Francesco Alessandro Palermo

Variation of the genetic expression pattern after exposure to estradiol-17β and 4-nonylphenol in male zebrafish (Danio rerio)

There is much concern about the increasing presence in the environment of synthetic chemicals that are able to disrupt the endocrine system. Among these compounds, 4-nonylphenol (4-NP) is one of the most studied xenoestrogens, due to its widespread accumulation in water sediment and consequent presence in fatty acid of aquatic organisms. Here, we have used a zebrafish microarray representing 16,399 genes to study the effects of 4-NP and estradiol-17beta (E2) in adult male zebrafish in order to elucidate the mechanism of action of 4-NP compared with that of E2. The microarray results showed that both 4-NP and E2 induced a strong expression of vitellogenin (VTG), the sex related precursor of the yolk proteins in oviparous vertebrates. Both treatments induced elevated protein turnover upregulating genes involved in proteolysis and those that are constituents of the ribosome. Many genes regulated by 4-NP and E2 are involved in energy metabolism, oxidative stress defense mechanisms, xenobiotic metabolism, and lipid metabolism. A different pattern of expression in the two treatments was found for genes involved in oxidative stress, since E2 seems to induce the mechanism of detoxification, while 4-NP seems to inhibit this protective mechanism of the cell. Overall, these findings demonstrate that the microarray approach can contribute significantly to the understanding of expression patterns induced by E2 and 4-NP in male zebrafish. The results also demonstrate that 4-NP is able to act through an alternative pattern to that of estradiol-17beta, modulating the expression of the same genes in a different manner.

Modulation of cortisol levels, endocannabinoid receptor 1A, proopiomelanocortin and thyroid hormone receptor alpha mRNA expressions by probiotics during sole (Solea solea) larval development.

In the present study, we investigated whether the use of Enterococcus faecium IMC 511 as a probiotic can modulate neuroendocrine system responses during the larval rearing of Solea solea; to this end, the gene expression patterns of proopiomelanocortin (POMC), endocannabinoid receptor 1A (CB1A), and thyroid receptor alpha (TRα) were quantified, and whole-body cortisol levels were measured. Probiotic treatment up-regulated transcription of all selected genes and cortisol concentrations on day 10 post hatch (ph), while on day 30 ph experimental groups showed significantly lower levels of both POMC and CB1A compared to those of the control group. These changes were no longer evident on day 60 ph, when POMC, CB1A, TRα gene expression and cortisol titers were found to be similar in all experimental groups. Our results suggest that metabolic responses to probiotic treatment can be modulated through the activation of genes selected for functional interaction between the hypothalamic-pituitary-thyroid (HPT) axis and the melanocortin and the endocannabinoid systems. Furthermore, the observed (30 ph) down-regulation of both POMC and CB1A gene expression coupled with up-regulation of TRα mRΝΑ levels suggest the activation of a compensatory mechanism that promotes growth and development and perhaps modulates food intake.

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.

Malnutrition may affect common sole (Solea solea L.) growth, pigmentation and stress response: Molecular, biochemical and histological implications

In the last decades there have been several evidences that traditionally used live preys like rotifers and Artemia salina have nutritional deficiencies that result in a general decrease of fish health, causing anomalies in the development, in growth and in pigmentation. In this study a partial of total replacement of traditional live preys with preserved copepods that represent the natural food of the larvae was evaluated during Solea solea culture. In this study a positive effect of co-feeding preserved copepods in sole larviculture was observed since larvae fed this diet growth and survived better, showed a better tolerance to captive conditions and had a better response to the final thermal/density stress-test with respect to larvae fed a traditional diet. Morphometric data were fully supported by molecular and biochemical ones. Moreover, liver histological investigations, revealed that the inclusion of preserved copepods in the larval diet was able to improve lipid assimilation. In conclusion, preserved copepods may be considered a suitable food for sole when used as a supplement to the traditional diet based on rotifers and Artemia nauplii.

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.

4-nonylphenol triggers apoptosis and affects 17-β-estradiol receptors in calvarial osteoblasts.

The present research examines the effects of 4-nonylphenol (4-NP) on mouse primary calvarial osteoblasts (COBs). Incubation of the cells with 4-NP at 10(-5)M and 10(-6)M striking decreased osteoblasts viability and phosphatidylserine (PS) exposure, measured by Annexin V, was greatly enhanced. In addition, an up-regulation of Bax/Bcl2 ratio with a drop in ΔΨm and an increase of cleaved caspase 9 and 3 was found, suggesting that the alkylphenol induced osteoblast death via the mitochondrial-dependent apoptotic pathway. Interestingly, treatment with 4-NP was also able to increase cleaved caspase 8 in parallel with the truncated active Bid (t-Bid) suggesting that 4-NP-mediated apoptosis depends on cross talk between the extrinsic and intrinsic pathways. It is of relevance, that the apoptotic effects of 4-NP overcame 17-β-Estradiol (17-β E(2)) induced-survival on osteoblasts. Also, the alkylphenol interfered with 17-β E(2) regulated estrogen receptors expression.

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.

Involvement of Endocannabinoid CB1 Receptor in the Modulation of Stress Responses Related to Xenoestrogen Exposure

The endocannabinoid system, through the cannabinoid receptor CB1, is involved in the modulation of adaptive responses to environmental conditions. However, little is known about the role of the cannabinergic system, particularly CB1 receptor expression, in relation to the effects induced by xenoestrogens concerning the reproductive axis. Our results demonstrate that only 10−8 mol/L of 17β‐estradiol was able to induce significantly higher levels of CB1A mRNA, while no effects were found after treatment with 4‐nonylphenol (10−8 or 10−6 mol/L); moreover, mRNA expression titers of CB1B did not show any significant change. The estrogenic effects of treatments were evidenced by a dose‐dependent induction of plasma hepatic vitellogenin titers. It can be concluded that low doses of estrogens, and possibly of xenoestrogens, may increase endocannabinoid signaling pathways.

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.