Giovanna Elvira Granato

The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK) cells where no signs of apoptosis were observed, but herein, analysis of MDBK cell morphology, in a large number of exposed cells, revealed some alterations, as expanded cytoplasm, an increase of intercellular spaces and many pyknotic nuclei. Hence, the aim of the current study was to elucidate the influences of dioxin on cell proliferation and cell death. We found that dioxin increased proliferation, as well as, activated cell death with autophagy, as we detected by increased amount of LC3-II, an autophagosome marker. Furthermore, formation of acidic vesicular organelles was observed by fluorescence microscopy following staining with the lysosomotropic agent acridine orange. These results were accompanied by down-regulation of telomerase activity, bTERT and c-Myc. Key tumor-suppressor protein p53 and expression of cell cycle inhibitor p21Waf1/Cip1 were activated after TCDD exposure. These changes occurred with activation of ATM phosphorylation in the presence of a decrease in Mdm2 protein levels. Taken together, these results support the idea that TCDD in MDBK cells, may exert its action, in part, by enhancing cell proliferation, but also by modulating the incidence of induced cell death with autophagy.

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) influences infection of kidney cells (MDBK) with Bovine Herpesvirus 1 (BHV-1) through an increase in virus replication and an acceleration of BHV-1-induced apoptosis. Previously our group demonstrated that BHV-1, in the early stages of infection, significantly up-regulates telomerase activity in MDBK cells, while, in the late phases of infection, when BHV-1-induced apoptosis occurred, a down-regulation of telomerase activity was detected. Hence, herein, for the first time, we described the influences of TCDD on telomerase activity during virus infection. In kidney cells (MDBK) infected with BHV-1 and exposed to different doses of TCDD we explored telomerase activity by TRAP assay. Concomitantly, we examined protein levels of both bTERT and c-Myc by Western blot analysis. In all groups, TCDD induced an acceleration in down-regulation of telomerase activity. Particularly, TCDD drastically and significantly decreased telomerase activity when virus-induced apoptosis took place. This result was accompanied from an accelerated down-regulation of bTERT and c-Myc. Finally, in the presence of TCDD, we evidenced a dose-dependent overexpression of aryl hydrocarbon receptor. Hence, our data suggest that TCDD, through a significant acceleration in down-regulation of telomerase activity, bTERT and c-Myc, may contribute to accelerated BHV-1-induced apoptosis.

Feline herpesvirus 1 (FHV-1) is a widespread cat pathogen inducing rhinitis, conjunctivitis and corneal ulcers. To alleviate acute FHV-1-induced disease, antiviral agents are used often with antibiotics. But sometimes, these treatments, as well as conventional doses of cytokines have moderate efficacy and/or collateral effects. Herein we have investigated the effects of low dose interleukin (IL)-12 plus interferon (IFN)-gamma, prepared by Sequential Kinetic Activated (SKA), on the treatment of FHV-1 infection. Twenty-five, unvaccinated FHV-1-positive cats were recruited into a prospective, randomized, placebo-controlled, double-blinded clinical trial. Fifteen cats were treated for 6 months with oral low doses of SKA IL-12 plus IFN-gamma and 10 cats were treated with placebo. At 1, 6 and 12 months (follow-up) after the beginning of treatment, clinical assessment, PCR assay and blood count were carried out. At follow-up, in treated group, we observed significant (p < 0.05) improvements in clinical signs and PCR became negative in 12/15 cats (80%). In placebo, 10/10 cats were PCR-positive, with improvements (30%) or worsening (70%) in clinical signs. Blood values were normal in both groups. Our results show that the low dose therapy, based on activated solutions of IL-12 plus IFN-gamma, represents a novel approach to treat FHV-1 infection in cats.

Abstract Our previous studies have demonstrated that 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) increases Bovine Herpesvirus 1 (BHV‐1) replication through a dose‐dependent increase in cytopathy and increased viral titer. Furthermore, TCDD was able to trigger BHV‐1‐induced apoptosis by up‐regulating the activation of initiator caspases 8 and 9, as well as of effector caspase 3. Since TCDD activates caspase 3 after 4 h of infection, we have hypothesized an involvement of BHV‐1 infected cell protein 0 (bICP0) in this process. Such protein, the major transcriptional regulatory protein of BHV‐1, has been shown to indirectly induce caspase 3 activation and apoptosis. In order to elucidate the role of bICP0 in this apoptotic pathway, here we have analyzed the effects of TCDD on bICP0 expression. Following infection of bovine cells with BHV‐1, we detected apoptotic features already at 12 h after infection, only in TCDD exposed groups. Furthermore, in the presence of different doses of TCDD, we observed a time‐dependent modulation and increase of bICP0 gene expression levels, as revealed by RT‐PCR analysis. Western blot analysis and immunocytochemistry revealed that TCDD induced an increase of bICP0 protein levels in a dose‐dependent manner, compared to unexposed groups. Moreover, Western blot analysis of nuclear and cytosolic fractions of infected cells revealed that TCDD anticipated the presence of bICP0 protein in the cytoplasm. In conclusion, both the increase of replication of BHV‐1 and anticipation of BHV‐1‐induced apoptosis could be the result of a relationship between TCDD and bICP0. J. Cell. Biochem. 111: 333–342, 2010. © 2010 Wiley‐Liss, Inc.

Bovine herpesvirus type 4 (BoHV-4), like other herpesviruses, induces a series of alterations in the host cell that modify the intracellular environment in favor of viral replication, survival and spread. This research examined the impact of BoHV-4 infection on autophagy in BoHV-4 infected Madin Darby bovine kidney (MDBK) cells. Protein extracts of BoHV-4 infected and control MDBK cells were subjected to Western blot. The concentrations of the autophagy and apoptosis-related proteins Beclin 1, p21, PI3 kinase, Akt1/2, mTOR, phospho mTOR, p62 and the light chain three (LC3) were normalized to the actin level and expressed as the densitometric ratio. Western blot analysis of virus-infected cells revealed that autophagic degradation pathway was induced in the late phase of BoHV-4 infection. After 48 h post-infection the protein LC3II, which is essential for autophagy was found to be markedly increased, while infection of MDBK cells with BoHV-4 resulted in a depletion of p62 levels. Becline 1, PI3 kinase, Akt1/2 and p21 expression increased between 24 and 48 h post-infection. Surprisingly, mTOR and its phosphorylated form, which are negative regulators of autophagy, also increased after 24 h post-infection. In conclusion, our findings suggest that BoHV-4 has developed mechanisms for modulation of autophagy that are probably part of a strategy designed to enhance viral replication and to evade the immune system. Additional studies on the relationship between autophagy and BoHV-4 replication and survival, in both lytic and latent replication phases, are needed to understand the role of autophagy in BoHV-4 pathogenesis.

Abstract Cyclosporin A (CsA) is the prototype of immunosuppressant drugs that have revolutionized the management of all transplantation and autoimmune diseases. Side effects of CsA mainly affecting the kidney but also observed in liver and heart, limit the therapeutic use of this drug after organ transplantation. The renal toxicity of CsA is attributed to reduced renal blood flow which leads to hypoxia‐reoxygenation injury accompanied by excessive generation of oxygen‐derived free radicals. In several therapeutic protocols, CsA is used in association with corticosteroids to obtain better therapeutic results. Recently, our studies showed that hydrocortisone (HY) has a protective effect on CsA‐induced cardiotoxicity. In fact our previous results demonstrated that in rat cardiomyocytes, CsA toxicity is due to a calcium overload, which in turn induce lipid peroxidation and determines oxidative stress‐induced cell injury. Treatment with HY effectively inhibits CsA‐induced toxicity, decreasing lipid peroxidation as well as calcium intracellular concentration. In this study we evaluated in vivo the effects of CsA, used alone or in association with HY, on some parameters of renal dysfunction (blood urea nitrogen; BUN, creatinine, and cholesterol), malondialdheyde (MDA) levels, antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and apoptosis. CsA administration for 24 days resulted in a marked renal oxidative stress, which significantly deranged the renal functions. Treatment with CsA in association with HY significantly improved the renal dysfunction and renal oxidative status. This study clearly suggests the role of oxidative stress in the pathogenesis of CsA‐induced nephrotoxicity. J. Cell. Biochem. 113: 997–1004, 2012. © 2011 Wiley Periodicals, Inc.

Infection of kidney cells (MDBK) with Bovine Herpesvirus 1 (BoHV-1) is affected by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which accelerates BoHV-1-induced apoptosis and increases virus replication. Herein, to elucidate the mechanism through TCDD modifies BoHV-1 infection, we analyzed the modulation of a members of Sirtuin proteins family in MDBK cells. We found that mitochondrial SIRT3 was upregulated during infection. This change was accompanied by cytoskeletal rearrangements and cell extensions. All these trends were drastically modified by TCDD. We hypothesize that, taken together, these results might further clarify the processes responsible for the action of TCDD on the BoHV-1 replication, resulting in enhanced virus production.

Abstract Introduction: Prostate cancer (PC) is the most commonly diagnosed cancer in males in the Western world and the third leading cause of cancer in men. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shows a wide range of severe adverse effects in animal models, including immunotoxicity, developmental and reproductive toxicity, teratogenicity, and carcinogenicity. The exposure to TCDD early in development may increase susceptibility to PC and the exposure to TCDD in aged rats can cause greater epithelial proliferation, accompanied by morphological alterations manifested as lobe-specific prostatic hyperplasia. The administration of TCDD to a variety of cultured cells may alter their ability to proliferate and die. Autophagy is an evolutionarily conserved cellular pathway playing a critical role in cellular homeostasis. Defects of autophagy are involved in the pathogenesis of different diseases, including cancer. One of the well-known autophagy markers is the microtubule associated light chain protein 3 (LC3). We have previously demonstrated that exposure of Madin-Darby Bovine Kidney cells to TCDD increased cell proliferation and autophagy. The aim of this study is to evaluate the biological effects of TCDD exposure on a highly metastatic prostate cancer cell line (PC-3). Methods: PC3 cells were exposed to different concentrations of TCDD (D1=0.1 pg/ml, D2=1 pg/ml and D3=100 pg/ml) and cell viability was assessed by Trypan Blue exclusion after 24, 48, and 72 hours of exposure. Autophagy was evaluated using the following: (a) detection of acidic vesicular organelles (AVOs) by acridine orange staining; (b) immunoflurescence analysis (IF) of LC3; (c) LC3 gene expression by real-time PCR; and (d) study of autophagic flux by chloroquine (CQ) autophagy inhibitor (10μM). Results: When compared with their relative controls, only D3 exposure at 24, 48, and 72 hours caused the following: (1) statistically significant increase of cell proliferation (CP) (CP24h =20.3%, CP48h =54.5% and CP72h =52.4%); (2) detection of AVOs; (3) LC3 IF positivity; and (4) LC3 over expression (expression ratio24h =1.4, expression ratio48h =6.2 and expression ratio72h =11.4). When PC-3 cells were D3 exposed and treated with CQ at 24, 48, and 72 hours, LC3 expression ratios compared with controls (dioxin treatment) decreased (CQ24h =-12.8, CQ48h =-28.1 and CQ72h =-2.8). Conclusions: While increased incidence of PC in men exposed to TCDD is known, the molecular mechanisms of carcinogenesis of this compound have not been fully investigated. Our preliminary study demonstrated that treatment of PC-3 with TCDD results in enhanced cell proliferation and activation of autophagy, potentiating the transformed phenotype of these cancer cells. Comparison of the normal primary prostate epithelial cells with PC-3 will further clarify the oncogenic mechanisms of TCDD in PC. Citation Format: Giovanna Elvira Granato, Roberto Ciarcia, Filomena Fiorito, Salvatore Florio, Ugo Pagnini, Luisa De Martino, Antonio Giordano, Giuseppe Russo. Enhanced cell proliferation and induced autophagy in PC-3 prostate cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1675. doi:10.1158/1538-7445.AM2013-1675

Dioxins are commonly known as highly toxic compounds that are persistent organic pollutants. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin congener, and dioxin-like products are formed during the incomplete combustion of organic compounds in the presence of chlorine (waste incineration, burning of various fuels and poorly controlled combustion sources). In 1997, the International Agency for Research on Cancer (IARC) classified TCDD as carcinogen. Prostate cancer (PC) is an extremely serious disease in dogs. PC also represents the most commonly diagnosed cancer in males in the Western world. In general, animal models of human cancer have evolved in attempts to capture the complexity of the human disease. In particular, as humans, the dogs are the only other mammals that develop PC. The canine prostate gland shares many morphological and functional similarities with the human prostate, so this specie represents thus an attractive model for the study of the prostatic disease in dogs. Consequently, to evaluate the biological effects of TCDD on prostate cancer, in this study we used human prostate cancer cell line, PC3. Until now, very few data are present in literature about PC3 and TCDD exposure. It is known that in PC3 cell line, a hormone-independent prostate cancer cell line, TCDD induces cytochrome P450 (CYP) 1A1 and CYP1B1 via the aryl hydrocarbon receptor (AhR). This effect of TCDD could result in higher elimination rates of concomitant drugs metabolized by these particular CYP isoenzymes. Autophagy is a tightly regulated process playing a normal part in cell growth, development, and homeostasis and helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products through the degradation via the lysosome. Defects of autophagy machinery are responsible for pathogenesis of different diseases, including cancer. The role of autophagy in cancer is controversial. There is evidence that autophagy may play a critical role in cancer progression at later stages, such as dissemination and metastasis, which account for most cancer-associated deaths, whereas in other cases it clearly contributes to tumor suppression by inducing tumor cell death. So, the aim of this study is to evaluate the biological effects of TCDD exposure to a highly metastatic prostate cancer cell line, PC3. The prostate cancer cell line PC3 was exposed to different concentrations of TCDD (0.1, 1 and 100 pg/ml) and after 24, 48, and 72 hours of exposure cell proliferation and viability, cell morphology and cell cycle analysis were performed. Also, autophagy was evaluated using the following: (a) detection of acidic vesicular organelles (AVOs) by acridine orange staining; (b) immunofluorescence analysis (IF) of LC3 (microtubule associated light chain protein 3), one of the well-known autophagy markers; (c) LC3 gene expression by real-time PCR; (d) study of autophagic flux by chloroquine (CQ) (10µM), autophagy inhibitor, by western blot; (e) study the expression of multiple genes involved in autophagy machinery by real-time PCR. When compared with their relative controls, TCDD (100 pg/ml) exposure at 24, 48, and 72 hours caused the following: (1) significant increase of cell proliferation (CP) (CP24h =20.3%, CP48h =54.5% and CP72h =52.4%); (2) significant increase of cell population at S phase; (3) significant increase of autophagy demonstrated by (a) detection of AVOs; (b) LC3 IF positivity; (c) LC3 over expression (expression ratio24h =1.4, expression ratio48h =6.2 and expression ratio72h =11.4); (d) CQ increased LC3-II accumulation; (e) moreover, we demonstrated that the induction of autophagy by TCDD in PC3 cells was accompanied by an increase in the mRNA levels of certain genes that are present in different subnetworks composing the autophagy interactive network, such as: PIK3C3, BECN-1, AMBRA1, MAP1LC3B, ATG4A, ATG4C, ATG5, ATG7, ATG10, ATG16L1, GABARAPL1, PRKAA, WIPI-1. Also, we showed that TCDD negatively influenced genes that are autophagy inhibitor, such as: AKT1 and BCL2. These data suggested a multiple effect of TCDD on autophagy machinery. Furthermore, we revealed that TCDD exposure upregulated TNF whose high levels have been related to prostate cancer progression via stimulation of proliferation, survival of malignant cells and increased resistance to chemotherapeutic agents. From the 1980s, several illegal and uncontrolled sites of urban, toxic, and industrial waste disposal have been active in Campania region, Southern Italy, with the highest concentration reached in Naples and Caserta provinces. In 1994, Campania region is under a declared State of Emergency, because of the saturation of regional waste treatment facilities. There are growing national and international evidences that the accumulation of waste, illegal and legal, urban and industrial, has contaminated soil, water, and the air with a range of toxic pollutants including dioxins. A high correlation between incidences of cancer, respiratory illnesses, and genetic malformations and the presence of industrial and toxic waste landfills was also found. In Spring 2002, a dioxin emergency emerged in this region, as result of National Control Program of Residues in Foodstuff carried out by Health Minister in 2001, indeed, levels of TCDD exceeding the European Union tolerance were detected in dairy products and milk from cow and water buffalo, raised on in some areas of Campania Region. Cancer mortality descriptive studies demonstrated an increase of incidents rates of the prostate cancer in the same areas where dioxin levels were elevated. Furthermore, studies performed in animal model suggested that TCDD exposure is associated with abnormal prostate development, altered prostate pathology and increase susceptibility to prostate cancer. The administration of TCDD to a variety of cultured cells may alter their ability to proliferate and die. In a previous study we demonstrated that TCDD induced proliferation in Madin-Darby Bovine Kidney (MDBK), an epithelial cell line, in which analysis of MDBK cell morphology revealed some alterations in a large number of exposed cells, where neither signs of apoptosis nor necrosis were detected, but we found that TCDD activated cell death with autophagy. Herein, TCDD exposure in PC3, a cancer cell line, induced no signs of cell death. So, taken together, our results support the idea that TCDD, may induce the progression of prostate cancer enhancing cell proliferation, inducing autophagy, deregulating the expression of genes related to the autophagy machinery, and upregulating TNF resulting in an increased risk for both animal and human health.

Autosomal dominant polycystic kidney disease (ADPKD) affects millions of people worldwide. Vasopressin promotes disease progression.A randomized controlled trial with equal (1:1) allocation.This trial examined the effect of combining a low-osmolar (low-sodium [1,500 mg/d], low-protein [0.8 g per kilogram of body weight]) diet and adjusted water intake on vasopressin secretion in 34 patients with ADPKD.Participants were randomly assigned to receive a low-osmolar diet followed by adjusted water intake to achieve urine osmolality ≤ 280 mOsm/kg water versus no intervention for 2 weeks.The primary outcome of the study was change (delta) in copeptin levels and urine osmolality between the intervention and control groups from baseline to 2 weeks.Fasting plasma copeptin level, 24-hour urine osmolality, and total solute intake.Baseline characteristics of the 2 groups were similar. Mean plasma copeptin levels and urine osmolality declined from 6.2 ± 3.05 (SD) to 5.3 ± 2.5 pmol/L (P = 0.02) and from 426 ± 193 to 258 ± 117 mOsm/kg water (P = 0.01), respectively, in the intervention group compared to a nonsignificant change in the control group (from 4.7 ± 3.6 to 5.07 ± 4 pmol/L [P = 0.2] and 329 ± 159 to 349 ± 139 mOsm/kg water [P = 0.3], respectively). The change in copeptin levels (primary outcome) and urine osmolality was statistically significant between the intervention and control groups (delta copeptin, −0.86 ± 1.3 vs +0.39 ± 1.2 pmol/L [P = 0.009]; delta urine osmolality, −167 ± 264 vs +20 ± 80 mOsm/kg water [P = 0.007], respectively). Total urinary solute decreased in only the intervention group and significantly differed between groups at week 1 (P = 0.03), reducing mean water prescription from 3.2 to 2.6 L/d.Small sample size and short follow-up.We developed a stepwise dietary intervention that led to a significant reduction in vasopressin secretion in patients with ADPKD. Furthermore, this intervention led to a reduction in water required for vasopressin reduction.

Constituting an integral part of a heme's porphyrin ring, iron is essential for supplying cells and tissues with oxygen. Given tight links between oxygen delivery and iron availability, it is not surprising that iron deprivation and oxygen deprivation (hypoxia) have very similar consequences at the molecular level. Under hypoxia, the expression of major iron homeostasis genes including transferrin, transferrin receptor, ceruloplasmin, and heme oxygenase-1 is activated by hypoxia-inducible factors to provide increased iron availability for erythropoiesis in an attempt to enhance oxygen uptake and delivery to hypoxic cells. Iron-response proteins (IRP1 and IRP2) and “cap-n-collar” bZIP transcriptional factors (NE-F2 p45; Nrf1, 2, and 3; Bach1 and 2) also control gene and protein expression of the key iron homeostasis proteins. In this article, we give an overview of the mechanisms by which iron pathways are regulated by hypoxia at multiple levels. In addition, potential clinical benefits of manipulating iron pathways in the hypoxia-related conditions anemia and ischemia are discussed.