Transcriptional analyses reveal different mechanism of toxicity for a chronic exposure to fluoxetine and venlafaxine on the brain of the marine fish Dicentrarchrus labrax.

Selective serotonin reuptake inhibitor (SSRI) and serotonin norepinephrine reuptake inhibitor (SNRI) are prescribed for clinical depression and detected in aquatic ecosystems. The main aim of this study was to explore and evaluate transcriptional responses of neurotransmitter genes in the brain of a marine fish species, European seabass, and to analyze global brain transcriptomic changes by a RNA-seq technology (MACE, massive analysis of cDNA ends). The juveniles were exposed to two psychopharmaceuticals: (i) fluoxetine (FLX) at the concentration of 0.5 µg/L and 50 µg/L; (ii) venlafaxine (VENX) at the concentration of 0.01 µg/L and 1 µg/L. The exposures were performed for 21 days, followed by a 7-day recovery period to assess the reversibility of effects. Both psychopharmaceuticals affected differentially the neurotransmitter mRNA expression analyzed by RT-qPCR (serotonin receptors: 5-ht3a, 5-ht3b; dopamine receptors: d2, d3; neurotransmitter transporter: sert, vmat; degrading enzyme: mao). Transcriptomic analyses after 21 days of exposure revealed 689 and 632 significant different transcripts by FLX at 0.5 and 50 µg/L, respectively, and 432 and 1250 by VENX at 0.01 and 1 µg/L, respectively, and confirmed different mechanism of toxicity between both compounds. At environmental concentrations, more general pathways including energy metabolism were affected, while at the higher concentration effects on neurotransmitter pathways were observed (FLX: exocytosis and vesicle formation; VENX: small molecule catabolism regulating dopamine and tyrosine level). These results provided new insights into the chronic effects of psychopharmaceutical compounds on marine fish and suggest the need of a separate ecotoxicological risk analysis.

The DNA methyltransferase DNMT3A contributes to autophagy long-term memory.

Macroautophagy/autophagy is a conserved catabolic pathway that targets cytoplasmic components for their degradation and recycling in an autophagosome-dependent lysosomal manner. Under physiological conditions, this process maintains cellular homeostasis. However, autophagy can be stimulated upon different forms of cellular stress, ranging from nutrient starvation to exposure to drugs. Thus, this pathway can be seen as a central component of the integrated and adaptive stress response. Here, we report that even brief induction of autophagy is coupled in vitro to a persistent downregulation of the expression of MAP1LC3 isoforms, which are key components of the autophagy core machinery. In fact, DNA-methylation mediated by de novo DNA methyltransferase DNMT3A of MAP1LC3 loci upon autophagy stimulation leads to the observed long-term decrease of MAP1LC3 isoforms at transcriptional level. Finally, we report that the downregulation of MAP1LC3 expression can be observed in vivo in zebrafish larvae and mice exposed to a transient autophagy stimulus. This epigenetic memory of autophagy provides some understanding of the long-term effect of autophagy induction and offers a possible mechanism for its decline upon aging, pathological conditions, or in response to treatment interventions.Abbreviations: ACTB: actin beta; ATG: autophagy-related; 5-Aza: 5-aza-2'-deoxycytidine; BafA1: bafilomycin A1; CBZ: carbamazepine; CDKN2A: cyclin dependent kinase inhibitor 2A; ChIP: chromatin immunoprecipitation; Clon.: clonidine; CpG: cytosine-guanine dinucleotide: DMSO: dimethyl sulfoxide; DNA: deoxyribonucleic acid; DNMT: DNA methyltransferase; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase alpha; DNMT3B: DNA methyltransferase beta; dpf: days post-fertilization; EBSS: Earle's balanced salt solution; EM: Zebrafish embryo medium; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GRO-Seq: Global Run-On sequencing; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP1LC3B2: microtubule-associated protein 1 light chain 3 beta 2; MEM: minimum essential medium; MEF: mouse embryonic fibroblasts; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction; SQSTM1/p62: sequestosome 1; Starv.: starvation; Treh.: trehalose; ULK1: unc-51 like autophagy activating kinase 1.

Effects of norfluoxetine and venlafaxine in zebrafish larvae: Molecular data.

The data presented herein relates to the article entitled "Norfluoxetine and venlafaxine in zebrafish larvae: single and combined toxicity of two pharmaceutical products relevant for risk assessment" [1]. Recent studies have shown the occurrence of active metabolites of human and veterinary pharmaceuticals in surface and wastewaters. Besides their biological activity, some are predicted to interact with the same molecular targets of their parental compounds, thus showing the potential to elicit detrimental effects on animals. Despite this, limited investigation on their effects on aquatic animals has been done. Genomic material resulting from zebrafish (Danio rerio) larvae exposed to the psychoactive compounds norfluoxetine (main fluoxetine metabolite), venlafaxine, or their mixture was collected for gene expression analysis of a determined pool of genes potentially involved in their mode-of-action and metabolism. Molecular parameters are a cost-effective and reliable way to understand modes-of-action and the potential risk of micropollutants, such as pharmaceutical products, in non-target organisms. Moreover, gene expression patterns can provide crucial complementary information to improve risk assessment, and monitoring of affected systems. The data reported in this article was used to depict the effects of single or combined exposure to norfluoxetine and venlafaxine and identify biomarkers of exposure to these compounds of interest to diagnose exposure and routine monitoring.

Mixture effects of oxygenated PAHs and benzo[a]pyrene on cardiovascular development and function in zebrafish embryos.

Polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs (oxy-PAHs), are common environmental pollutants known to cause health effects in humans and wild-life. In particular, vertebrate cardiovascular development and function are sensitive to PACs. However, the interactive effects of PAHs and oxy-PAHs on cardiovascular endpoints have not been well studied. In this study, we used zebrafish embryos (ZFEs) as a model to examine developmental and cardiovascular toxicities induced by the three environmental oxy-PAHs benzo[a]fluorenone (BFLO), 4H-cyclopenta[def]phenanthren-4-one (4H-CPO) and, 6H-benzo[cd]pyren-6-one (6H-BPO), and the PAH benzo[a]pyrene (BaP) either as single exposures or binary oxy-PAH + PAH mixtures. 6H-BPO induced developmental and cardiovascular toxicity, including reduced heartbeat rate and blood flow, at lower doses compared to the other compounds. Exposure to binary mixtures generally caused enhanced toxicity and induction of aryl hydrocarbon receptor (AhR)-regulated gene expression (ahr2 and cyp1a) compared to single compound exposure. This was associated with differential expression of genes involved in cardiovascular development and function including atp2a2, myh6, tbx5 and zerg. AhR-knock-down significantly reduced the cardiovascular toxicity of 6H-BPO and its binary mixture with BaP indicating a significant AhR-dependence of the effects. Measurements of internal concentrations showed that the toxicokinetics of BaP and 6H-BPO were altered in the binary mixture compared to the single compound exposure, and most likely due to CYP1 inhibition by 6H-BPO. Altogether, these data support that similar to interactions between PAHs, mixtures of PAHs and oxy-PAHs may cause increased developmental and cardiovascular toxicity in ZFEs through an AhR-dependent mechanism.

Similar polycyclic aromatic hydrocarbon and genotoxicity profiles of atmospheric particulate matter from cities on three different continents.

The extractable organic material (EOM) from atmospheric total suspended particles (TSP) contains several organic compounds including non-substituted polycyclic aromatic hydrocarbons (PAHs), alkyl-PAHs, and nitro-PAHs. These chemicals seem to be among the key drivers of TSP genotoxicity. We have shown previously that the mutagenic potencies of the EOM from Limeira, Stockholm, and Kyoto, cities with markedly different meteorological conditions and pollution sources are similar. Here we compare the profiles of non-substituted PAHs (27 congeners), alkyl-PAHs (15 congeners), and nitro-PAHs (7 congeners) from the same EOM samples from these cities. We also compared the genotoxicity profiles using comet and micronucleus assays in human bronchial epithelial cells. The profiles of PAHs, as well as the cytotoxic and genotoxic potencies when expressed in EOM, were quite similar among the studied cities. It seems that despite the differences in meteorological conditions and pollution sources of the cities, removal, mixing, and different atmospheric transformation processes may be contributing to the similarity of the PAHs composition and genotoxicity profiles. More studies are required to verify if this would be a general rule applicable to other cities. Although these profiles were similar for all three cities, the EOM concentration in the atmospheres is markedly different. Thus, the population of Limeira (∼10-fold more EOM/m3 than Stockholm and ∼6-fold more than Kyoto) is exposed to higher concentrations of genotoxic pollutants, and Kyoto's population is 1.5-fold more exposed than Stockholm's. Therefore, to reduce the risk of human exposure to TSP genotoxins, the volume of emissions needs to be reduced.

In vitro and in vivo genotoxicity of oxygenated polycyclic aromatic hydrocarbons.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of environmental pollutants found in complex mixtures together with PAHs. In contrast to the extensively studied PAHs, which have been established to have mutagenic and carcinogenic properties, much less is known about the effects of oxy-PAHs. The present work aimed to investigate the genotoxic potency of a set of environmentally relevant oxy-PAHs along with environmental soil samples in human bronchial epithelial cells (HBEC). We found that all oxy-PAHs tested induced DNA strand breaks in a dose-dependent manner and some of the oxy-PAHs further induced micronuclei formation. Our results showed weak effects in response to the oxy-PAH containing subfraction of the soil sample. The genotoxic potency was confirmed in both HBEC and HepG2 cells following exposure to oxy-PAHs by an increased level of phospho-Chk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro. We further exposed zebrafish embryos to single oxy-PAHs or a binary mixture with PAH benzo[a]pyrene (B[a]P) and found the mixture to induce comparable or greater effects on the induction of DNA strand breaks compared to the sum of that induced by B[a]P and oxy-PAHs alone. In conclusion, oxy-PAHs were found to elicit genotoxic effects at similar or higher levels to that of B[a]P which indicates that oxy-PAHs may contribute significantly to the total carcinogenic potency of environmental PAH mixtures. This emphasizes further investigations of these compounds as well as the need to include oxy-PAHs in environmental monitoring programs in order to improve health risk assessment.

Histopathological lesions, P-glycoprotein and PCNA expression in zebrafish (Danio rerio) liver after a single exposure to diethylnitrosamine.

The presence of carcinogenic compounds in the aquatic environment is a recognized problem. ABC transporters are well known players in the multidrug-resistance (MDR) phenomenon in mammals associated with resistance to chemotherapy, however little is known in fish species. Thus, the aim of this study was to induce hepatic tumours and evaluate long-term effects on P-glycoprotein (P-gp) and proliferating cell nuclear antigen (PCNA) proteins in Danio rerio liver, after exposure to diethylnitrosamine (DEN). Several hepatic histopathological alterations were observed in zebrafish after exposure to DEN including pre-neoplastic lesions 6 and 9 months post-exposure. After 3, 6 and 9 months of exposure to DEN, P-gp and PCNA proteins expression were up-regulated. In conclusion, this study has shown that zebrafish ABC transporters can play a similar role as in human disease, hence zebrafish can be used also as a biological model to investigate in more deep mechanisms involved in disease processes.

Obesity and prostate cancer: gene expression signature of human periprostatic adipose tissue.

BACKGROUND: Periprostatic (PP) adipose tissue surrounds the prostate, an organ with a high predisposition to become malignant. Frequently, growing prostatic tumor cells extend beyond the prostatic organ towards this fat depot. This study aimed to determine the genome-wide expression of genes in PP adipose tissue in obesity/overweight (OB/OW) and prostate cancer patients. METHODS: Differentially expressed genes in human PP adipose tissue were identified using microarrays. Analyses were conducted according to the donors' body mass index characteristics (OB/OW versus lean) and prostate disease (extra prostatic cancer versus organ confined prostate cancer versus benign prostatic hyperplasia). Selected genes with altered expression were validated by real-time PCR. Ingenuity Pathway Analysis (IPA) was used to investigate gene ontology, canonical pathways and functional networks. RESULTS: In the PP adipose tissue of OB/OW subjects, we found altered expression of genes encoding molecules involved in adipogenic/anti-lipolytic, proliferative/anti-apoptotic, and mild immunoinflammatory processes (for example, FADS1, down-regulated, and LEP and ANGPT1, both up-regulated). Conversely, in the PP adipose tissue of subjects with prostate cancer, altered genes were related to adipose tissue cellular activity (increased cell proliferation/differentiation, cell cycle activation and anti-apoptosis), whereas a downward impact on immunity and inflammation was also observed, mostly related to the complement (down-regulation of CFH). Interestingly, we found that the microRNA MIRLET7A2 was overexpressed in the PP adipose tissue of prostate cancer patients. CONCLUSIONS: Obesity and excess adiposity modified the expression of PP adipose tissue genes to ultimately foster fat mass growth. In patients with prostate cancer the expression profile of PP adipose tissue accounted for hypercellularity and reduced immunosurveillance. Both findings may be liable to promote a favorable environment for prostate cancer progression.

Performance of an adipokine pathway-based multilocus genetic risk score for prostate cancer risk prediction.

Few biomarkers are available to predict prostate cancer risk. Single nucleotide polymorphisms (SNPs) tend to have weak individual effects but, in combination, they have stronger predictive value. Adipokine pathways have been implicated in the pathogenesis. We used a candidate pathway approach to investigate 29 functional SNPs in key genes from relevant adipokine pathways in a sample of 1006 men eligible for prostate biopsy. We used stepwise multivariate logistic regression and bootstrapping to develop a multilocus genetic risk score by weighting each risk SNP empirically based on its association with disease. Seven common functional polymorphisms were associated with overall and high-grade prostate cancer (Gleason≥7), whereas three variants were associated with high metastatic-risk prostate cancer (PSA≥20 ng/mL and/or Gleason≥8). The addition of genetic variants to age and PSA improved the predictive accuracy for overall and high-grade prostate cancer, using either the area under the receiver-operating characteristics curves (P<0.02), the net reclassification improvement (P<0.001) and integrated discrimination improvement (P<0.001) measures. These results suggest that functional polymorphisms in adipokine pathways may act individually and cumulatively to affect risk and severity of prostate cancer, supporting the influence of adipokine pathways in the pathogenesis of prostate cancer. Use of such adipokine multilocus genetic risk score can enhance the predictive value of PSA and age in estimating absolute risk, which supports further evaluation of its clinical significance.

Human periprostatic adipose tissue promotes prostate cancer aggressiveness in vitro.

BACKGROUND: Obesity is associated with prostate cancer aggressiveness and mortality. The contribution of periprostatic adipose tissue, which is often infiltrated by malignant cells, to cancer progression is largely unknown. Thus, this study aimed to determine if periprostatic adipose tissue is linked with aggressive tumor biology in prostate cancer. METHODS: Supernatants of whole adipose tissue (explants) or stromal vascular fraction (SVF) from paired fat samples of periprostatic (PP) and pre-peritoneal visceral (VIS) anatomic origin from different donors were prepared and analyzed for matrix metalloproteinases (MMPs) 2 and 9 activity. The effects of those conditioned media (CM) on growth and migration of hormone-refractory (PC-3) and hormone-sensitive (LNCaP) prostate cancer cells were measured. RESULTS: We show here that PP adipose tissue of overweight men has higher MMP9 activity in comparison with normal subjects. The observed increased activities of both MMP2 and MMP9 in PP whole adipose tissue explants, likely reveal the contribution of adipocytes plus stromal-vascular fraction (SVF) as opposed to SVF alone. MMP2 activity was higher for PP when compared to VIS adipose tissue. When PC-3 cells were stimulated with CM from PP adipose tissue explants, increased proliferative and migratory capacities were observed, but not in the presence of SVF. Conversely, when LNCaP cells were stimulated with PP explants CM, we found enhanced motility despite the inhibition of proliferation, whereas CM derived from SVF increased both cell proliferation and motility. Explants culture and using adipose tissue of PP origin are most effective in promoting proliferation and migration of PC-3 cells, as respectively compared with SVF culture and using adipose tissue of VIS origin. In LNCaP cells, while explants CM cause increased migration compared to SVF, the use of PP adipose tissue to generate CM result in the increase of both cellular proliferation and migration. CONCLUSIONS: Our findings suggest that the PP depot has the potential to modulate extra-prostatic tumor cells' microenvironment through increased MMPs activity and to promote prostate cancer cell survival and migration. Adipocyte-derived factors likely have a relevant proliferative and motile role.

Tumor cell-educated periprostatic adipose tissue acquires an aggressive cancer-promoting secretory profile.

BACKGROUND/AIMS: The microenvironment produces important factors that are crucial to prostate cancer (PCa) progression. However, the extent to which the cancer cells stimulate periprostatic adipose tissue (PPAT) to produce these proteins is largely unknown. Our purpose was to determine whether PCa cell-derived factors influence PPAT metabolic activity. METHODS: Primary cultures of human PPAT samples from PCa patients (adipose tissue organotypic explants and primary stromal vascular fraction, SVF) were stimulated with conditioned medium (CM) collected from prostate carcinoma (PC3) cells. Cultures without CM were used as control. We used multiplex analysis and ELISA for protein quantification, qPCR to determine mitochondrial DNA (mtDNA) copy number and zymography for matrix metalloproteinase activity, in order to evaluate the response of adipose tissue explants and SVFs to PC3 CM. RESULTS: Stimulation of PPAT explants with PCa PC3 CM induced adipokines associated with cancer progression (osteopontin, tumoral necrosis factor alpha and interleukin-6) and reduced the expression of the protective adipokine adiponectin. Notably, osteopontin protein expression was 13-fold upregulated. Matrix metalloproteinase 9 activity and mitochondrial DNA copy number were higher after stimulation with cancer CM. Stromovascular cells from PPAT in culture were not influenced by tumor-derived factors. CONCLUSION: The modulation of adipokine expression by tumor CM indicates the pervasive extent to which tumor cells command PPAT to produce factors favorable to their aggressiveness.

IL-6/IL-6R as a potential key signaling pathway in prostate cancer development.

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in prostate regulation and in prostate cancer (PC) development/progression. IL-6 acts as a paracrine and autocrine growth stimulator in benign and tumor prostate cells. The levels of IL-6 and respective receptors are increased during prostate carcinogenesis and tumor progression. Several studies reported that increased serum and plasma IL-6 and soluble interleukin-6 receptor levels are associated with aggressiveness of the disease and are associated with a poor prognosis in PC patients. In PC treatment, patients diagnosed with advanced stages are frequently submitted to hormonal castration, although most patients will eventually fail this therapy and die from recurrent castration-resistant prostate cancer (CRPC). Therefore, it is important to understand the mechanisms involved in CRPC. Several pathways have been proposed to be involved in CRPC development, and their understanding will improve the way to more effective therapies. In fact, the prostate is known to be dependent, not exclusively, on androgens, but also on growth factors and cytokines. The signaling pathway mediated by IL-6 may be an alternative pathway in the CRPC phenotype acquisition and cancer progression, under androgen deprivation conditions. The principal goal of this review is to evaluate the role of IL-6 pathway signaling in human PC development and progression and discuss the interaction of this pathway with the androgen recepto pathway. Furthermore, we intend to evaluate the inclusion of IL-6 and its receptor levels as a putative new class of tumor biomarkers.The IL-6/IL-6R signaling pathway may be included as a putative molecular marker for aggressiveness in PC and it may be able to maintain tumor growth through the AR pathway under androgen-deprivation conditions. The importance of the IL-6/IL-6R pathway in regulation of PC cells makes it a good candidate for targeted therapy.