Paolo Cocci

Effects of Diisodecyl Phthalate on PPAR:RXR-Dependent Gene 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.

Effects of 4-nonylphenol on hepatic gene expression of peroxisome proliferator-activated receptors and cytochrome P450 isoforms (CYP1A1 and CYP3A4) in juvenile sole (Solea solea)

The objective of the present study was to investigate the modulatory effects of the xenoestrogen 4-nonylphenol (4-NP) on hepatic peroxisome proliferator-activated receptor (PPAR) α and β gene expression patterns in relation to the detoxification pathways mediated by cytochrome P450 isoforms (CYP1A1 and CYP3A4). Waterborne 4-NP-induced effects were compared with those of 10(-8)M 17β-estradiol (E2) by using in vivo dose-response experiments carried out with juvenile sole (Solea solea). Compared to the controls, significantly higher levels of PPARα mRNAs were found in fish treated with E2 or 4-NP (10(-6)M) 3 d after exposure; the highest dose of 4-NP also caused up-regulation of retinoid X receptor α (RXRα) transcript levels. On the contrary, PPARβ gene expression was not modulated by E2 or 4-NP. Our data show that 4-NP-induced PPARα mRNA levels coincide with suppression of CYP1A1 and CYP3A4 expression similarly to E2. The results from these in vivo studies suggest the presence of cross-talk between nuclear receptor-mediated signaling pathways and PPARα that may result in modulation of CYP450 isoforms expression following 4-NP treatment in sole liver.

Xenoestrogens elicit a modulation of endocannabinoid system and estrogen receptors in 4NP treated goldfish, Carassius auratus.

Based on pharmacological, behavioral and neuroanatomical studies, the endocannabinoids appear to be pivotal in some important neuroendocrine regulations of both vertebrates and invertebrates. Interestingly, a well developed endocannabinoid system was recently demonstrated by us in different bonyfish brain areas which control reproduction, energy balance and stress. Fish in particular are very sensitive to different types of stressors which can heavily affect their reproductive activity and negatively reverberate on aquaculture. Since recent new data have been reported on endocrine disruptors (EDs) impact on zebrafish receptor CB1 expression, in the present research we have investigated the response of the endocannabinoid system to acute treatment with an environmental stressor such as the xenoestrogen nonylphenol (4NP) in the brain and peripheral tissues of the goldfish Carassius auratus. First of all the estrogenic effects induced by 4NP were demonstrated by a dose-dependent increase of plasma levels and gene expression of the biomarker vitellogenin, then changes in cannabinoid receptors and anandamide degradative enzyme, the fatty acid amide hydrolase (FAAH), were analysed by means of Real Time PCR. As the exposure to EDs may lead to an activation of estrogen receptors and affects the Aromatase (AROB) transcription, changes in mRNA levels for ER subtypes and AROB were also evaluated. Our results confirm in goldfish the effect of 4NP on ERα and ERβ1 receptors and point out a different sensitivity of CB1 and CB2 for this compound, suggesting distinct roles of these cannabinoid receptors in some adaptive processes to contrast stress induced by xenoestrogen exposure.

Cortisol response to waterborne 4-nonylphenol exposure leads to increased brain POMC and HSP70 mRNA expressions and reduced total antioxidant capacity in juvenile sole (Solea solea)

4-Nonylphenol (4-NP) is a breakdown product of alkylphenolpolyethoxylates and can be found in almost all environmental water matrices. 4-NP can act as environmental stressor on fish, typically causing modulation of hypothalamic-pituitary-interrenal axis (HPI). To examine the effects of the xenoestrogen 4-NP or 17β-estradiol (E2) on induction of stress response mechanisms by evaluating the levels of proopiomelanocortin (POMC) mRNA, heat shock protein 70 (HSP70) mRNA and plasma cortisol, we exposed juvenile sole (Solea solea), under static condition for 7 day, to either 10(-6) or 10(-8) M 4-NP, or 10(-8) M E2. In addition, plasma cortisol titers were correlated to the total antioxidant capacity (TAC), one of the oxidative stress parameters. 4-NP treatments resulted in high levels of POMC mRNA, HSP70 mRNA and plasma cortisol. On the contrary, E2 basically down-regulated POMC expression. Moreover, elevated cortisol levels in fish exposed to the highest dose of 4-NP were accompanied by low TAC. These results suggest that 4-NP modulates the sole HPI axis inducing a cortisol-mediated stress response. Specifically, we suggest that 4-NP affects brain POMC mRNA levels via non-estrogen receptor (ER)-mediated mechanism further supporting the ability of 4-NP to target multiple receptor systems.

Alterations of gene expression indicating effects on estrogen signaling and lipid homeostasis in seabream hepatocytes exposed to extracts of seawater sampled from a coastal area of the central Adriatic Sea (Italy).

Recent evidences suggest that the toxicological effects of endocrine disrupting chemicals (EDCs) involve multiple nuclear receptor-mediated pathways, including estrogen receptor (ER) and peroxisome proliferator-activated receptor (PPAR) signaling systems. Thus, our objective in this study was to detect the summated endocrine effects of EDCs with metabolic activity in coastal waters of the central Adriatic Sea by means of a toxicogenomic approach using seabream hepatocytes. Gene expression patterns were also correlated with seawater levels of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). We found that seawater extracts taken at certain areas induced gene expression profiles of ERα/vitellogenin, PPARα/Stearoyl-CoA desaturase 1A, cytochrome P4501A (CYP1A) and metallothionein. These increased levels of biomarkers responses correlated with spatial distribution of PAHs/PCBs concentrations observed by chemical analysis in the different study areas. Collectively, our data give a snapshot of the presence of complex EDC mixtures that are able to perturb metabolic signaling in coastal marine waters.

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) and 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-PCR.

Effects of dietary nucleotides on acute stress response and cannabinoid receptor 1 mRNAs in sole, Solea solea

The objective of the present study was to evaluate the modulation of acute stress response by dietary nucleotides (NT) in sole, Solea solea. A basal diet was supplemented with levels of 0 (normal diet), or 0.4 g NT/kg dry diet for 8 weeks. At the end of feeding trial, fish fed the normal and NT-supplemented diet were subjected to a standardized protocol of disturbance and sampled over a 24h recovery after the stressor exposure. Modulatory effects of NT on acute stress response (cortisol and glucose), proopiomelanocortin (POMC) and cannabinoid receptor 1 splice variants (CB1A and CB1B) mRNA levels were studied. Both plasma cortisol and glucose levels of fish fed NT-supplemented diet were significantly lower than fish fed the control diet at 1 and 4h post-stress time-points. There are no significant effects of dietary NT on POMC and HSP70 mRNA levels. In our study, both CB1A and CB1B trascript levels were induced in fish fed the normal diet at 1 and 4h post-stress intervals. Collectively, the results obtained suggest that dietary NT modulates the CB1-like receptor mRNA expressions leading to attenuation in stressor-induced plasma cortisol level in sole.

Partial cloning, tissue distribution and effects of epigallocatechin gallate on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase mRNA transcripts in goldfish (Carassius auratus).

Epigallocatechin gallate (EGCG), the major active component of the green tea, has recently been found to inhibit 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR) activity in vitro and to modulate lipogenesis in vivo. In this study we have evaluated the effects of short-term in vivo exposure to EGCG (6 μg g(-1) BW or 9 μg g(-1) BW) on hepatic HMGCoAR gene expression of goldfish (Carassius auratus). We initially characterized a partial sequence of goldfish HMGCoAR suggesting that the obtained fragment shares high similarity (>92%) with other fish HMGCoAR sequences. Further, the HMGCoAR transcript was detected in all goldfish tissues (except muscle) but primarily in liver, brain and gonads; on the contrary, low expression levels were found in intestine, heart, gill, and kidney. Both EGCG doses significantly decreased hepatic HMGCoAR mRNA levels 180 min post-injection. HMGCoAR was also significantly down-regulated at 90 min after injection in fish treated with the highest dose of EGCG. Our results demonstrate that hepatic HMGCoAR gene expression is acutely responsive to short-term EGCG exposure in goldfish. This finding suggests a potential role of EGCG in transcriptional regulation of the rate-limiting enzyme in cholesterol synthesis.

Investigating the potential impact of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) on gene biomarker expression and global DNA methylation in loggerhead sea turtles (Caretta caretta) from the Adriatic Sea

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) are priority contaminants that bioaccumulate through the food webs and affect the biology of a variety of resident and migratory species, including sea turtles. Few studies have evaluated toxicological biomarkers of exposure to PAHs and PCBs in these animals. The present paper reports the results of an initial field study to quantify the association between plasma concentrations of PAHs/PCBs and whole blood cell expression of gene biomarkers in juvenile loggerhead sea turtles (Caretta caretta) rescued along the Italian coasts of the northern and central areas of the Adriatic Sea. While detectable levels of PAHs were found in all plasma samples examined, only three PCB congeners (PCB52, PCB95, and PCB149) were noted, with detection percentages ranging between 48% and 57%. A significant correlation was found between 3 of the 6 gene biomarkers assessed (HSP60, CYP1A and ERα) and plasma levels of some PAH congeners. In contrast, no significant association between PCB burden and gene expression was observed. The global DNA methylation levels were significantly and positively correlated with the concentrations of most of the PAHs and only one of the PCB congeners (PCB52). The relation between PAH concentration and gene expression in whole blood cells suggests that these genes may respond to environmental contaminant exposure and are promising candidates for the development of biomarkers for monitoring sea turtle exposure to persistent organic pollutants (POPs).

Effects of endocrine disrupting chemicals on estrogen receptor alpha and heat shock protein 60 gene expression in primary cultures of loggerhead sea turtle (Caretta caretta) erythrocytes

Abstract The loggerhead turtle (Caretta caretta) can be considered a good indicator species for studying the ecological impact of endocrine disrupting chemicals (EDCs) on wildlife. However, the effect of these environmental pollutants on nuclear steroid hormone signaling has not yet been addressed in sea turtles mainly due to the legal constraints of their endangered status. Here we describe the use of primary erythrocyte cell cultures as in vitro models for evaluating the effects of different EDCs on the expression of estrogen receptor &agr; (ER&agr;). In addition, we evaluated erythrocyte toxicity caused by EDCs using Alamar Blue assay and heat shock proteins 60 (HSP60) expression. Primary cultures of erythrocytes were exposed to increasing concentrations of 4‐nonylphenol (4NP), Diisodecyl phthalate (DiDP), Tri‐m‐cresyl phosphate (TMCP) and Tributyltin (TBT) for 48 h. Alamar Blue demonstrated that exposure of erythrocytes to each contaminant for up to 48 h led to a significant impairment of cellular metabolic activity at 100 &mgr;M, with the exception of TBT. Moreover, our data indicate that loggerhead erythrocytes constitutively express ER&agr; and HSP60 at the transcript level and respond to EDCs by up‐regulating their expression. In this regard, ER&agr; was up‐regulated in a dose‐dependent manner after 48 h exposure to both 4NP and TMCP. Interestingly, the dosage‐dependent effects of DiDP on ER&agr; expression were opposite in comparison to that obtained following exposure to the other tested compounds. This work provides the first indication regarding the potential of primary erythrocytes as study models for evaluating the effects of EDCs on sea turtles. HighlightsLoggerhead primary erythrocytes as in vitro model for evaluating the effects of EDCs.48 h exposure to EDCs led to an impairment of erythrocyte metabolic activity.Loggerhead erythrocytes constitutively express ER&agr; and HSP60 at transcript level.Erythrocytes respond to EDCs by up‐regulating the expression of both ER&agr; and HSP60.ER&agr; expression seems to be more sensitive to a wide variety of EDCs than HSP60.