Effects of Diisodecyl Phthalate on PPAR:RXR-Dependent Expression Pathways in Sea Bream Hepatocytes

Evidence that endocrine-disrupting chemicals (EDCs) may target metabolic disturbances, beyond interference with the functions of the endocrine systems has recently accumulated. Among EDCs, phthalate plasticizers like the diisodecyl phthalate (DiDP) are commonly found contaminants of aquatic environments and have been suggested to function as obesogens by activating peroxisome proliferator activated receptors (PPARs), a subset of nuclear receptors (NRs) that act as metabolic sensors, playing pivotal roles in lipid homeostasis. However, little is known about the modulation of PPAR signaling pathways by DiDP in fish. In this study, we have first investigated the ligand binding efficiency of DiDP to the ligand binding domains of PPARs and retinoid-X-receptor-α (RXRα) proteins in fish using a molecular docking approach. Furthermore, in silico predictions were integrated by in vitro experiments to show possible dose-relationship effects of DiDP on PPAR:RXR-dependent gene expression pathways using sea bream hepatocytes. We observed that DiDP shows high binding efficiency with piscine PPARs demonstrating a greater preference for RXRα. Our studies also demonstrated the coordinate increased expression of PPARs and RXRα, as well as their downstream target genes in vitro. Principal component analysis (PCA) showed the strength of relationship between transcription of most genes involved in fatty acid metabolism and PPAR mRNA levels. In particular, fatty acid binding protein (FABP) was highly correlated to all PPARs. The results of this study suggest that DiDP can be considered an environmental stressor that activates PPAR:RXR signaling to promote long-term changes in lipid homeostasis leading to potential deleterious physiological consequences in teleost fish

Ghrelin induces apoptosis in colon adenocarcinoma cells via proteasome inhibition and autophagy induction.

Ghrelin is a metabolism-regulating hormone recently investigated for its role in cancer survival and progression. Controversially, ghrelin may act as either anti-apoptotic or pro-apoptotic factor in different cancer cells, suggesting that the effects are cell type dependent. Limited data are currently available on the effects exerted by ghrelin on intracellular proteolytic pathways in cancer. Both the lysosomal and the proteasomal systems are fundamental in cellular proliferation and apoptosis regulation. With the aim of exploring if the proteasome and autophagy may be possible targets of ghrelin in cancer, we exposed human colorectal adenocarcinoma cells to ghrelin. Preliminary in vitro fluorimetric assays evidenced for the first time a direct inhibition of 20S proteasomes by ghrelin, particularly evident for the trypsin-like activity. Moreover, 1 μM ghrelin induced apoptosis in colorectal adenocarcinoma cells by inhibiting the ubiquitin-proteasome system and by activating autophagy, with p53 having an "interactive" role

Plasticizers used in food-contact materials affect adipogenesis in 3T3-L1 cells

Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01-25 μM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein β (Cebpβ) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase of adipogenesis. DEGDB increased the expression of Lpl only, while the phthalate DiDP did not change the expression of either late-phase marker genes Fabp4 and Lpl. Taken together, our results suggest that exposure to low, environmentally relevant doses of the plasticizers DiNP, DiDP, DEGDB and TMCP increase lipid accumulation in 3T3-L1 adipocytes, an effect likely mediated through activation of PPARγ and interference at different levels with the transcriptional cascade driving adipogenesis

In silico prediction and in vivo analysis of antiestrogenic potential of 2-isopropylthioxanthone (2-ITX) in juvenile goldfish (Carassius auratus)

Previous studies have shown both anti-estrogenic and anti-androgenic activities of 2-isopropylthioxanthone (2-ITX), a well known food contaminant, in in vitro assays. However, no data are available on the anti-estrogenic potentials and risks of 2-ITX in aquatic organisms. This work evaluated the potential endocrine disrupting effects of 2-ITX at the level of estrogen receptor (ER) signaling cascade using juvenile goldfish (Carassius auratus) as model. Firstly, we investigated the ligand binding efficiency of 2-ITX to the ligand binding domains (LBD) of goldfish ER subtypes using a molecular docking approach. Secondly, we assessed the effects of 2-ITX on E2-induced hepatic expression of ERα1, ERβ1, ERβ2, and vitellogenin (VTG) in vivo. Crosstalk between ER-VTG and aryl hydrocarbon receptor 2 (AhR2)-cytochrome P4501A (CYP1A) was also investigated. Fish were injected with increasing doses of 2-ITX ranging from 2 to 10µg/g BW, and results were compared to the effect of tamoxifen, a well-known ER modulator. We observed that compared to ERβ, the interaction potentials of 2-ITX to goldfish ERα1 LBD was more stable in the inactive receptor conformation. The in silico docking simulation analysis also revealed that 2-ITX acted as agonist for the goldfish AhR2 LBDs suggesting the ability of this compound to activate the cross-talk between the ERα- and AhR-signaling pathways. In vivo experiments confirm in silico simulation predictions demonstrating that 2-ITX reduced the estrogenicity of E2 at both transcriptional and post-transcriptional levels, indicating a clear anti-estrogenic effect. Co-exposure of E2 and 2-ITX also resulted in a significant decrease of CYP1A gene expression with respect to 2-ITX alone. Results from these studies collectively revealed that the antiestrogenic property of 2-ITX can be ascribed to a combination of effects on multiple signaling pathways suggesting the potential for this environmental contaminant to affect the hormonal control of reproductive processes in fish

Differential tissue regulation of peroxisome proliferator-activated receptor α (PPARα) and cannabinoid receptor 1 (CB1) gene transcription pathways by diethylene glycol dibenzoate (DEGB): preliminary observations in a seabream (Sparus aurata) in vivo model

Today a variety of endocrine disrupting chemicals (EDCs) are recognized in the group of metabolic disruptors, a wide range of environmental contaminants that alter energy balance regulation by affecting the peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) pathway. Herein, we investigated the effect of diethylene glycol dibenzoate (DEGB), a dibenzoate-based plasticizer used as alternative to phthalates, on the expression of key genes involved in lipid metabolism and energy balance by using Sparus aurata juveniles as models. We also evaluated the correlation between cannabinoid receptor 1 (CB1) and PPARα transcriptional patterns in both liver and brain tissues. Exposure to the highest DEGB concentration differentially modulated PPARα/CB1 transcriptional pathways in liver/brain tissues of seabream. We hypothesize that, at peripheral level (i.e. liver), DEGB acts as PPARα agonist resulting in a potential stimulation of key lipolytic genes and a concomitant down-regulation of endocannabinoid metabolic enzyme genes

Tri-m-cresyl phosphate and PPAR/LXR interactions in seabream hepatocytes: revealed by computational modeling (docking) and transcriptional regulation of signaling pathways

The interactions between tri-m-cresyl phosphate (TMCP; an organophosphate flame retardant) and peroxisome proliferator activated receptors (PPARs) or liver X receptor α (LXRα) were investigated in seabream hepatocytes. The study was designed to characterize the binding of TMCP to PPARα, PPARγ and LXRα by computational modeling (docking) and transcriptional regulation of signaling pathways. TMCP mainly established a non-polar interaction with each receptor. These findings reflect the hydrophobic nature of this binding site, with fish LXRα showing the highest binding efficiency. Further, we have investigated the ability of TMCP to activate PPAR and LXR controlled transcriptional processes involved in lipid/cholesterol metabolism. TMCP induced the expression of all the target genes measured. All target genes were upregulated at all exposure doses, except for fatty acid binding protein 7 (FABP7) and carnitine palmitoyltransferase 1B. Collectively, our data indicate that TMCP can affect fatty acid synthesis/uptake and cholesterol metabolism through LXRα and PPARs, together with interactions between these transcription factors in seabream liver

Environmental pollutants directly affect the liver X receptor alpha activity: kinetic and thermodynamic characterization of binding

Liver X receptor is a ligand-activated transcription factor, which is mainly involved in cholesterol homeostasis, bile acid and triglycerides metabolism, and, as recently discovered, in the glucose metabolism by direct regulation of liver glucokinase. Its modulation by exogenous factors, such as drugs, industrial by-products and chemicals is documented. Owing to the abundance of these synthetic molecules in the environment, and to the established target role of this receptor, a number of representative compounds of phthalate, organophosphate and fibrate classes were tested as ligands/modulators of human liver X receptor, using an integrated approach, combining an in silico molecular docking technique with an optical SPR biosensor binding study. The compounds of interest were predicted and proved to target the oxysterols-binding site of human LXRα with measurable binding kinetic constants and with affinities ranging between 4.3×10-7-4.3×10-8 M. Additionally, non-cytotoxic concentration of these chemicals induced relevant changes in the LXRα gene expression levels and other target genes (SREBP-1c and LGK) in human liver hepatocellular carcinoma cell line (HepG2), as demonstrated by q-RT-PC

Effects of Tart Cherry supplementation in Diet-Induced Obesity (DIO) rats.

Excessive accumulation of adipose tissue correlates with metabolic changes that increase the risk of cardiovascular and cerebrovascular disease. Hypertension, impaired glucose tolerance, insulin resis-tance and dyslipidemia often accompanied obesity. The excess of fat storage in the abdomen is the prime cause of the metabolic abnormalities and therefore represents an important target in the treatment of obesity. Fruit and vegetable intake is inversely correlated with cardiovascular morbidity and mortality [1,2]. Furthermore, fruits and vegetables contain non-nutritive phytochemicals that may contribute to their health-promoting effects. Anthocyanins are phytochemical flavonoids principally found in red-, blue- and purple-pigmented fruits and vegetables. Several studies have suggested that anthocyanin-rich plant extracts can modify lipid metabolism in vitro and can reduce hyperlipidemia in vivo [3,4]. Tart cherries (Prunus cerasus L.) are a rich source of anthocyanins [5]. This study was designed to evaluate the effects of anthocyanin-rich tart cherries extract and seeds powder on Diet-Induced Obesity (DIO) rats. DIO rats of 7 weeks of age, expose to caloric-dense diet ad-libitum, provide a useful animal model sharing several common features with human obesity. DIO rats were studied for 17 weeks of hyper-caloric diet with the supplementation of tart cherries seeds powder (DS) and seeds powder plus tart cherries extract, containing 1mg of anthocyanins (DES). DIO rats were compared to the control rats with not fat diet (Chow).To determine the systemic effects of caloric dense expousure we examined food consumption, fat mass content and fasting glycemia, insulin levels, cholesterol and triglycerides. Ultrasonographic (US) and computed tomography (CT) evaluations were performed to detect adi-pose tissue deposition. In CT, also fat infarction of the liver was investigated. For the assessment, the difference in attenuation values between liver and spleen as well as the calculation of the spleen-to-liver attenuation ratio were taken into account. The results confirmed the development of obesity after 5 weeks of the fat diet. After 17 weeks, rats increased significantly their body weight in comparison to the control group. Glycaemia and insulin levels were higher in DIO rats, while no differences in values of total cholesterol and triglycerides were observed. Systolic blood pressure was higher in DIO rats after 17 weeks of high-fat diet com-pared to age-matched CHOW rats. The US and CT analysis indicated an increase of deposition of visceral adipose tissue and evidenced a decrease of hepatic attenuation in DIO rats. The increase of spleen-to-liver attenuation ratio in DIO rats suggests a moderate hepatic steatosis. No difference in body weight was found in DS and DES rats compared to age-matched DIO rats. Supplementation of tart cherries in DS and DES induce a decrease of the blood pressure and the glycemia. Furthermore, the serum levels of thiobarbituric reactive substances decreased without changes of the antioxidant properties. CT analysis revealed a decrease of spleen-to-liver attenuation ratio, in DES rats. The evidence of the CT was confirmed by histological analysis of liver. Sections of DIO rats present a distinctive pattern of hepatic injury characterized by steatosis with hepatocytic ballooning degeneration at the perivenular areas. The steatosis elements decrease in DS and DES rats. The preliminary findings of this study indicate that supplementation with tart cherries, although did not reduce the body weight in DIO rats, but prevent the development of related risk factors. Further studies are needed to better clarify the benefits of tart cherry supplementation on health and disease prevention. [1] P.J. Mink, C.G. Scrafford, L.M. Barraj, et al. Flavonoid intake and cardiovascular disease mor-tality: a prospective study in postmenopausal women. Am J Clin Nutr 2007, 85, 895. [2] E.J. Brunner, A. Mosdol, D.R. Witte, et al. Dietary patterns and 15-y risks of major coronary events, diabetes, and mortality. Am J Clin Nutr 2008;87:1414. [3] X. Xia, W. Ling, J. Ma, et al. An anthocyanin-rich extract from black rice enhances atheroscle-rotic plaque stabilization in apolipoprotein E-deficient mice. J Nutr 2006,136, 2220. [4] E.M. Seymour, S.K. Lewis, D. E. Urcuyo-Llanes et al. Regular Tart Cherry Intake Alters Ab-dominal Adiposity, Adipose Gene Transcription, and Inflammation in Obesity-Prone Rats Fed a High Fat Diet. J Med Food 2009, 12, 935. [5] F Blando, C Gerardi, I Nicoletti: Sour cherry (Prunus cerasus L) anthocyanins as ingredients for functional foods. J Biomed Biotechnol 2004, 2004, 253–258