The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was timeand dose-dependent, with an apparent K m of 22 M and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.

The orientation dependence of the backscattered yield of 1.0-MeV helium ions has been used to investigate the lattice characteristics of silicon and germanium implanted at room temperature with 40-keV heavy ions (Ga, As, Sb, In, P). The method gives directly the number of substrate atoms displaced from their lattice sites by more than the Thomas–Fermi screening distance (≈ 0.2 Å) and also the location of the implanted atoms.Each heavy ion is found to displace several thousand lattice atoms in a localized region around its track. At doses where the individual disordered regions do not overlap (~1013 ions/cm2, or less), the silicon lattice reorders at 260 °C and the germanium lattice at 180 °C. At doses greater than ~1014 ions/cm2, a completely amorphous layer is formed in both silicon and germanium. During annealing, the reordering of the lattice is initiated at the interface between the amorphous region and the underlying crystal. The reordering of this amorphous layer occurs at significantly higher temperatures (570 °C in Si and 380 °C in germanium) than those required for the isolated disordered regions. At doses much greater than 1015/cm2, even higher anneal temperatures are required.Following a room-temperature implantation, each implanted atom is embedded in a highly disordered region (at least, for doses > 1014/cm2) and consequently the backscattered spectra from these atoms do not show orientation effects. As the lattice reorders during annealing, the number of implanted atoms lying along the [Formula: see text] lattice row increases. Following anneal, ~75% of the As atoms and ~90% of the Sb atoms are found on substitutional sites in silicon, while in germanium, ~80% of the In atoms are found on substitutional sites. Annealing to higher temperatures (700–900 °C in the case of silicon) often causes the number of substitutional atoms to decrease again, owing to the effect of internal precipitation or diffusion to the surface.

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxRoverexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1-or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.Thioredoxin reductases (TrxRs) 1 are members of the nucleotide-disulfide oxidoreductase family and are ubiquitously found in mammalian tissues. Two main isoforms of thioredoxin reductases exist in mammals: the classical cytosolic form (TrxR1, the TXNRD1 gene product) (1, 2), and the mitochondrial form (TrxR2) (3, 4). A third form, thioredoxin and glutathione reductase, is mainly expressed in testis (5). All mammalian TrxRs are homodimeric selenocysteine-containing enzymes (6) that share high sequence homology between each other and contain an NADPH binding domain and a FAD binding domain per subunit (7). TrxRs reduce and thereby activate thioredoxins (Trxs), which are small ubiquitous proteins with a conserved active site sequence (-Trp-Cys-Gly-ProCys-) that catalyze many redox reactions through the reversible oxidation of the active site dithiol to a disulfide. Trxs are involved in a variety of reactions, such as redox regulation of transcription factors (8, 9), reduction of hydroperoxides (4, 10), and modulation of cell growth (11) and apoptosis (12, 13). The selenocysteine residue (Sec) that is found at the C terminus in TrxRs (-Gly-Cys-Sec-Gly-COOH) is encoded by a UGA codon that is normally identified as a stop codon, but which can be transcribed as Sec in the presence of a selenocysteine insertion sequence (SECIS) element in the 3Ј-UTR of the corresponding mRNA (14). The C-terminal Cys-Sec motif is located on a presumably flexible arm of the enzyme that is kept reduced by the N-terminal redox active motif (-Cys-Val-Asn-Val-Gly...

Insulin-like growth factor I (IGF-I) and IGF-II are peptides that presumably are required for normal fetal and postpubertal growth. The production of IGFs is developmentally regulated and the liver appears to be a major site of production. By analysing mRNA levels for IGF-I and IGF-II in the rat liver we have attempted to further study the expression of these growth factors during development and regeneration as well as during the course of hepatic carcinogenesis. Fetal livers are characterized by a high level of IGF-II mRNA and a low level of IGF-I mRNA, while in adult livers the opposite situations occur, i.e. a high level of IGF-I mRNA and a non-measurable level of IGF-II mRNA. During the course of experimentally induced hepatic cancer, IGF-I mRNA was consistently reduced and in a majority of cancers analysed (6/9) IGF-II mRNA was increased, i.e. a fetal type of IGF expression can be switched on in some experimentally induced hepatocellular carcinomas. The onset of IGF-II production during hepatic carcinogenesis appears to be a late phenomenon since liver nodules, preceding the development of hepatocellular cancer, were found not to contain IGF-II mRNA. Furthermore, during hepatic regeneration following partial hepatectomy no marked change in IGF-I or IGF-II mRNA levels was noted. The above results suggest that the fetal growth factor IGF-II could have a role in hepatic cancer.

Activation of the activator protein 1 (AP-1) transcription factor as well as increased serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-8 predict poor prognosis of patients with hepatocellular carcinomas (HCCs). Moreover, HCC patients display reduced selenium levels, which may cause lipid peroxidation and oxidative stress because selenium is an essential component of antioxidative glutathione peroxidases (GPx). We hypothesized that selenium-lipid peroxide antagonism controls the above prognostic markers and tumor growth. (1) In human HCC cell lines (HCC-1.2, HCC-3, and SNU398) linoleic acid peroxide (LOOH) and other prooxidants enhanced the expression of VEGF and IL-8. LOOH up-regulated AP-1 activation. Selenium inhibited these effects. This inhibition was mediated by glutathione peroxidase 4 (GPx4), which preferentially degrades lipid peroxides. Selenium enhanced GPx4 expression and total GPx activity, while knock-down of GPx4 by small interfering RNA (siRNA) increased VEGF, and IL-8 expression. (2) These results were confirmed in a rat hepatocarcinogenesis model. Selenium treatment during tumor promotion increased hepatic GPx4 expression and reduced the expression of VEGF and of the AP-1 component c-fos as well as nodule growth. (3) In HCC patients, increased levels of LOOH-related antibodies (LOOH-Ab) were found, suggesting enhanced LOOH formation. LOOH-Ab correlated with serum VEGF and IL-8 and with AP-1 activation in HCC tissue. In contrast, selenium inversely correlated with VEGF, IL-8, and HCC size (the latter only for tumors smaller than 3 cm). Conclusion: Reduced selenium levels result in accumulation of lipid peroxides. This leads to enhanced AP-1 activation and consequently to elevated expression of VEGF and IL-8, which accelerate the growth of HCC. Selenium supplementation could be considered for investigation as a strategy for chemoprevention or additional therapy of early HCC in patients with low selenium levels. (HEPATOLOGY 2012;55:1112-1121 H epatocellular carcinoma (HCC) is one of the most common cancers worldwide. Solid tumor growth depends on neoangiogenesis. Microvessel density within the tumor is an independent prognostic marker and predictor of HCC recurrence.1 A variety of cytokines is involved in neoangiogenesis, but clinical relevance in HCC was shown only for interleukin (IL)-8 and vascular endothelial Abbreviations: 2-AAF, 2-acetylaminofluoren; AP-1, activator protein 1; CXCL1, chemokine (C-X-C motif) ligand 1; DEN, diethylnitrosamin; DHFC, 2 0 ,7 0 -dichlorofluorescin diacetate; IL-8, interleukin 8; HCC, hepatocellular carcinoma; HIF-1a, hypoxia inducible factor 1a; LH, linoleic acid; LOOH, linoleic acid hydroperoxides; LOOH-Ab, linoleic acid hydroperoxide related antibodies; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.From the

Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min 21 in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mm and was even further increased at 0.5 mm. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP 1 ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.

Previous studies in animals and humans have shown that selenium compounds can prevent cancer development. In this work we studied the tumor preventive effect of selenium supplementation, administrated as selenite, in the initiation, promotion and progression phases in a synchronized rat model for chemically induced hepatocarcinogenesis, the resistant hepatocyte model. Selenite in supra-nutritional but subtoxic doses (1 and 5 p.p.m.) was administrated to the animals through the drinking water. Such supplementation during the initiation phase did not have a tumor preventive effect. However, selenite treatment during the promotion phase decreased the volume fraction of pre-neoplastic liver nodules from 38% in control animals to 25 (1 p.p.m.) and 14% (5 p.p.m.) in the selenite-supplemented groups. In addition the cell proliferation within the nodules decreased from 42% in the control to 22 (1 p.p.m.) and 17% (5 p.p.m.). Immunohistochemical staining for the selenoenzyme thioredoxin reductase 1 revealed an increased expression of the enzyme in liver nodules compared with the surrounding tissue. The activity was reduced to 50% in liver homogenates from selenium-treated animals but the activity of the selenoenzyme glutathione peroxidase was essentially unaltered. Selenite treatment (5 p.p.m.) during the progression phase resulted in a significantly lower volume fraction of liver tumors (14 compared with 26%) along with a decrease in cell proliferation within the tumors (34 compared with 63%). Taken together our data indicate that the carcinogenetic process may be prevented by selenium supplementation both during the promotion and the progression phase.

The implantation behavior of S b in silicon has been investigated by studying the orientation dependence of the baclcscattering yield of a 1.0-MeV helium bearn. This orientation dependence ("clianneling") provides quantitative information on the location of iinplanted atoms in the lattice and also on the ainount of lattice disorder accompanying the implantation. The general principles of the channeling technique and the advantages of using a beam of I-Ie+ rather than I-I+ are discussed. Results for 25 "C and 450 "C irnplantations are presented and compared. The hot implant introduces very little lattice disorder, and a large fraction (up t o 95%) of the S b atoms is found on substitutional sites. I11 the 25 "C implants, however, a heavily damaged region is formed around the traclc of each iinplanted Sb. At doses greater than -10" ions/cm2, these damaged regions overlap to produce an essentially a m o r p h o~~s layer, which then requires an annealing temperature of -650 "C t o reorder the lattice and to achieve a high level of substitutional Sb. "I-Iot implant" studies have also been made for several other dopants-Ga, As, In, and -Ye; however, unlilce Sb, the substit~~tional level in all these cases never exceeded 60%. This unique behavior of Sb implants in silicon has also been found in a corresponding study of the electrical characteristics (Paper 11). A preliminary investigation of boron-doped silicon, sing the orientation dependence of 610-1ceV protons, indicates that substitutional boron atoms are removed from lattice sites by relatively small doses of energetic protons. This enhanced sensitivity t o radiation does not occur for the Sb-doped samples.