An increase in O-GlcNAcylation of Sp1 down-regulates the gene expression of pi class glutathione S-transferase in diabetic mice.

Oxidative stress is a key factor contributing to the development of diabetes complications. Glutathione S-transferases (GSTs) protect against products of oxidative stress by conjugating glutathione to electrophilic substrates, producing compounds that are generally less reactive and more soluble. The expression and activity of GSTs during diabetes have been extensively studied, but little is known about regulation mechanisms of Pi-class GST (GSTP). The aim of the present study was to evaluate how GSTP is regulated in a Streptozotocin (STZ)-induced murine diabetes model. GST activity and GSTP expression were determined in adult male mice diabetized with STZ. Specificity protein 1 (Sp1) expression and O-glycosylation, as well as the role of AP-1 members Jun and Fos in the regulation of GSTP expression, were also assessed. The results showed that GST total activity and GSTP mRNA and protein levels were decreased in the diabetic liver, and returned to normal values after insulin administration. The insulin-mimetic drug vanadate was also able to restore GST activity, but failed to recover GSTP mRNA/protein levels. In diabetic animals, O-glycosylated Sp1 levels were increased, whereas, in insulin-treated animals, glycosylation values were similar to those of controls. After vanadate administration, Sp1 expression levels and glycosylation were lower than those of controls. Our results suggest that hyperglycemia could lead to the observed increase in Sp1 O-glycosylation, which would, in turn, lead to a decrease in the expression of Sp1-dependent GSTP in the liver of diabetic mice.

Effects of volatile anaesthetics on heme metabolism in a murine genetic model of Acute Intermittent Porphyria. A comparative study with other porphyrinogenic drugs.

BACKGROUND: Acute Intermittent Porphyria (AIP) is an inherited disease produced by a deficiency of Porphobilinogen deaminase (PBG-D). The aim of this work was to evaluate the effects of Isoflurane and Sevoflurane on heme metabolism in a mouse genetic model of AIP to further support our previous proposal for avoiding their use in porphyric patients. A comparative study was performed administering the porphyrinogenic drugs allylisopropylacetamide (AIA), barbital and ethanol, and also between sex and mutation using AIP (PBG-D activity 70% reduced) and T1 (PBG-D activity 50% diminished) mice. METHODS: The activities of 5-Aminolevulinic synthetase (ALA-S), PBG-D, Heme oxygenase (HO) and CYP2E1; the expression of ALA-S and the levels of 5-aminolevulinic acid (ALA) were measured in different tissues of mice treated with the drugs mentioned. RESULTS: Isoflurane increased liver, kidney and brain ALA-S activity of AIP females but only affected kidney AIP males. Sevoflurane induced ALA-S activity in kidney and brain of female AIP group. PBG-D activity was further reduced by Isoflurane in liver male T1; in AIP male mice activity remained in its low basal levels. Ethanol and barbital also caused biochemical alterations. Only AIA triggered neurological signs similar to those observed during human acute attacks in male AIP being the symptoms less pronounced in females although ALA-S induction was greater. Heme degradation was affected. DISCUSSION: Biochemical alterations caused by the porphyrinogenic drugs assayed were different in male and female mice and also between T1 and AIP being more affected the females of AIP group. GENERAL SIGNIFICANCE: This is the first study using volatile anaesthetics in an AIP genetic model confirming Isoflurane and Sevoflurane porphyrinogenicity.

Effects of iron polymaltose complex, ferrous fumarate and ferrous sulfate treatments in anemic pregnant rats, their fetuses and placentas.

Although oral iron preparations are widely prescribed to prevent and to treat iron deficiency anemia in pregnancy, comparative data on their effects to the mother, fetus and placenta are limited. In this study, the effects of oral iron polymaltose complex (IPC), ferrous fumarate (FF) and ferrous sulfate (FS) were compared in anemic pregnant rats, their fetuses and placentas. Hematological variables and oxidative stress markers in the liver, heart and kidneys of the dams and fetuses as well as the markers for oxidative stress, inflammation and hypoxia in placentas were assessed. Pregnancy outcome was measured by number of fetuses, and by neonate and placental weight. All therapies were comparably effective in correcting anemia. FS and FF, but not IPC, resulted in liver damage in dams and oxidative stress in dams, fetuses and placentas. FS group presented the highest catalase and GPx levels in dams, fetuses and placentas. IPC, but not FF or FS, restored normal TNF-α and IL6 expression levels in placentas whereas FS-treated animals presented the highest cytokine levels, suggesting a local inflammatory reaction. Anemia-induced high levels of HIF-1α were partially lowered by IPC and FF but further elevated by FS. Most of the negative effects associated with IDA were resolved by IPC treatment. Especially FS treatment was found to elicit hepatic damage in the dams, oxidative stress in the dams, fetuses and placenta as well as inflammation and high levels of HIF-1α in the placenta. Pregnancy outcome of FFand FS-treated animals was worse than that of IPC-treated animals.

Comparison of oxidative stress and inflammation induced by different intravenous iron sucrose similar preparations in a rat model.

Iron sucrose originator (IS(ORIG)) has been used to treat iron deficiency and iron deficiency anemia for decades. Iron sucrose similars (ISSs) have recently entered the market. In this non-clinical study of non-anemic rats, five doses (40 mg iron/kg body weight) of six ISSs marketed in Asian countries, IS(ORIG) or saline solution (control) were administered intravenously over four weeks to compare their toxicologic effects. Vasodilatory effects, impaired renal function and hepatic damage were only observed in the ISS groups. Significantly elevated serum iron and transferrin saturation levels were observed in the ISS groups suggesting a higher release of iron resulting in higher amounts of non-transferrin bound (free) iron compared to IS(ORIG). This might explain the elevated oxidative stress and increased levels of inflammatory markers and antioxidant enzymes in the liver, heart and kidneys of ISS-treated animals. Physico-chemical analyses showed that the molecular structure of most of the ISSs differed greatly from that of the IS(ORIG). These differences may be responsible for the organ damage and oxidative stress observed in the ISS groups. Significant differences were also found between different lots of a single ISS product. In contrast, polarographic analyses of three different IS(ORIG) lots were identical, indicating that the molecular structure and thus the manufacturing process for IS(ORIG) is highly consistent. Data from this study suggest that ISSs and IS(ORIG) differ significantly. Therefore, before widespread use of these products it would be prudent to evaluate additional non-clinical and/or clinical data proving the safety, therapeutic equivalence and interchangeability of ISSs with IS(ORIG).

ALAS1 gene expression is down-regulated by Akt-mediated phosphorylation and nuclear exclusion of FOXO1 by vanadate in diabetic mice.

Porphyrias are diseases caused by partial deficiencies of haem biosynthesis enzymes. Acute porphyrias are characterized by a neuropsychiatric syndrome with intermittent induction of hepatic ALAS1 (δ-aminolaevulinate synthase 1), the first and rate-limiting enzyme of the haem pathway. Acute porphyria attacks are usually treated by the administration of glucose; its effect is apparently related to its ability to inhibit ALAS1 by modulating insulin plasma levels. It has been shown that insulin blunts hepatocyte ALAS1 induction, by disrupting the interaction of FOXO1 (forkhead box O1) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α). We evaluated the expression of ALAS1 in a murine model of diabetes and determined the effects of the insulinomimetic vanadate on the enzyme regulation to evaluate its potential for the treatment of acute porphyria attacks. Both ALAS1 mRNA and protein content were induced in diabetic animals, accompanied by decreased Akt phosphorylation and increased nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. Vanadate reversed ALAS1 induction, with a concomitant PI3K (phosphoinositide 3-kinase)/Akt pathway activation and subsequent reduction of nuclear FOXO1, PGC-1α and FOXO1-PGC-1α complex levels. These findings support the notion that the FOXO1-PGC-1α complex is involved in the control of ALAS1 expression and suggest further that a vanadate-based therapy could be beneficial for the treatment of acute porphyria attacks.

Evaluation of toxicity and oxidative stress induced by intravenous iron isomaltoside 1000 in a nonclinical model.

The physicochemical characteristics of intravenous iron complexes affect the extent of weakly-bound iron and thus the degree of oxidative stress. The new preparation iron isomaltoside 1000 (IIM) was compared to iron sucrose (IS) and a control group in terms of biochemistry, oxidative stress, inflammatory markers and iron deposition in the liver, heart and kidneys of healthy rats. Renal function was significantly impaired in the IIM group versus both IS and controls. Liver enzymes were also significantly higher in IIM-treated animals versus the other groups, indicative of hepatic injury. Systolic blood pressure was significantly lower following IIM administration compared to IS or control animals. Oxidative stress in the liver, heart and kidneys was greater in the IIM group, as indicated by significantly increased levels of malondialdehyde and antioxidant enzyme activity, accompaniedby a significantly lower ratio of reduced to oxidized glutathione. Microscopy demonstrated more extensive positive staining for iron, and a smaller area of ferritin staining, in the liver, heart and kidneys of rats treated with IIM versus IS.Levels of the inflammatory markers TNF-alpha and IL6 were both significantly higher in the IIM group versus IS in all assessed tissues. These findings indicate that IIM has a less favorable safety profile than IS in healthy rats, adversely affecting iron deposition, oxidative stress and inflammatory responses, with impaired liver and renal function.

Assessment of the extent of oxidative stress induced by intravenous ferumoxytol, ferric carboxymaltose, iron sucrose and iron dextran in a nonclinical model.

Intravenous (i.v.) iron is associated with a risk of oxidative stress. The effects of ferumoxytol, a recently approved i.v. iron preparation, were compared with those of ferric carboxymaltose, low molecular weight iron dextran and iron sucrose in the liver, kidneys and heart of normal rats. In contrast to iron sucrose and ferric carboxymaltose, low molecular weight iron dextran and ferumoxytol caused renal and hepatic damage as demonstrated by proteinuria and increased liver enzyme levels. Higher levels of oxidative stress in these tissues were also indicated, by significantly higher levels of malondialdehyde, significantly increased antioxidant enzyme activities, and a significant reduction in the reduced to oxidized glutathione ratio. Inflammatory markers were also significantly higher with ferumoxytol and low molecular weight iron dextran rats than iron sucrose and ferric carboxymaltose. Polarographic analysis suggested that ferumoxytol contains a component with a more positive reduction potential, which may facilitate iron-catalyzed formation of reactive oxygen species and thus be responsible for the observed effects. Only low molecular weight iron dextran induced oxidative stress and inflammation in the heart.

Differences between original intravenous iron sucrose and iron sucrose similar preparations.

Iron sucrose (Venofer; reference) has a good safety record and is prescribed in patients with anaemia and chronic kidney disease worldwide, but various iron sucrose similar (ISS) preparations are now utilized in clinical practice. This study evaluates possible differences between iron sucrose and ISS preparations on haemodynamic and oxidative stress markers in normal rats. 60 male and 60 female Sprague Dawley rats were divided into four groups and assigned to receive commercially available ISS test 1, ISS test 2, reference or isotonic saline solution (control). A single i.v. dose of iron (40 mg/kg) or saline (equivalent volume) was administered after 24 h and every 7 days for 4 weeks. Blood samples were collected for biological assessment of haemoglobin (Hb), serum iron and percentage transferrin saturation (TSAT), and urine samples were collected to investigate creatinine clearance and proteinuria. Animals were sacrificed after receiving an i.v. dose on days 1, 7 and 28, and kidney, liver, and heart homogenates were then collected to determine antioxidant enzyme levels. Tissues were processed using Prussian blue and immmunohistochemistry techniques to identify iron deposits, tissue ferritin and pro-inflammatory markers. Systolic blood pressure was significantly reduced in the ISS groups relative to the reference and control groups after 24 h and on days 7, 14 and 21 (p < 0.05). Creatinine clearance was reduced (p < 0.01) and proteinuria marked (p < 0.01) in the ISS groups at 24 h and on days 7 and 28 relative to the reference and control groups which did not differ throughout the study. Liver enzymes were also increased in the ISS groups at 24 h and on days 7 and 28. Both ISS test 1 and ISS test 2 groups presented a significant increase in catalase, thiobarbituric reactive species, Cu, Zn-superoxide dismutase (CuZnSOD) and glutathione peroxidase activity, and a decrease in glutathione levels (p < 0.01) in the liver, heart and kidney at 24 h and on day 7 relative to the reference and control groups. Serum iron and percentage TSAT were elevated in all groups (except control) (p < 0.01) but no differences in Hb concentration were observed between them. Finally, levels of the proinflammatory markers TNF-alpha and IL6 were significantly elevated in the ISS groups (liver, heart and kidney) compared with the reference and control groups on day 28 (p < 0.01). These findings suggest significant differences between the reference and ISS test 1/ISS test 2 regarding oxidative stress and the inflammatory responses of liver, heart and kidneys in normal rats. A possible explanation for these observations could be the stability of the iron complex.