Glyphosate Exposure Induces Cytotoxicity, Mitochondrial Dysfunction and Activation of ERα and ERß Estrogen Receptors in Human Prostate PNT1A Cells.

Glyphosate, the active ingredient of several broad-spectrum herbicides, is widely used throughout the world, although many adverse effects are known. Among these, it has been recognized as an endocrine disruptor. This work aimed to test the effects and potential endocrine disrupting action of glyphosate on PNT1A human prostate cells, an immortalized non-tumor epithelial cell line, possessing both ERα and ERß estrogen receptors. The results showed that glyphosate induces cytotoxicity, mitochondrial dysfunction, and rapid activation of ERα and ERß via nuclear translocation. Molecular analysis indicated a possible involvement of apoptosis in glyphosate-induced cytotoxicology. The apoptotic process could be attributed to alterations in mitochondrial metabolism; therefore, the main parameters of mitochondrial functionality were investigated using the Seahorse analyzer. Impaired mitochondrial function was observed in glyphosate-treated cells, with reductions in ATP production, spare respiratory capacity, and proton leakage, along with increased efficiency of mitochondrial coupling. Finally, the results of immunofluorescence analysis demonstrated that glyphosate acts as an estrogen disruptor determining the nuclear translocation of both ERs. Nuclear translocation occurred independent of dose, faster than the specific hormone, and persisted throughout treatment. In conclusion, the results collected show that in non-tumor prostate cells glyphosate can cause cell death and acts as a xenoestrogen, activating estrogen receptors. The consequent alteration of hormonal functions can have negative effects on the reproductive health of exposed animals, compromising their fertility.

Investigating the Effect of Surface Hydrophilicity on the Destiny of PLGA-Poloxamer Nanoparticles in an In Vivo Animal Model.

This study aimed to examine the impact of different surface properties of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (P NPs) and PLGA-Poloxamer nanoparticles (PP NPs) on their in vivo biodistribution. For this purpose, NPs were formulated via nanoprecipitation and loaded with diphenylhexatriene (DPH), a fluorescent dye. The obtained NPs underwent comprehensive characterization, encompassing their morphology, technological attributes, DPH release rate, and thermodynamic properties. The produced NPs were then administered to wild-type mice via intraperitoneal injection, and, at scheduled time intervals, the animals were euthanized. Blood samples, as well as the liver, lungs, and kidneys, were extracted for histological examination and biodistribution analysis. The findings of this investigation revealed that the presence of poloxamers led to smaller NP sizes and induced partial crystallinity in the NPs. The biodistribution and histological results from in vivo experiments evidenced that both, P and PP NPs, exhibited comparable concentrations in the bloodstream, while P NPs could not be detected in the other organs examined. Conversely, PP NPs were primarily sequestered by the lungs and, to a lesser extent, by the kidneys. Future research endeavors will focus on investigating the behavior of drug-loaded NPs in pathological animal models.

Effects of Dibutylphthalate and Steroid Hormone Mixture on Human Prostate Cells.

Phthalates are a family of aromatic chemical compounds mainly used as plasticizers. Among phthalates, di-n-butyl phthalate (DBP) is a low-molecular-weight phthalate used as a component of many cosmetic products, such as nail polish, and other perfumed personal care products. DBP has toxic effects on reproductive health, inducing testicular damage and developmental malformations. Inside the male reproductive system, the prostate gland reacts to both male and female sex steroids. For this reason, it represents an important target of endocrine-disrupting chemicals (EDCs), compounds that are able to affect the estrogen and androgen signaling pathways, thus interfering with prostate homeostasis and inducing several prostate pathologies. The aim of this project was to investigate the effects of DBP, alone and in combination with testosterone (T), 17ß-estradiol (E2), and both, on the normal PNT1A human prostate cell-derived cell line, to mimic environmental contamination. We showed that DBP and all of the tested mixtures increase cell viability through activation of both estrogen receptor α (ERα) and androgen receptor (AR). DBP modulated steroid receptor levels in a nonmonotonic way, and differently to endogenous hormones. In addition, DBP translocated ERα to the nucleus over different durations and for a more prolonged time than E2, altering the normal responsiveness of prostate cells. However, DBP alone seemed not to influence AR localization, but AR was continuously and persistently activated when DBP was used in combination. Our results show that DBP alone, and in mixture, alters redox homeostasis in prostate cells, leading to a greater increase in cell oxidative susceptibility. In addition, we also demonstrate that DBP increases the migratory potential of PNT1A cells. In conclusion, our findings demonstrate that DBP, alone and in mixtures with endogenous steroid hormones, acts as an EDC, resulting in an altered prostate cell physiology and making these cells more prone to cancer transformation.

Cocaine Effects on Reproductive Behavior and Fertility: An Overview.

Cocaine is one of the most widely used drugs that, due to its molecular properties, causes various behavioral alterations, including sexual behavior. In vivo and in vitro studies conducted mainly in mammals have shown various disorders of sexual activity and morpho-functional dysfunctions of the gonads in both sexes. Although the modalities are still unclear, cocaine has been shown to alter the cell cycle, induce apoptosis, and alter sperm motility. In females, this drug alters the formation of the meiotic spindle as well as may obstruct the ovulation mechanism of mature oocytes. The data provided in this review, in addition to reviewing the current literature on the main effects of cocaine on spermatogenesis and oogenesis mainly in mammals, will hopefully provide a basic overview that may help and support further future studies on the molecular interaction of cocaine and its metabolites with germ cells.

VT-1161-A Tetrazole for Management of Mono- and Dual-Species Biofilms.

VT-1161 is a novel tetrazole antifungal agent with high specificity for fungal CYP51 (compared to human CYP enzymes) which has been proven to have fewer adverse effects and drug-drug interaction profiles due to fewer off-target inhibitors. In this study, we evaluated the anti-biofilm potential of VT-1161 against mono- and dual-species biofilms of Candida albicans, Klebsiella pneumoniae and Staphylococcus aureus. VT-1161 inhibited planktonic growth of all three strains, with an MIC value of 2 µg mL-1 for C. albicans and 0.5 µg mL-1 for K. pneumoniae and S. aureus, and killed 99.9% of the microbial populations, indicating a cytocidal action. Additionally, VT-1161 showed an excellent anti-biofilm action, since it inhibited mono-microbial biofilms by 80% at 0.5 µg mL-1, and dual-species biofilms of C. albicans/K. pneumoniae and C. albicans/S. aureus by 90% at the same concentration. Additionally, the eradication of mature biofilms after 24 h of VT-1161 exposure was excellent, reaching 90% at 2 µg mL-1 for both mono- and dual-species biofilms. In such mixed biofilms, the use of VT-1161 was revealed to be an alternative treatment because it was able to reduce the number of cells of each species during both inhibition and eradication. Since long-term therapy is necessary for most fungal biofilm infections due to their recurrence and obstinacy, VT-1161 showed low cytotoxicity against normal human cell lines and also against the invertebrate model Caenorhabditis elegans. Considering the excellent anti-biofilm potential and its GRAS (generally recognized as safe) status, VT-1161 may find use in the prevention or therapeutic treatment of mono- or poly-microbial biofilms.

Antiinflammatory Activities of Curcumin and Spirulina: Focus on Their Role against COVID-19.

Nutraceuticals have for several years aroused the interest of researchers for their countless properties, including the management of viral infections. In the context of the COVID-19 pandemic, studies and research on the antiviral properties of nutraceuticals have greatly increased. More specifically, over the past two years, researchers have focused on analyzing the possible role of nutraceuticals in reducing the risk of SARS-CoV-2 infection or mitigating the symptoms of COVID-19. Among nutraceuticals, turmeric, extracted from the rhizome of the Curcuma Longa plant, and spirulina, commercial name of the cyanobacterium Arthrospira platensis, have assumed considerable importance in recent years. The purpose of this review is to collect, through a search of the most recent articles on Pubmed, the scientific evidence on the role of these two compounds in the fight against COVID-19. In the last two years many hypotheses, some confirmed by clinical and experimental studies, have been made on the possible use of turmeric against COVID-19, while on spirulina and its possible role against SARS-CoV-2 infection information is much less. The demonstrated antiviral properties of spirulina and the fact that these cyanobacteria may modulate or modify some mechanisms also involved in the onset of COVID-19, lead us to think that it may have the same importance as curcumin in fighting this disease and to speculate on the possible combined use of these two substances to obtain a synergistic effect.

Transmission Electron Microscopy: A Method for Studying the Adrenal Chromaffin Cells.

Transmission electron microscopy and the use of glutaraldehyde-osmium fixation allow to distinguish norepinephrine from epinephrine granules in the adrenochromaffin cells, a difficult distinction with histochemical methods if both types of granules are present in the same cell. Here we describe all the steps necessary to process the adrenochromaffin tissue for the transmission electron microscopy; this protocol is suitable for any kind of adrenal tissue, and personally we used it in mammals, reptiles, and amphibians.

The Arylamidine T-2307 as a Novel Treatment for the Prevention and Eradication of Candida tropicalis Biofilms.

Candida tropicalis is an emerging pathogen with a high mortality rate due to its virulence factors, including biofilm formation, that has important repercussions on the public health system. The ability of C. tropicalis to form biofilms, which are potentially more resistant to antifungal drugs and the consequent increasing antimicrobial resistance, highlights an urgent need for the development of novel antifungal. The present study analyzed the antibiofilm capacity of the arylamidine T-2307 on two strains of Candida tropicalis. Antimicrobial activity and time-killing assays were performed to evaluate the anticandidal effects of T-2307, the antibiofilm ability on biomass inhibition and eradication was evaluated by the crystal violet (CV) method. Furthermore, in Galleria mellonella infected larvae an increased survival after pre-and post- treatment with T-2307 was observed. The MTT test was used to determine the viability of immortalized human prostate epithelial cells (PNT1A) after exposure to different concentrations of T-2307. Levels of interleukin IL-4, IL-8, IL-10 were quantified after Candida infection of PNT1A cells and treatment. Active doses of T-2307 did not affect the viability of PNT1A cells, and drug concentrations of 0.005 or 0.01 µg mL-1 inhibited the secretion of inflammatory cytokines. Taken together, these results provide new information on T-2307, indicating this drug as a new and promising alternative therapeutic option for the treatment of Candida infections.

Effect of Myrtenol and Its Synergistic Interactions with Antimicrobial Drugs in the Inhibition of Single and Mixed Biofilms of Candida auris and Klebsiella pneumoniae.

The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.

Toxic Effects on Thyroid Gland of Male Adult Lizards (Podarcis Siculus) in Contact with PolyChlorinated Biphenyls (PCBs)-Contaminated Soil.

Skin exposure is considered a potentially significant but little-studied pathway for PolyChlorinated Biphenyls uptake in terrestrial reptiles. In this study, a native Italian lizard, Podarcis siculus, was exposed to PCBs-contaminated soil for 120 days. Tissues distribution of PCBs, thyroid hormone levels, and thyroid histo-physiopathology were examined. The accumulation of PCBs in skin, plasma, liver, kidney, and brain were highest at 120 days. The alteration of triiodothyronine (T3) and thyroxine (T4) levels after different concentrations and times to exposure of PCBs was accompanied by the changes in the hormones involved in the hypothalamus-pituitary-thyroid (HPT) axis, namely Thyrotropin Releasing Hormone (TRH) and Thyroid Stimulating Hormone (TSH). Moreover, hepatic levels of deiodinase II (5'ORDII) and content of T3 were positively correlated to exposure to PCBs. These results indicated that in lizards, PCBs exposure through the skin has the potential to disrupt the thyroid endocrine system. Overall, the observed results indicate that PCBs could be associated with changes in thyroid homeostasis in these reptiles, through direct interactions with the metabolism of T4 and T3 through the HPT axis or indirect interactions with peripheral deiodination.

Combined Effects of Different Endocrine-Disrupting Chemicals (EDCs) on Prostate Gland.

Endocrine-disrupting chemicals (EDCs) belong to a heterogeneous class of environmental pollutants widely diffused in different aquatic and terrestrial habitats. This implies that humans and animals are continuously exposed to EDCs from different matrices and sources. Moreover, pollution derived from anthropic and industrial activities leads to combined exposure to substances with multiple mechanisms of action on the endocrine system and correlated cell and tissue targets. For this reason, specific organs, such as the prostate gland, which physiologically are under the control of hormones like androgens and estrogens, are particularly sensitive to EDC stimulation. It is now well known that an imbalance in hormonal regulation can cause the onset of various prostate diseases, from benign prostate hyperplasia to prostate cancer. In this review, starting with the description of normal prostate gland anatomy and embryology, we summarize recent studies reporting on how the multiple and simultaneous exposure to estrogenic and anti-androgenic compounds belonging to EDCs are responsible for an increase in prostate disease incidence in the human population.

Alkyphenol Exposure Alters Steroidogenesis in Male Lizard Podarcis siculus.

BACKGROUND: Nonylphenol (NP) and Octylphenol (OP) are persistent and non-biodegradable environmental contaminants classified as endocrine disruptor chemicals (EDCs). These compounds are widely used in several industrial applications and present estrogen-like properties, which have extensively been studied in aquatic organisms. The present study aimed to verify the interference of these compounds alone, and in mixture, on the reproductive cycle of the male terrestrial vertebrate Podarcis siculus, focusing mainly on the steroidogenesis process. METHODS: Male lizards have been treated with different injections of both NP and OP alone and in mixture, and evaluation has been carried out using a histological approach. RESULTS: Results obtained showed that both substances are able to alter both testis histology and localization of key steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß- hydroxysteroid dehydrogenase (17ß-HSD) and P450 aromatase. Moreover, OP exerts a preponderant effect, and the P450 aromatase represents the major target of both chemicals. CONCLUSIONS: In conclusion, NP and OP inhibit steroidogenesis, which in turn may reduce the reproductive capacity of the specimens.

OctylPhenol (OP) Alone and in Combination with NonylPhenol (NP) Alters the Structure and the Function of Thyroid Gland of the Lizard Podarcis siculus.

Different environmental contaminants disturb the thyroid system at many levels. AlkylPhenols (APs), by-products of microbial degradation of AlkylPhenol Polyethoxylates (APEOs), constitute an important class of Endocrine Disrupting Chemicals (EDCs), the two most often used environmental APs being 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP). The purpose of the present study was to investigate the effects on the thyroid gland of the bioindicator Podarcis siculus of OP alone and in combination with NP. We used radioimmunoassay to determine their effects on plasma 3,3',5-triiodo-L-thyronine (T3), 3,3',5,5'-L-thyroxine (T4), thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH) levels in adult male lizards. We also investigated the impacts of AP treatments on hepatic 5'ORD (type II) deiodinase and hepatic content of T3 and T4. After OP and OP + NP administration, TRH levels increased, whereas TSH, T3, and T4 levels decreased. Lizards treated with OP and OP + NP had a higher concentration of T3 in the liver and 5'ORD (type II) activity, whereas T4 concentrations were lower than that observed in the control group. Moreover, histological examination showed that the volume of the thyroid follicles became smaller in treated lizards suggesting that that thyroid follicular epithelial cells were not functionally active following treatment. This data collectively suggest a severe interference with hypothalamus-pituitary-thyroid axis and a systemic imbalance of thyroid hormones.

Emerging endocrine disruptors in two edible fish from the Persian Gulf: Occurrence, congener profile, and human health risk assessment.

The occurrence of endocrine disrupting chemicals (EDCs) has been determined in two widely consumed fish species from Persian Gulf i.e., Epinephelus coioides and Platycephalus indicus by applying a validated analytical for the simultaneous detection of fourteen EDCs. The concentrations of all detected EDCs were greater in the liver than in the muscle (except for bisphenol A in P. indicus), suggesting a prolonged exposure of the fishes to these pollutants in the Persian Gulf. Specifically, the results showed that di (2-ethylhexyl) phthalate (DEHP) was the compound detected most frequently and at the highest concentration in both species. DEHP levels in ranged from 6.68 to 297.48 µg g-dw-1 and from 13.32 to 350.52 µg g-dw-1, in muscle and in liver, respectively. A risk assessment study was conducted, and demonstrated that consuming two fish based- meals per week may result in a moderate risk especially for vulnerable population groups.

Adrenal gland response to endocrine disrupting chemicals in fishes, amphibians and reptiles: A comparative overview.

The adrenal gland is an essential component of the body stress response; it is formed by two portions: a steroidogenic and a chromaffin tissue. Despite the anatomy of adrenal gland is different among classes of vertebrates, the hormones produced are almost the same. During stress, these hormones contribute to body homeostasis and maintenance of ion balance. The adrenal gland is very sensitive to toxic compounds, many of which behave like endocrine-disruptor chemicals (EDCs). They contribute to alter the endocrine system in wildlife and humans and are considered as possible responsible of the decline of several vertebrate ectotherms. Considering that EDCs regularly can be found in all environmental matrices, the aim of this review is to collect information about the impact of these chemical compounds on the adrenal gland of fishes, amphibians and reptiles. In particular, this review shows the different behavior of these "sentinel species" when they are exposed to stress condition. The data supplied in this review can help to further elucidate the role of EDCs and their harmful impact on the survival of these vertebrates.

Effects of alkylphenols mixture on the adrenal gland of the lizard Podarcis sicula.

Alkylphenols (AP) are widespread environmental compounds belonging to the large family of substances known as Endocrine Disrupting Chemicals (EDCs). The present study was carried out to assess the effects of Octylphenol (OP) alone and in combination with Nonylphenol (NP) on the hypothalamus-pituitary-adrenal gland (HPA) axis of the lizard Podarcis sicula. Lizards are good bioindicators due to their features such as wide distribution, large population and good sensitivity to contaminants. Results obtained showed a time and dose-dependent stimulation of the HPA together with a high variation of both catecholamine plasma levels and greater vascularization and hypertrophy of steroidogenic cord of adrenal gland after both OP and OP + NP treatments. Interestingly, the OP + NP mixture treatment has provoked a state of stress of the adrenal gland which in fact appeared to be characterized by the presence of a marked macrophage infiltration which can be seen especially close to the connective capsule surrounding the gland. This macrophage infiltration could be an evidence of a particularly pronounced inflammatory state to indicate, probably, an animal's response to a non-physiological situation.

Differential expression of several factors involved in placental development in normal and abnormal condition.

The placenta, a temporary organ that forms during pregnancy, is the largest fetal organ and the first to develop. It is recognized as an organ that plays a vital role as a metabolic and physical barrier in the fetoplacental unit; throughout fetal development it acts as the lungs, gut, kidneys, and liver of the fetus. When its two components, the fetal and the maternal one, successfully interact, pregnancy proceeds healthily. However, in some cases there may be pregnancy disorders, such as preeclampsia (PE) and gestational diabetes mellitus (GDM), which can lead to a different outcome for the mother and the newborn. In recent years, several studies have been conducted to try to understand how the expression of factors involved in the development of the placenta varies under pathological conditions compared with normal conditions. The purpose of this review is to summarize recent discoveries in this field.

Immunolocalization of aromatase P450 in the epididymis of Podarcis sicula and Rattus rattus.

The goal of this study was to evaluate P450 aromatase localization in the epididymis of two different vertebrates: the lizard Podarcis sicula, a seasonal breeder, and Rattus rattus, a continuous breeder. P450 aromatase is a key enzyme involved in the local control of spermatogenesis and steroidogenesis and we proved for the first time that this enzyme is represented in the epididymis of both P. sicula and R. rattus. In details, P450 aromatase was well represented in epithelial and myoid cells and in the connective tissue of P. sicula epididymis during the reproductive period; instead, during autumnal resumption this enzyme was absent in the connective tissue. During the non-reproductive period, P450 aromatase was localized only in myoid cells of P. sicula epididymis, whereas in R. rattus it was localized both in myoid cells and connective tissue. Our findings, the first on the epididymis aromatase localization in the vertebrates, suggest a possible role of P450 aromatase in the control of male genital tract function, particularly in sperm maturation.

Intrauterine exposure to diethylhexyl phthalate disrupts gap junctions in the fetal rat testis.

Fetal exposure to certain phthalate esters can disrupt testis development in rodents and lead to male reproductive disorders, but with a causal link less certain in humans. Di(2-ethylhexyl) phthalate (DEHP) is one of the most common phthalates found in the environment and in rodents it is known to induce serious testis toxicity, as well as male reproductive disorders including cryptorchidism, hypospadias, impaired spermatogenesis and reduced fertility. In this study, we show that perinatal DEHP exposure disrupts gap junction localization in fetal and postnatal rat testis and correlate these findings to morphological changes. The protein Connexin 43 (CX43), normally expressed strongly in testicular gap junctions, was markedly downregulated in Leydig cells of DEHP-exposed fetal testes. In the postnatal testes, CX43 expression was recovered in the DEHP-exposed animals, even though Leydig cell clusters and malformed cords with intratubular Leydig cells were still present.

Nonylphenol acts on prostate adenocarcinoma cells via estrogen molecular pathways.

Estrogens play a role in the patho-physiology of the prostate. In the present work we studied the effects of nonylphenol (NP), a xenoestrogen, on human adenocarcinoma prostate cells (LNCaP). In order to understand molecular and cellular involvement, we observed the effects on cell cycle and we investigated the expression and the cellular localization of estrogen receptors and gene expression of cyclin D1, ki-67, c-myc, IL-8, IL-1ß. We performed the same experiments with 17ß-estradiol (E2), the most abundant estrogen circulating in nonpregnant humans in order to compare these two different substances. We demonstrated the ability of 1 × 10-10 M NP to induce proliferation of LNCaP, S-phase progression, increase of ERα expression and its translocation from the cytoplasm to the nucleus. Moreover, we observed an up-regulation of key target genes involved in cell cycle and inflammation process. Particularly, after NP treatment, IL-8 and IL-1ß mRNA levels are increased more than 50% indicating a major NP involvement in inflammation processes than E2. These data suggest the proliferative effects of NP on prostate adenocarcinoma cells and highlight some aspects of molecular pathways involved in prostate responses to NP.