Paola I. Ingaramo

Hyperglycemia induces apoptosis in rat liver through the increase of hydroxyl radical: new insights into the insulin effect

In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated by hyperglycemia through the Bax-caspase pathway and the effects of insulin protection against the apoptosis induced by hyperglycemia. Male adult Wistar rats were randomized in three groups: control (C) (sodium citrate buffer, i.p.), streptozotocin (STZ)-induced diabetic (SID) (STZ 60 mg/kg body weight, i.p.), and insulin-treated SID (SID+I; 15 days post STZ injection, SID received insulin s.c., twice a day, 15 days). Rats were autopsied on day 30. In liver tissue, diabetes promoted a significant increase in hydroxyl radical production which correlated with lipid peroxidation (LPO) levels. Besides, hyperglycemia significantly increased mitochondrial BAX protein expression, cytosolic cytochrome c levels, and caspase-3 activity leading to an increase in apoptotic index. Interestingly, the treatment of diabetic rats with desferoxamine or tempol (antioxidants/hydroxyl radical scavengers) significantly attenuated the increase in both hydroxyl radical production and in LPO produced by hyperglycemia, preventing apoptosis by reduction of mitochondrial BAX and cytosolic cytochrome c levels. Insulin treatment showed similar results. The finding that co-administration of antioxidants/hydroxyl radical scavengers together with insulin did not provide any additional benefit compared with those obtained using either inhibitors or insulin alone shows that it is likely that insulin prevents oxidative stress by reducing the effects of hydroxyl radicals. Importantly, insulin significantly increased apoptosis inhibitor protein expression by induction of its mRNA. Taken together, our studies support that, at least in part, the hydroxyl radical acts as a reactive intermediate, which leads to liver apoptosis in a model of STZ-mediated hyperglycemia. A new anti-apoptosis signal for insulin is shown, given by an increase of apoptosis inhibitor protein.

Tumor necrosis factor alpha pathways develops liver apoptosis in type 1 diabetes mellitus.

We analyzed the contribution of TNF-α intracellular pathway in the development of apoptosis in the liver of streptozotocin-induced diabetic rats. In liver tissue, diabetes promoted a significant increase of TNF-α/TNF-R1, and led to the activation of caspase-8, of nuclear factor kappa B (NFκB), and JNK signaling pathways. The activation of NFκB led to an induction of iNOS and consequent increase in NO production. As a consequence of such changes a significant increase of caspase-3 activity and of apoptotic index were observed in the liver of diabetic animals. Importantly, the treatment in vivo of diabetic rats with etanercept (TNF-α blocking antibody) or aminoguanidine (selective iNOS inhibitor) significantly attenuated the induction of apoptosis by reduction of caspase-3 activity. Overall, we demonstrated that in the diabetes enhances TNF-α in the liver, which may be a fundamental key leading to apoptotic cell death, through activation of caspase-8, NFκB and JNK pathways.

Interferon-α2b and transforming growth factor-β1 treatments on HCC cell lines: Are Wnt/β-catenin pathway and Smads signaling connected in hepatocellular carcinoma?

Wnt/β-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated β-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-β1 (TGF-β1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/β-catenin and TGF-β1/Smads pathways is the association of Smads with β-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-β1 treatments on Wnt/β-catenin pathway, Smads proteins levels, β-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-β1 attenuated Wnt/β-catenin signal by decreasing β-catenin and Frizzled7 receptor proteins contents and the interaction of β-catenin with TCF4. Truncated β-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates β-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-β1 proved to be effective as modulators of Wnt/β-catenin pathway in HCC cell lines holding both wild-type and truncated β-catenin. Since the inhibition of β-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-β1 could be useful as potential treatments in patients with HCC.

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis. In this work, we evaluate inflammation and the proteins involved in apoptotic process in liver of infected mice and the role of TNFalpha. C57BL6/mice were infected subcutaneously with 100 viable trypomastigotes of Tulahuén strain of T cruzi. One set of these animals were treated with 375 microg of antihuman TNFalpha blocking antibody. Animals were sacrificed at 14 days post-infection (p.i).The analyses of Bcl-2 family proteins revealed an increase of the pro-apoptotic proteins Bax and tBid in T. cruzi-infected mice. Compared with control animals, cytochrome c release was increased. Apoptosis was also induced in infected mice. Anti-TNFalpha treatment decreases hepatic apoptosis. Our results suggest that T. cruzi infection induces programmed cell death in the host liver by increase of TNFalpha production, associated with TNF-R1 over-expression, that set in motion the Bid cleavage and mitochondrial translocation, Bax mitochondrial translocation, cytochrome c release, and ultimately apoptosis induction.

Cyclooxygenase-2 over-expression inhibits liver apoptosis induced by hyperglycemia.

Increased expression of COX‐2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX‐2 protects hepatocytes from several pro‐apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX‐2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX‐2 (hCOX‐2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post‐injection, Wt diabetic animals showed a decrease in PI3K activity and P‐Akt levels, an increase of P‐JNK, P‐p38, pro‐apoptotic Bad and Bax, release of cytochrome c and activities of caspases‐3 and ‐9, leading to an increased apoptotic index. This situation was improved in diabetic COX‐2 Tg. In addition, SID COX‐2 Tg showed increased expression of anti‐apoptotic Mcl‐1 and XIAP. Pro‐apoptotic state in the liver of diabetic animals was improved by over‐expression of COX‐2. We also analyzed the roles of high glucose‐induced apoptosis and hCOX‐2 in vitro. Non‐transfected and hCOX‐2‐transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non‐transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX‐2‐transfected cells was suppressed by addition of DFU (COX‐2 selective inhibitor), and mimicked by addition of PGE2 in non‐transfected cells. Taken together, these results demonstrate that hyperglycemia‐induced hepatic apoptosis is protected by hCOX‐2 expression. J. Cell. Biochem. 114: 669–680, 2013.

Neonatal exposure to a glyphosate based herbicide alters the development of the rat uterus

Glyphosate-based herbicides (GBHs) are extensively used to control weeds on both cropland and non-cropland areas. No reports are available regarding the effects of GBHs exposure on uterine development. We evaluated if neonatal exposure to a GBH affects uterine morphology, proliferation and expression of proteins that regulate uterine organogenetic differentiation in rats. Female Wistar pups received saline solution (control, C) or a commercial formulation of glyphosate (GBH, 2mg/kg) by sc injection every 48h from postnatal day (PND) 1 to PND7. Rats were sacrificed on PND8 (neonatal period) and PND21 (prepubertal period) to evaluate acute and short-term effects, respectively. The uterine morphology was evaluated in hematoxylin and eosin stained sections. The epithelial and stromal immunophenotypes were established by assessing the expression of luminal epithelial protein (cytokeratin 8; CK8), basal epithelial proteins (p63 and pan cytokeratin CK1, 5, 10 and 14); and vimentin by immunohistochemistry (IHC). To investigate changes on proteins that regulate uterine organogenetic differentiation we evaluated the expression of estrogen receptor alpha (ERα), progesterone receptor (PR), Hoxa10 and Wnt7a by IHC. The GBH-exposed uteri showed morphological changes, characterized by an increase in the incidence of luminal epithelial hyperplasia (LEH) and an increase in the stromal and myometrial thickness. The epithelial cells showed a positive immunostaining for CK8, while the stromal cells for vimentin. GBH treatment increased cell proliferation in the luminal and stromal compartment on PND8, without changes on PND21. GBH treatment also altered the expression of proteins involved in uterine organogenetic differentiation. PR and Hoxa10 were deregulated both immediately and two weeks after the exposure. ERα was induced in the stromal compartment on PND8, and was downregulated in the luminal epithelial cells of gyphosate-exposed animals on PND21. GBH treatment also increased the expression of Wnt7a in the stromal and glandular epithelial cells on PND21. Neonatal exposure to GBH disrupts the postnatal uterine development at the neonatal and prepubertal period. All these changes may alter the functional differentiation of the uterus, affecting the female fertility and/or promoting the development of neoplasias.

Role of reactive oxygen species in the early stages of liver regeneration in streptozotocin-induced diabetic rats

Abstract Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.

Effects of neonatal exposure to a glyphosate-based herbicide on female rat reproduction

In this study, we investigated whether neonatal exposure to a glyphosate-based herbicide (GBH) alters the reproductive performance and the molecular mechanisms involved in the decidualization process in adult rats. Newborn female rats received vehicle or 2 mg/kg/day of a GBH on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate (i) the reproductive performance on gestational day (GD) 19 and (ii) the ovarian steroid levels, uterine morphology, endometrial cell proliferation, apoptosis and cell cycle regulators, and endocrine pathways that regulate uterine decidualization (steroid receptors/COUP-TFII/Bmp2/Hoxa10) at the implantation sites (IS) on GD9. The GBH-exposed group showed a significant increase in the number of resorption sites on GD19, associated with an altered decidualization response. In fact, on GD9, the GBH-treated rats showed morphological changes at the IS, associated with a decreased expression of estrogen and progesterone receptors, a downregulation of COUP-TFII (Nr2f2) and Bmp2 mRNA and an increased expression of HOXA10 and the proliferation marker Ki67(Mki67) at the IS. We concluded that alterations in endometrial decidualization might be the mechanism of GBH-induced post-implantation embryo loss.

Endosulfan affects uterine development and functional differentiation by disrupting Wnt7a and β-catenin expression in rats

Neonatal exposure to a low dose of endosulfan may disrupt the expression of Wnt7a and β-catenin during uterine development leading to the failure of uterine functional differentiation during implantation. New-born female Wistar rats were treated with vehicle, endosulfan (600 μg/kg/d, E600) or diethylstilbestrol (0.2 μg/kg/d, DES) on postnatal days (PNDs) 1, 3, 5 and 7. Subsequently, uterine histomorphology and the protein expression of Wnt7a and β-catenin were evaluated on PND8, PND21 and gestational day (GD) 5 (pre-implantation period). In the E600 rats, Wnt7a and β-catenin protein expression was increased in the epithelium on PND8, and Wnt7a expression was decreased in the endometrial glands on PND21. On GD5, the number of uterine glands was decreased in the E600-and DES-treated rats. In addition, Wnt7a expression was decreased in all uterine compartments, and β-catenin expression was increased in the luminal and glandular epithelia of the E600-and DES-treated rats. Disruption of Wnt7a and β-catenin uterine expression in the prepubertal and adult females altered the uterine preparation for embryo implantation, which could be associated with the subfertility triggered by endosulfan.

Neonatal exposure to a glyphosate-based herbicide alters uterine decidualization in rats

We investigated whether defective modulation of uterine signaling may cause decidualization failure in rats neonatally exposed to a glyphosate-based herbicide (GBH). Female pups received vehicle or 2mg/kg of GBH from postnatal day (PND) 1 to PND7. On PND8 and PND21, Wnt5a and β-catenin expression was evaluated in uterine samples. On gestational day (GD) 9, Wnt5a, Wnt7a and β-catenin expression and Dkk1 and sFRP4 mRNA were evaluated on implantation sites. On PND8, GBH-exposed rats showed increased Wnt5a and β-catenin expression in luminal epithelium (LE), whereas on PND21, they showed increased Wnt5a and β-catenin expression in subepithelial stroma but decreased β-catenin expression in glandular epithelium. On GD9, GBH-exposed rats showed decreased Wnt5a and Wnt7a expression in the antimesometrial zone and LE respectively, without changes in β-catenin expression, while Dkk1 and sFRP4 were up- and down-regulated respectively. We concluded that neonatal GBH exposure may lead to embryo losses by disturbing uterine signaling.