PBCs environmental pollution and epigenome: a new role for androgen receptor-dependent modulation?

Epigenome is an important target of environmental effects, modulating disease susceptibility during the whole life. In our previous studies we have demonstrated that prenatal exposure to polychlorinated biphenyls (PCBs), an important class of endocrine disruptors, alters in liver some histone post-translational modifications (H3K4me3/H4K16Ac) and the expression of the corresponding modulating enzymes (Jarid1b/SirtT1) and reduces the androgen receptor (AR) levels1. Furthermore, it is known that steroid receptors could act also as co-regulator of histone modification enzymes. It is also remarkable that AR and Jarid1b (demethylating enzyme) interact each other and that Jarid1b is able to potentiate the transcriptional activity of AR2. The AR down-regulation, shown by our data, is not directly related to the reduction of H3K4me3 levels in the AR promoter, as our ChIP experiments have indicated1. The observed AR reduction might be related to the down-regulation of AR induced by its own activation3. The aim of this work was to characterize the complex scenario of AR involvement in regulating histone modifications. First of all, we investigated if a reconstituted mixture of PCBs is able to modulate AR transcriptional activity; we observed that PCBs are able to induce AR mediated transcription in a dose dependent way and that Jarid1b presence potentiates the PCB effect on AR transcriptional activity. Furthermore we showed that the localization of PCB- activated AR is influenced by Jarid1b presence. Finally, using AR promoter fused with a reporter gene, we found that PCBs are able to auto-downregulate AR, especially at the presence of Jarid1b. In conclusion it is possible to hypothesize that AR modulation exerted by PCB pass through chromatin structure remodelling. It remains to clarify if AR is involved in mediating PCB induced disruption of Jarid1b

Epigenome and Environment: effects of a PCB exposure on epigenome during early development in the rat

Introduction: Epigenetic represents the genome programming to express appropriate set of genes in a temporal and spatial way during life. Epigenetic signature is dynamic and responsive to environment, especially in critical periods of development. Deregulation of epigenetic mechanisms may be responsible for incorrect transcription patterns, resulting in stable modifications of key physiological processes. Exposure to PCBs (polychlorinated biphenyls) during gestation and lactation, modifies the gene expression of important mediators of sex steroid action and of their receptors. Androgen receptor (AR) could also act as co-regulator of histone modifications enzymes. Aim: to evaluate whether prenatal administration of a reconstituted PCB mixture is able to influence epigenome Results: PCBs reduce histone post-translational modifications (H3K4me3 and H4K16Ac) in a dimorphic way, possibly as the result of a decreased expression of the histone modification enzymes, Jarid1b and SirtT1. AR gene and protein expression is also reduced, especially in females. ChIP performed against H3K4me3 shows a correlation between the expression of some genes involved in embryonic differentiation (e.g., TSHR and GabaRr3) and histone post- translational gene modifications. Conclusion: the influence of PCB exposure during differentiation on epigenome is dimorphic and affects chromatin packaging, probably through an impairment of interaction between androgen receptor and histone modification enzymes

In vitro chronic administration of ERbeta selective ligands and prostate cancer cell growth : hypotheses on the selective role of 3beta-adiol in AR-positive RV1 cells

Prostate cancer (PC) progression from androgen-dependent (AD) to castration-resistant (CR) disease is a process caused by modifications of different signal transduction pathways within tumor microenvironment. Reducing cell proliferation, estrogen receptor beta (ERbeta) is emerging as a potential target in PC chemoprevention. Among the known selective ERbeta ligands, 3beta-Adiol, the endogenous ligand in the prostate, has been proved to counteract PC progression. This study compares the effects of chronic exposure (1-12 weeks) to different ERbeta selective ligands (DPN, 8beta-VE2, 3beta-Adiol) on proliferation of human androgen-responsive CWR22Rv1 cells, representing an intermediate phenotype between the AD- and CR-PC. 3beta-Adiol (10 nM) is the sole ligand decreasing cell proliferation and increasing p21 levels. In vitro transcriptional activity assays were performed to elucidate different behavior between 3beta-Adiol and the other ligands; in these experiments the endogenous and the main ERbeta subtype activation were considered. It is concluded that ERbeta activation has positive effects also in androgen-responsive PC. The underlying mechanisms are still to be clarified and may include the interplay among different ERbeta subtypes and the specific PC microenvironment. ERbeta agonists might be useful in counteracting PC progression, although the final outcome may depend upon the molecular pattern specific to each PC lesion

Dimorphic expression of testosterone metabolizing enzymes in the hypothalamic area of developing rats

Androgen transformation into estrogens through the aromatase enzyme, occurring in the rat hypothalamus during fetal life, leads to male-specific sexual differentiation of brain. Aromatase shows a peak of expression and activity in a limited period during late gestation; however, the possible dimorphism in its expression during embryogenesis is unclear. One of the mechanisms controlling tissue-specific aromatase expression might be the formation of transcript variants, that differ in the 5'-untranslated regions (5'-UTR). Exon If is the major 5'-UTR used in rodent hypothalamic-preoptic area, with low amounts of other variants encoded by different exons I also present. Another enzymatic conversion, possibly involved in brain differentiation, is the 5 alpha-reduction of Testosterone to DHT, catalyzed by two 5 alpha-reductases (5 alpha-R type1 and 2). Aim of the present study is to evaluate, in parallel, by semiquantitative RT-PCR, the dimorphic profile of the three enzymes and the pattern of the brain-specific aromatase expression in male and female rats from gestation-day 16 to postnatal-day 5 (or 15 only for 5 alpha-R1). It has been observed that, in both sexes, 5 alpha-R1 is significantly higher around birth than prenatally, and that 5 alpha-R2 expression appears to be higher in males than in females, particularly just after birth. Moreover, aromatase has two expression peaks, that are male-specific, before and after birth; only exon If is used in males, while different transcripts might be present in females postnatally. It is concluded that rodent brain sexual differentiation probably involves the activation of both 5 alpha-R2 and aromatase enzymes in a sex- and time-specific pattern

Prenatal Aroclor 1254 exposure and brain sexual differentiation: effect on the expression of testosterone metabolizing enzymes and androgen receptors in the hypothalamus of male and female rats

Polychlorinated biphenyls (PCBs) are industrial pollutants detected in human milk, serum and tissues. They readily cross the placenta to accumulate in fetal tissues, particularly the brain. These compounds affect normal brain sexual differentiation by mechanisms that are incompletely understood. The aim of this study was to verify whether a technical mixture of PCBs (Aroclor 1254) would interfere with the normal pattern of expression of hypothalamic aromatase and 5-alpha reductase(s), the two main enzymatic pathways involved in testosterone activation and of androgen receptor (AR). Aroclor 1254 was administered to pregnant rats at a daily dose of 25mg/kg by gavage from days 15 to 19 of gestation (GD15-19). At GD20 the expression of aromatase, 5-alpha reductase types 1 and 2 and androgen receptor (AR) and aromatase activity were evaluated in the hypothalamus of male and female embryos. The direct effect of Aroclor was also evaluated on aromatase activity adding the PCB mixture to hypothalamic homogenates or to primary hypothalamic neuronal cultures. The data indicate that aromatase expression and activity is not altered by prenatal PCB treatment; 5-alpha reductase type 1 is similarly unaffected while 5-alpha reductase type 2 is markedly stimulated by the PCB exposure in females. Aroclor also decreases the expression of the AR in females. The observed in vivo effects are indicative of a possible adverse effect of PCBs on the important metabolic pathways by which testosterone produces its brain effects. In particular the changes of 5-alpha reductase type 2 and AR in females might be one of the mechanisms by which Aroclor exposure during fetal development affects adult sexual behavior in female rat

PLATELET-ENRICHED PLASMA (PRP) AND CYTOSKELETON REARRANGEMENT IN HUMAN OSTEOBLASTS

Aim: The use of platelet-enriched plasma (PRP) has been proposed as a source of growth factors in enhancing new bone formation after fractures. However, the molecular mechanism at the basis of PRP action in tissue regeneration is not fully understood yet. The recent finding that PRP is a potent chemoattractive agent of osteoblasts \u201cin vitro\u201d (Celotti at al. 2006), prompted us to investigate whether the increased cell migration might be due to cytoskeleton rearrangements. Methods: Human osteoblastic cells (SaOS-2), grown to semiconfluence, were pre-treated with different PRP dilutions (1/1000 or 1/100) for different times (30, 60 or 150 minutes). The microchemotaxis assay was performed in Boyden chamber using FCS1% as chemotactic stimulus. The migrated cells were counted under optical microscope; F-actin was labelled with FITC-phalloidin and examined under laser confocal microscope. Results: PRP pre-treatment stimulates cell migration both in absence (DMEM) and in presence of FCS 1% in a dose- and time-dependent manner. Interestingly, PRP pre-treatment induces cytoskeleton reorganization of the actin microfilaments, promoting the formation of F-actin bundles. The extent of the rearrangement is dependent on the concentration and the lenght of the pre-treatment: PRP 1/100 is more effective in early F-actin organization (60 min.), while PRP 1/1000 needs a longer time to have the same effect. Conclusions: The pre-treatment with PRP is able to increase the migratory and chemokinesis capacity of osteoblastic cells by cytoskeleton reorganization. The molecular pathway linking PRP to actin microfilaments rearrangement is under investigation

Platelet-enriched plasma (PRP) and cytoskeleton rearrangement in human osteoblasts

Aim: The use of platelet-enriched plasma (PRP) has been proposed as a source of growth factors in enhancing new bone formation after fractures. However, the molecular mechanism at the basis of PRP action in tissue regeneration is not fully understood yet. The recent finding that PRP is a potent chemoattractive agent of osteoblasts \u201cin vitro\u201d (Celotti at al. 2006), prompted us to investigate whether the increased cell migration might be due to cytoskeleton rearrangements. Methods: Human osteoblastic cells (SaOS-2), grown to semiconfluence, were pre-treated with different PRP dilutions (1/1000 or 1/100) for different times (30, 60 or 150 minutes). The microchemotaxis assay was performed in Boyden chamber using FCS1% as chemotactic stimulus. The migrated cells were counted under optical microscope; F-actin was labelled with FITC-phalloidin and examined under laser confocal microscope. Results: PRP pre-treatment stimulates cell migration both in absence (DMEM) and in presence of FCS 1% in a dose- and time-dependent manner. Interestingly, PRP pre-treatment induces cytoskeleton reorganization of the actin microfilaments, promoting the formation of F-actin bundles. The extent of the rearrangement is dependent on the concentration and the lenght of the pre-treatment: PRP 1/100 is more effective in early F-actin organization (60 min.), while PRP 1/1000 needs a longer time to have the same effect. Conclusions: The pre-treatment with PRP is able to increase the migratory and chemokinesis capacity of osteoblastic cells by cytoskeleton reorganization. The molecular pathway linking PRP to actin microfilaments rearrangement is under investigation