Fighting bisphenol a-induced male infertility: The power of antioxidants

Bisphenol A (BPA), a well-known endocrine disruptor present in epoxy resins and poly-carbonate plastics, negatively disturbs the male reproductive system affecting male fertility. In vivo studies showed that BPA exposure has deleterious effects on spermatogenesis by disturbing the hypothalamic–pituitary–gonadal axis and inducing oxidative stress in testis. This compound seems to disrupt hormone signalling even at low concentrations, modifying the levels of inhibin B, oestradiol, and testosterone. The adverse effects on seminal parameters are mainly supported by studies based on urinary BPA concentration, showing a negative association between BPA levels and sperm concentration, motility, and sperm DNA damage. Recent studies explored potential approaches to treat or prevent BPA-induced testicular toxicity and male infertility. Since the effect of BPA on testicular cells and spermatozoa is associated with an increased production of reactive oxygen species, most of the pharmacological approaches are based on the use of natural or synthetic antioxidants. In this review, we briefly describe the effects of BPA on male reproductive health and discuss the use of antioxidants to prevent or revert the BPA-induced toxicity and infertility in men.This research was funded by Institute for Biomedicine—iBiMED, grant number UID/BIM/04501/2020 and by individual grant from FCT of the Portuguese Ministry of Science and Higher Education to J.S. (SFRH/BD/136896/2018)

Multi-method Active Learning Approach: improving the educational experience in Pharmaceutical Drug Development

Improve educational experience, teaching effectiveness and learning outcomes remains one of the major educational challenges nowadays. Accordingly, our aim is to determine the effect of organizing and teaching the Pharmaceutical Drug Development unit using a Multi-method Active Learning Approach, MALA, as a novel teaching/learning strategy. MALA involved several different activities about pharmaceutical legislation, medicines production and validation process, Common Technical Document, and others, and the evaluation of this active learning approach was made in two ways: students’ performance (grades), and students’ evaluation regarding the curricular unit and the teachers’ performance. Results revealed two main important aspects of the implementation of MALA: (i) the implementation of MALA showed high rates of students’ satisfaction regarding the curricular unit and teachers’ performance; (ii) student’s performance (grades) were very high, revealing excellent teaching/ learning results. In conclusion, the present study fostered the concept that the MALA- learning approach should contribute to knowledge- enhancing in the pharmaceutical practice and could encourage integration of the students´ learning skills in the future career, thus stimulating the flexibility in learning.publishe

A ruthenium(II)-trithiacyclononane curcuminate complex: synthesis, characterization, DNA-interaction, and cytotoxic activity

The coordination of ruthenium(II) complexes to anionic oxygen-based donors are very rare. This study describes a simple, one-pot method for obtaining [ruthenium(II)(trithiacyclononane)(curcumin)(S-DMSO)]Cl (1) in 37% yield. The structural characterization of complex 1 by elemental analysis, FT-IR, 1-D and 2-D NMR, ESI+-MS as well as UV–vis and fluorescence spectroscopies are presented. The DNA-melting temperature (Tm) assay shows that salmon sperm DNA (smDNA) in the presence of complex 1 has a higher melting temperature, with ΔTm = 7.4 °C, while in the presence of curcumin the melting temperature remains unaltered. The in vitro cytotoxic activities of curcumin and complex 1 were investigated using the tumor human prostate cell line, PC-3, and the healthy cell line, PNT-2. Complex 1 is innocuous toward normal prostate epithelial cells and, whereas curcumin is toxic, with inhibition rates of ca. 35 and 65% at 50 and 80 μM, respectively. On the tumor cell line PC-3, complex 1 did not cause viability changes, whereas curcumin exhibited dose-dependent inhibition, with ca. 73% inhibition at the highest concentration tested, i.e. 80 μM. This study suggests that coordination with the trithiacyclononane ruthenium(II) scaffold stabilizes the photochemical properties of curcumin and strongly changes its biologic activity.publishe

Rat prostate: practical tips for ultrasonographic monitoring

Background: Prostate is the largest accessory gland of the male reproductive tract. The prostate of men over 40 years- old is frequently affected by several pathologies, like benign prostate hyperplasia and cancer. Rats have been used as model to study prostate cancer. This study intended to address the usefulness of ultrasonography for rat prostate monitoring. Materials and Methods: Eight male Wistar Unilever rats were acquired from Charles River Laboratories and main- tained under controlled conditions of temperature, humidity, air system filtration and light/dark cycle. The prostate was evaluated by ultrasonography in awake animals. The animals were restrained by a researcher and placed in supine position. The skin of the inguinal region was shaved using a machine clipper (AESCULAP® GT420 Isis, USA). A real-time scan- ner (Logic P6®, GE, USA) and a 12 MHz linear transducer were used. Acoustic gel (Parker Laboratories Inc., USA) was applied. A complete transverse scan using B mode was per- formed from the cranial to the caudal region of the prostate, and a sagittal scan was performed moving the probe from the right to the left side. Procedures were approved by the Portuguese Ethics Committee (no.021326). Results: Prostate was easily evaluated by ultrasonography in all animals. In the transverse scan, the urinary bladder presents as a round to oval shape filled with urine (anechoic structure) and the prostate lobes were visible around it. The ventral prostate lobes appear as hypoechoic elongated struc- tures (one right and one left) with a hyperechoic capsule, placed ventrally to the urinary bladder. In this scan, the dorsal prostate was observed close to the urinary bladder neck, as a round hypoechoic structure with a hyperechoic capsule, dor- sally to the urinary bladder. In the sagittal scan, the urinary bladder was observed as an elongated structure filled with urine (anechoic content). The ventral prostate lobes were occasionally observed ventrally to the neck of the urinary bladder, as previously described. The dorsal prostate was ob- served dorsally to the neck of the urinary bladder, presenting as a round to elongated shape, with a hypoechoic appearance and a hyperechoic capsule.Conclusions: The ultrasonography is a non-invasive and ac- cessible tool for prostate monitoring in the rat model. Acknowledgments: This work was supported by European Investment Funds by FEDER/ COMPETE/POCI - Operational Competitiveness and Internationalization Program and National Funds by FCT - Portuguese Foundation for Science and Technology, under the projects Project RUNawayPCa (POCI-01-0145-FEDER-016728 and PTDC/DTP-DES/6077/2014), UIDB/04033/2020, UIDB/ CVT/00772/2020 and UIDB/50006/2020 (LAQV)

Anatomy and imaging of rat prostate: practical monitoring in experimental cancer-induced protocols

The rat has been frequently used as a model to study several human diseases, including cancer. In many research protocols using cancer models, researchers find it difficult to perform several of the most commonly used techniques and to compare their results. Although the protocols for the study of carcinogenesis are based on the macroscopic and microscopic anatomy of organs, few studies focus on the use of imaging. The use of imaging modalities to monitor the development of cancer avoids the need for intermediate sacrifice to assess the status of induced lesions, thus reducing the number of animals used in experiments. Our work intends to provide a complete and systematic overview of rat prostate anatomy and imaging, facilitating the monitoring of prostate cancer development through different imaging modalities, such as ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI).publishe

The nuclear envelope protein, LAP1B, is a novel protein phosphatase 1 substrate

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.publishe

An intriguing shift occurs in the novel protein phosphatase 1 binding partner, TCTEX1D4: evidence of positive selection in a pika model

T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif 90RVSF93 is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif 96NLS98, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The dN/dS for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms.publishe

Novel indole-based tambjamine-analogues induce apoptotic lung cancer cell death through p38 mitogen-activated protein kinase activation

Lung cancer has become the leading killer cancer worldwide, due to late diagnosis and lack of efficient anticancer drugs. We have recently described novel natural-derived tambjamine analogues that are potent anion transporters capable of disrupting cellular ion balance, inducing acidification of the cytosol and hyperpolarization of cellular plasma membranes. Although these tambjamine analogues were able to compromise cell survival, their molecular mechanism of action remains largely unknown. Herein we characterize the molecular cell responses induced by highly active indole-based tambjamine analogues treatment in lung cancer cells. Expression changes produced after compounds treatment comprised genes related to apoptosis, cell cycle, growth factors and its receptors, protein kinases and topoisomerases, among others. Dysregulation of BCL2 and BIRC5/survivin genes suggested the apoptotic pathway as the induced molecular cell death mechanism. In fact, activation of several proapoptotic markers (caspase-9, caspase-3, and PARP) and reversion of the cytotoxic effect upon treatment with an apoptosis inhibitor (Z-VAD-FMK) were observed. Moreover, members of the Bcl-2 protein family suffered changes after tambjamine analogues treatment, with a concomitant protein decrease towards the prosurvival members. Besides this, it was observed cellular accumulation of ROS upon compound treatment and an activation of the stress-kinase p38 MAPK route that, when inhibited, reverted the cytotoxic effect of the tambjamine analogues. Finally, a significant therapeutic effect of these compounds was observed in subcutaneous and orthotopic lung cancer mice models. Taken together, these results shed light on the mechanism of action of novel cytotoxic anionophores and demonstrate the therapeutic effects against lung cancer.Spanish Government and EU funds through the Fondo de Investigaciones Sanitarias (FIS, project PI13/ 00089) and from La Marat o de TV3 Foundation (project 20132730) to R. P erez-Tom as. R. Ramos was supported by the Sociedad Espa~nola de Neumología y Cirugía Tor acica (SEPAR, Project 017/2013), R. Quesada by the Consejería de Educación de la Junta de Castilla y León (project BU340U13) and by the La Marat o de TV3 Foundation (project 20132732) and A. Villanueva by the FIS (project PI13/01339)

Castanea sativa mill. flowers as potential chemopreventive agent against rat prostate cancer model

Introduction: Prostate cancer is one of the most common cancer among men, having a huge impact in their health [1]. This work aimed to evaluate the influence of a decoction extract obtained from C. sativa flowers (CF) on chemically and hormonally induced rat prostate cancer animal model. Material & Methods: All the animal experiments were approved by the Institutional Animals Ethics Committee and by Portuguese national authorities (DGAV no 021326). Forty male Wistar Unilever rats were randomly divided into four groups: control group (n=10), induced group (n=15), CF control group (n=5) and CF induced group (n=10). Animals from induced groups received a multistep induction protocol, which consisted of sequential administration of flutamide, testosterone propionate, the carcinogenic agent MNU and crystalline testosterone. The CF extract, rich in ellagitannins especially trigalloy-HHDP- glucose, was administered in the drinking water (3 mg/animal/day) for 49 weeks. Animals were sacrificed at 61 weeks of age and organs were collected, weighed and processed for light microscopy. Data were analysed using SPSS and GraphPad Prism software. Results: There were no significant differences in relative mean liver weight among groups exposed and not exposed to the CF extract and no animals developed severe hepatic changes. Animals from CF induced group developed less prostatic intraepithelial neoplasia than induced group. Also, animals exposed to the CF extract did not present areas of inflammation of the dorsolateral prostate lobe greater than 50% unlike the groups not exposed (p<0.05). The administration of CF in induced animals was able to decrease the activity of CAT and GST by 36% and 20%, respectively (p<0.05). Conclusions: These results suggest that CF extract was well tolerate by the animals and did not cause severe hepatic and renal toxicity. C. sativa flowers extract may be used as chemopreventive agent against prostate cancer and seems to have an antioxidant role