured. GPx4 responded to Se deficiency only by a non significant minor down-regulation of its mRNA (1.2 to 1.6-fold) and by a 1.5 to 2.0-fold loss of activity (Figure1 B, C). The mRNA expression data obtained by MA analysis could be confirmed by 2-step RT-PCR analysis. No influence of the supplemented Se compound (selenite or selenate) and of the dietary Se level (0.2 and 1.0 mg/kg) on GPx4 expression and activity could be observed. Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089 A c c e p t e d M a n u s c r i p t

  by selenate feeding. The distinct up-regulation of Agouti related protein precursor, Hypocretin orexin receptor 1, Neuropeptide y precursor, and Ob-receptor gene related protein suggests the involvement of Se in central processes of appetite-and body weight-regulation. Moreover a distinct influence of Se on carbohydrate-and lipid metabolism was indicated by the regulation of Acyl-coenzyme A:cholesterol acyltransferase 1, Fructose-6-phosphate-2kinase, and Phosphorylase-b-kinase. A strong influence of selenate was also observed for the regulation of genes involved in metabolic signal transduction pathways (80-k diacylglycerol kinase, and Protein tyrosine phosphatase 1B), the regulation of vascular tone

  Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089 A c c e p t e d M a n u s c r i p t Rapamycin-and FKBP 12 target 1 were distinctly up-regulated by selenate. p58 was also strongly affected by selenite feeding. In contrast, other regulated genes in area III (NAPOR-3, Granzyme k) indicated an antiapoptotic role of Se. Moreover, in area III the regulation of genes participating in growth and differentiation of various tumours (ADAMTS-1, PKCQ interacting protein picot, and Proliferin related protein) indicate a tumour-protective function of Se. The differential regulation of numerous genes related to inflammatory or immunomodulatory reactions (area IV) as well as of genes with other functions, such as structural protein function, hormonal function, receptor function, a function in signal transduction or a function as an ion channel (area V) clearly indicated biological functions of Se beyond its role in functional selenoproteins. Whereas the regulated genes of area IV indicate an important target function of Se in the modulation of immune response, numerous regulated genes of area V may give some new insight into the beneficial effects of Se in the prevention of Se deficiency diseases, such as muscular dystrophy and liver necrosis. Differentially expressed genes indicating such a response are e.g., Calcium transporting ATPase Serca 1a, Cardiac ankyrin repeat protein, Mu-calpain large subunit, Tissue inhibitor of metalloproteinase 3, Titin, and Cyclic-3',5'-monophosphate response element binding protein 1.

  on other selected genes of the functional areas I to V are shown in Tables1 to 5as online supporting material.Moreover the full microarray data sets are available as online supporting material. The assignment of the samples to the 6 microarrays can be seen from the first sheet in the Excel file containing the data of the microarrays 1 to 3. The second Excel file contains the original data of the microarrays 4 to 6. The red marked columns I and L are the backgroundcorrected expression data for the single genes which can be used to compare gene expression.DISCUSSIONIn recent years specific knowledge of the biological effects of Se beyond its role in functional selenoproteins has advanced. Both positive and critical functions of Se with regard to diseases of the Western World such as cancer, cardiac diseases, obesity, insulin resistance, diabetes and hyperlipidaemia have been investigated. To obtain information on cellular mechanisms underlying such specific biological effects of Se we used MA scanning as the Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089A c c e p t e d M a n u s c r i p t analytical tool. Moreover, we examined the effectiveness of the two inorganic selenocompounds selenite (Se oxidation state +IV) and selenate (Se oxidation state +VI) for differential gene expression. In order to produce as distinct effects for Se as possible, we compared the gene expression of Se supplemented rats with that of their companions during Se deficiency. In addition a direct comparison of selenite-and selenate-regulated genes was carried out. With regard to the regulation of functional selenoproteins our data (Table3) are in accordance with very recent findings on the influence of Se status on selenoprotein mRNA levels in mice and rats[33].The pronounced loss of GPx1 mRNA expression and activity due to Se deficiency and a maximization of both parameters by Se supply at the recommended dietary level (0.2 mg Se/kg as selenite or selenate, groups I and II) and at a moderate supranutritive supply (1.0 mg Se/kg as selenite or selenate) confirm the low rank of GPx1 in the hierarchy of selenoproteins. Conversely the high rank of GPx4 was confirmed by only a small loss of mRNA expression and enzyme activity as well as a low P value for regulation (Table3) due to a lack in dietary Se supply. Our results moreover suggest that selenite and selenate are similar in their effectiveness in influencing the expression and activity of GPx1, GPx4 and the expression of further selenoproteins.The counter-regulation of GPx1 and the phase II detoxification enzymes GSTa2, epoxide hydrolase and Afar (Table1, area I) was another central finding of our study. In the literature a few reports describe similar effects of Se on these so called xenobiotica metabolizing enzymes. Thus in a rat trial an increased aflatoxin toxicity due to Se supplementation was observed, while a moderate Se deficiency effected a distinctly better aflatoxin detoxification based on the up-regulation of the assigned Aflatoxin-B1-aldehyde reductase (Afar) and additionally of GSTA2[9]. Other studies reported an up-regulation of several glutathione-Stransferases and of Epoxide hydrolase in rat liver by Se deficiency. However, recent consolidated findings have shown the underlying mechanism for this phenomenon. In Se deficiency, the transcription of phase II detoxification enzymes increases via the activation of the Antioxidant Response Element (ARE), a DNA structure located upstream the coding DNA sequence of these enzymes. ARE activation is sensitive to changes in cellular redox status, an increase in peroxide tone, and to interaction with the transcription factor NF-E2related factor 2 (Nrf2). Different glutathione-S-transferases, various aldo-keto-reductases, such as Afar, and epoxide hydrolases possess an ARE. In our study the up-regulation of phase II detoxification enzymes by Se deficiency and vice versa the down-regulation with sufficient Se supply were supported by Heme oxygenase 3 regulation, representing another enzyme with an ARE in its DNA. That heme oxygenases respond to Se status is confirmed by the results of other trials, in which an up-regulation of heme oxygenase 1 due to a lack of Se supply was reported[10].

  A, B and C possible chemical reactions of selenite and selenate are summarized[17, 35, 36] which may explain the increased thiol reactivity of dietary selenate in peripheral tissues and represent one essential reason for its greater potential in influencing differential gene expression. The metabolic fates of different Se compounds still need intensive investigation using Se species analysis and protein mass spectrometry. Antidiabetic and insulin sensitizing effects of Se, restricted exclusively to the intermittent application of very high selenate doses, are based on the inhibition of Protein tyrosine phosphatase 1B (PTP1B). According to Figure 4 B and C selenious acid formation and superoxide anion generation during the peripheral reduction of larger selenate quantities provide a good example of the high reactivity of oral selenate towards cysteine residues in proteins such as PTP1B. For the interpretation of future data from Se studies, one should consider that orally Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.properties of selenite applied in cell cultures and in other in vitro studies [11, 17, 23].In our present MA experiment we have analyzed a distinct up-regulation of PTP1B mRNA due to Se supplementation at the recommended dietary level and at a slightly supranutritive concentration. PTP1B has recently been discussed as being a promising target in the treatment of obesity, insulin resistance and diabetes. Moreover, the enzyme is involved in growth and development of mammary tumours and in the activation of lipogenesis. Via the reduction of tyrosine phosphorylation of key signalling proteins such as IRS1 and Akt, an increased expression and activity of PTP1B may force the development of insulin resistance, obesity, hyperlipidemia, and diabetes[37, 38]. It may also be critical with regard to the growth of mammary cancer[39]. In contrast the reduced Akt phosphorylation mediated by increased PTP1B activity through dietary Se supplementation has been investigated as one mechanism explaining possible beneficial Se effects in prostate cancer treatment[12]. With regard to metabolic disorders recent large cross sectional studies and Se intervention trials support the PTP1B hypothesis by the finding that a significant positive correlation exists between Se status and the incidence of type II diabetes and increased blood lipids[15, 19- 21]. In this context the detailed regulation of PTP1B by dietary Se has recently been investigated by our group[22].

Bioscience

  Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089 A c c e p t e d M a n u s c r i p t From the comparison of gene expression related to Se deficiency, our data suggest that Se and, in particular, selenate is effective in influencing many other genes in the mammalian organism. Thus the regulation of genes that are also expressed in the liver and have specific functions for the integrity of the heart muscle and the skeletal muscle (e.g., Cardiac ankyrin repeat protein, Fibroblast Growth factor 13, Glial cell line-derived neurotrophic factor receptor beta, Mu-calpain large subunit, Tissue inhibitor of metalloproteinase 3, Titin, and Winglesstype MMTV integration site family) [51-56] may help to gain new insight into the beneficial effects of Se in the prevention of muscular dystrophy (

  Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089 A c c e p t e d M a n u s c r i p t

  Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089 A c c e p t e d M a n u s c r i p t

  FIGURE 1 A

  FIGURE 2 A

FIGURE 3 Figure 3

 33 FIGURE 3

  ), and (8) in selenate metabolism, starting from hydrogen selenide are identical with the pathways (2), (3), and (4) in selenite metabolism.*in Figure4A and B indicate that in selenite metabolism only one pathway leads to the formation of glutathionylated (inactivated) protein intermediates with SH-group containing proteins, whereas this modification can be formed by two pathways during selenate metabolism.C) Evidence for the higher thiol reactivity of selenate compared to selenite from diabetic dbdb mice, treated with supranutritional selenite-and selenate dosesThe higher protein glutathionylation effected by orally applied selenate compared to selenite, marked with* in Figure4A and B can clearly be proven by Western Blot Analysis using an "anti glutathione antibody". The Western Blot shows a distinctly higher glutathionylation of PTP1B, a protein with a catalytically active cystein SH-residue, in dbdb mice treated with supranutritional selenate compared to their selenite treated companions [unpublished results from 17]. Thus this reaction is clear proof of the higher peripheral thiol reactivity of selenate. Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089 A c c e p t e d M a n u s c r i p t

  ). Groups I and II were fed diets adequate in Se (0.20 mg Se/kg diet) either supplemented with sodium selenite (group I) or sodium selenate (group II). The diets of groups III and IV were supplemented with sodium selenite or sodium selenate to a supranutritive Se concentration of 1.0 mg Se/kg diet. The rats were kept individually in plastic cages with wood shavings as bedding under standard laboratory conditions (22°C, 55%

Validation of the MA data for selected differentially expressed genes (GPx1, GPx4, GSTa2, PTP1B) by two step reverse transcriptase PCR (2-step RT-PCR)

	For GPx1, GPx4, GSTa2 and PTP1B the validation of the MA data was carried out using 2-
	step reverse transcriptase PCR (2-step RT-PCR). The expression data were normalized to β
	actin expression. The 2-step RT-PCR method applied was elaborated and refined in our
	laboratory, and was described in detail previously [22]. New liver RNA pools were prepared
	for the RT-PCR experiments from the same 5 rats per group to exclude casual and
	systematic errors of the MA expression data. The gene bank accession numbers, the PCR
	primers used, and the length of the amplificates were as follows: Rat GPx1: NM 030826,
	primer forward: TCA TTG AGA ATG TCG CGT CT, primer reverse: CCC ACC AGG AAC
	TTC TCA AA, amplificate length: 388 bp; Rat GPx4: NM: 017165, primer forward: ATG CAC
	GAA TTC TCA GCC AAG, primer reverse: GGC AGG TCC TTC TCT AT, amplificate length:
	461 bp; Rat GSTa2: NM 0170131, primer forward: CAG GAG TGG AGT TTG ATG AGA,
	primer reverse: AGA GGG AAA GAG GTC AGA AG, amplificate length: 463 bp; Rat PTP1B:
	NM 012637, primer forward: GCA CTT CTG GGA GAT GGT GT, primer reverse: AAG AGG
	AAA GAC CCG TCC TC, amplificate length: 449; Rat β actin: NM 0311441, primer forward:
	TGT TAC CAA CTG GGA CGA CA, primer reverse: TCT CAG CTG TGG TGG TGA AG,
	amplificate length: 396 bp.

  to reduce excess sodium periodate to sodium iodide. 500 µL supernatant of this reaction were incubated at 70°C with 50 µL Schiffs reagent to form a red magenta dye. The extinction was read in a spectrophotometer at 560 nm and compared with a calibration curve prepared with 1-phenyl-1,2-ethanediol. 1U of epoxide hydrolase was defined as the formation of 1 µmol vicinal diol/min and related to 1 mg protein.

		Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089
		corresponding aldehyde and Statistical analysis
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089	A c c e p t e d M Data are given as means±standard deviation. For the enzymatic analyses and for the RT-PCR data, statistical differences between means (P<0.05) were analysed with "SPSS 14.0 for Windows" by one way ANOVA after ascertaining the normality of distribution (Shapiro Wilk test and Kolmogorov Smirnov test) and homogeneous variances (Levene test). Differences between means were analyzed with the LSD test if variances were a n u s c r i p t homogeneous or with the Games Howell test if not. Changes in gene expression (MA data)
		step, 45 µL of the liver homogenates were incubated in 630 µL of 50 mM phosphate buffer
		containing 10% DMSO and 10 mmol/L of the substrate styrene oxide for 2 h at 36°C. This
		reaction step allows the formation of the vicinal diol from the epoxide by the activity of
		epoxide hydrolase. Subsequently the samples were treated with 200 µL 100 mM sodium
		periodate and 135 µL 0.8 M sodium sulphite in order to oxidize the vicinal diol to the
		6

Table 2

 2

the final ratios originate from different regulatory effects (for details see table

legend

).

Table 1

 1

	increased risk of nonmelanoma skin cancer. In a smaller subtrial of the NPC study in which
	the participants received even 400 µg Se/day the cancer protective effect of the 200 µg dose
	was lost [16]. In contrast the Selenium and Vitamin E Cancer Prevention Trial (SELECT) in
	which healthy men were supplemented either with 200 µg Se/day in the form of
	selenomethionine or with vitamin E (400 I.U./day) or with a combination of both supplements
	compared to a placebo group was abandoned prematurely in autumn 2008 since the Se
	supplemented participants showed a 5% higher incidence of prostate cancer [15]. Due to the
	fact that tumour cells frequently possess a changed apoptosis program, the long term use of
	supranutritional Se supplements in cancer prevention and treatment should be reconsidered
	as well. This position is corroborated by our microarray data indicating both pro-and anti-
	apoptotic features of Se by modulation of the expression of NAPOR 3, BCL2-antagonist 1,

, area II) in particular indicates additional pathways by which Se may promote the development of insulin resistance, obesity, hyperlipidaemia and diabetes beside PTP1B regulation. Both the down-regulation of 80-k diacylglycerolkinase and the up-regulation of the Agouti related protein precursor, the Hypocretin orexin receptor and of the Neuropeptide y precursor open perspectives for future research concerning the role of Se in metabolic diseases [15, 18-21].

As mentioned above our data for the comparison of Se supplementation versus Se deficiency suggest a possible preventive mechanism of Se with regard to prostate cancer.

Similarly the regulation of genes involved in growth and differentiation of mammary cancer and lung cancer (ADAMTS-1), of colorectal cancer (PKCQ-interacting protein Picot), as well as of various cancer types (Proliferin related protein) indicated a cancer protective function of Se (Table

1

, area III)

[40][41][42]

. However these effects did not increase by increasing Se supplementation from the recommended dietary amount to the supranutritive level. The comparison of selenite-and selenate-regulated genes indicated a possible additional role of selenate with regard to the prevention of hepatocellular cancer (Alphab crystalline-related protein = heat shock protein 20, Table

2

)

[43]

.

Large human intervention trials carried out to investigate the preventive role of long term Se supplement use in cancer prevention revealed inconsistent results. Thus in the Nutritional Prevention of Cancer (NPC) study in which 1,312 high-risk dermatology patients received 200 µg Se/day in the form of selenized yeast or a placebo (matched control), a decreased risk of lung, colon, prostate, and total cancers was obtained by Se intervention, but an FOS related antigen, Granzyme k, Mast cell protease, and p58 (Table

1

, area III)

[44][45][46][47][48][49]

.

Very recent findings suggest that Se supplementation for cancer prevention is of particular interest for humans with selenoprotein P polymorphisms since this genetic constellation may reduce Se transfer to the peripheral organs and the subsequent sufficient synthesis of antioxidant selenoproteins

[50]

.

Table 1 ,

 1 area V). Similarly, the established effectiveness of Se in preventing liver necrosis is supported by the up-regulation of Alpha 7 c integrin, Mitogen activated protein kinase 12 ZPK, Plectin 1 (Table1, area III)

Table 1 Continued 612

 1612

	Gene Bank Assoc	Gene name	Selenite0.2	Selenite1.0	Selenate0.2	Selenate1.0
	Number		(P value)	(P value)	(P value)	(P

value) Area III: Genes related to the cell cycle, cell development, apoptosis or tumour metabolism

	NM053812	BCL 2 Antagonist 1	1.37↑*	1.72↑*	2.81↑	3.16↑
				(0.517)	(0.349)	(0.011)	(0.010)
	Z12298	Dermatan	Sulfate	2.87↑*	2.57↑*	3.98↑	3.63↑
		Proteoglycan II	(0.225)	(0.284)	(0.009)	(0.011)
	NM030585	FOS Related Antigen	3.51↑*	3.27↑*	6.54↑	4.67↑
				(0.122)	(0.186)	(0.009)	(0.004)
	NM017119	Granzyme K		1.79↓*	1.13↓*	2.48↓	2.46↓
				(0.071)	(0.731)	(0.005)	(0.009)
	NM017145	Mast Cell Protease 1	1.77↑*	2.47↑*	3.72↑	2.73↑
				(0.488)	(0.274)	(0.013)	(0.040)
	NM013055	Mitogen Protein Kinase 12 ZPK Activated	2.32↑* (0.351)	1.99↑* (0.357)	4.03↑ (0.050)	3.48↑ (0.040)
	U15425	P58		1.42↑*	1.81↑*	2.85↑	2.09↑
				(0.625)	(0.087)	(0.007)	(0.036)
	NM032614	PKCQ	Interacting	1.21↓*	1.03↓*	2.30↓	2.08↓
		Protein Picot		(0.582)	(0.914)	(0.006)	(0.008)
	U96275	Plectin 1		2.00↑*	3.28↑*	5.14↑	5.54↑
				(0.296)	(0.070)	(<0.001)	(<0.001)
	NM053364	Proliferin	Related	2.33↑*	2.68↑*	4.15↑	3.61↑
		Protein		(0.275)	(0.228)	(0.002)	(0.002)
	NM019906	Rapamycin	And	2.43↑*	2.95↑*	3.84↑	3.84↑
		FKBP12 Target-1	(0.095)	(0.174)	(0.010)	(<0.

001) Area IV: Genes related to inflammatory or immunomodulatory response

	NM021583	Ind.	Prostaglandine	1.73↑*	2.64↑*	3.33↑	4.58↑
		Synthase	(0.314)	(0.074)	(0.023)	(<0.001)
	NM057193	Interleukin 10 Receptor	1.94↑*	2.67↑*	2.81↑	2.84↑
				(0.220)	(0.065)	(0.010)	(0.009)
	NM020542	Macrophage Inflamm. Protein 1 Alpha Rec.	1.48↓* (0.299)	1.33↓* (0.485)	3.17↓ (0.004)	3.86↓ (0.003)
	M34097	Rat Natural Killer Cell Protease 1	1

.87↑* (0.334) 2.40↑* (0.018) 2.59↑ (0.005) 3.75↑ (0.004) Area V: Genes with other functions (hormonal function, receptor function, signal transduction) NM013220 Cardiac Ankyrin Repeat Protein 2.00↓* (0.115) 1.46↓* (0.319) 3.05↓ (0.043) 2.92↓ (0.045) S78641

		Cycl. Adenosine 3',5'-	1.86↑*	1.53↑*	3.29↑	2.58↑
		Monophosphate Resp. Elem. Binding Protein	(0.421)	(0.451)	(0.018)	(0.020)
	NM053428	Fibroblast	Growth	2.80↑*	3.14↑*	4.63↑	4.09↑
		Factor 13			(0.129)	(0.078)	(<0.001)	(<0.001)
	NM012750	Glial Cell Line Der.	1.57↑*	2.41↑*	3.88↑	3.66↑
		Neurotrophic Rec. Beta	Factor	(0.524)	(0.332)	(0.006)	(0.019)
	NM019152	MU	Calpain	Large	1.53↑*	2.69↑*	4.00↑	4.55↑
		Subunit			(0.430)	(0.227)	(0.013)	(0.006)
	NM012886	Tissue	Inhibitor	of	1.90↑*	4.44↑	3.76↑	2.68↑
		Metalloproteinase 3		(0.358)	(0.046)	(0.011)	(0.045)
	AJ401157 (Table 2)	Titin				1.00↑*	1.53↑*	2.72↑	3.62↑
						(0.999)	(0.204)	(0.016)	(0.012)
	NM053402	Wingless Type MMTV	2.91↑*	6.16↑	3.51↑	5.44↑
		Integration Site Family	(0.188)	(0.011)	(0.039)	(0.008)

*

indicate that selenite supplementation regulated most genes into the same direction as selenate 613 supplementation (up↑/down↓), but that these effects were not significant (P<0.05); genes occurring 614 additionally in Table 2 are highlighted on a grey background and a reference remark to Table 2 is given 615

  Bioscience Reports Immediate Publication. Published on 14 Aug 2009 as manuscript BSR20090089

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090089	A c c e p t e d M a n u s c r i p t

Table 2 Selection of genes differentially regulated in rat liver comparing selenate 616 feeding with selenite feeding at both dietary levels (0.2 and 1.0 mg/kg) 617 Gene Bank Assoc Number Gene name Selenate vs. Selenite [both levels] (P value) [Selenate0.2 vs. Selenite 0.2] [Selenate1.0 vs. Selenite1.0]

 2

	Selenite0.2	Selenite1.0	Selenate0.2	Selenate1.0
	vs.	vs.	vs.	vs.
	0Se	0Se	0Se	0Se
	(P value)	(P value)	(P value)	(P value)

Table 1 )

 1

	Titin	2.50↑	1.00↑	1.53↑	2.72↑	3.61↑
		(0.013)	(0.999)	(0.204)	(0.016)	(0.012)
		[2.72↑]
		[2.36↑]
	*D25543
	(

Table 1 )

 1

		Rat gcp360	2.67↑	1.59↑	2.26↑	4.67↑	5.64↑
			(0.004)	(0.498)	(0.178)	(<0.001)	(<0.001)
			[2.92↑]
			[1.74↑]
	AF100789	G protein-coupled receptor gpr55	2,74↑ (0.002)	1.37↓ (0.501)	1.27↑ (0.639)	2.61↑ (0.015)	2.86↑ (0.008)
			[3.59↑]
			[2.24↑]
	*D29960
	(

Table 1 )

 1

		Alphab crystallin-related protein	2.85↑ (0.004)	1.90↑ (0.219)	1.59↑ (0.453)	5.85↑ (0.002)	4.10↑ (0.005)
			[3.07↑]
			[2.57↑]
	S64315	Metabotropic glutamate	3
		receptor subtype
		5b

.72↑ (0.047) [6.82↑] [1.81↑] 1.45↓ (0.407) 1.18↑ (0.843) 4.68↑ (0.006) 2.20↑ (0.002)

  Genes regulated in an analogous manner by selenate and selenite are highlighted with * and appear on a dark grey 618 background. Moreover a reference to Table 1 is given. Genes regulated in a contrary manner by selenate and selenite are 619 highlighted with ** and appear on a light grey background. Genes regulated indifferently by selenite, but consistently by selenate

	NM024365	5-hydroxy-tryptamine receptor 6	3.81↑ (0.006)	2.04↑ (0.214)	1.10↓ (0.887)	4.14↑ (0.005)	7.12↑ (<0.001)
			[2.02↑]
			[7.84↑]
	**NM05378 1	Aldose-reductase-like (akr1-b7)	5.14↑ (0.014)	1.13↓ (0.823)	1.36↓ (0.637)	3.75↑ (0.017)	4.56↑ (0.007)
			[4.24↑]
			[6.20↑]
	**U56863 Ly-49.9 antigen	7.92↑ (0.003)	1.49↓ (0.296)	3.78↓ (0.033)	3.23↑ (0.004)	4.16↑ (0.020)
			[4.81↑]
			[15.8↑]

Table 3 Hierarchy of functional selenoproteins in rat liver as analyzed by microarray analysis Selenoprotein Selenite 0.2 vs. 0 Se Selenate 0.2 vs. 0 Se Selenite 1.0 vs. 0 Se Selenate 1.0 vs. 0 Se

 3

	Mean P
	value for
	extent of
	regulation

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

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