Horizontal Cell Biology: Monitoring Global Changes of Protein Interaction States with the Proteome-Wide Cellular Thermal Shift Assay (CETSA).

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.

Hirsutinolide- and Cadinanolide-type Sesquiterpene Lactones from Lessingianthus rubricaulis (Vernonieae, Asteraceae).

Sesquiterpene lactones are an important class of secondary metabolites frequently isolated from Lessingianthus genus that present a variety of biological properties, such as antimalarial, anti-inflammatory, antileishmanial, antitrypanosomal and anticancer. The limited phytochemical studies and the importance of this class of compounds isolated from Lessingianthus led us to study this genus. In this work, we focused on the phytochemical investigation and dereplication based on UHPLC-HRMS/MS and molecular networking of L. rubricaulis. Chemical investigation resulted in the isolation of several hirsutinolide-type sesquiterpene lactones including a new hirsutinolide derivative, 8,10α-hydroxy-1,13-bis-O-methylhirsutinolide, besides a cadinanolide and flavonoids. The dereplication study resulted in the identification of three known flavonoids, six known hirsutinolides and two known cadinanolides. Moreover, a fragmentation pathway for cadinanolide-type sesquiterpene lactones was proposed. These results contribute to chemotaxonomic studies and demonstrates the potential of Lessingianthus genus.

First Report of a Mastrevirus (Geminiviridae) Transmitted by the Corn Leafhopper.

Maize striate mosaic virus (MSMV; genus Mastrevirus) was recently reported in maize plants in Brazil and also detected by metagenomic analyses in the corn leafhopper, Dalbulus maidis (DeLong & Wolcott). Although these findings suggested that D. maidis is a potential vector, no transmission studies have been performed. Here, we tested the transmission of MSMV by D. maidis from field-collected infected plants and plants infected with MSMV via leafhopper-mediated transmission in the laboratory; all plants were confirmed positive for MSMV by PCR. In each one of three transmission replicates, aviruliferous D. maidis nymphs and adults were confined together on a source plant during a 4-day acquisition access period (AAP) and subsequently transferred to healthy maize seedlings (10 individuals per test plant) in a series of 4-day inoculation access periods (IAPs). We also tested transmission by the corn aphid, Rhopalosiphum maidis (Fitch) and by mechanical inoculation of healthy maize seedlings. Only D. maidis transmitted MSMV, with overall transmission rates of 29.4 and 39.5% on field-collected infected plants and 18.5% on infected plants in laboratory. D. maidis transmitted MSMV until the third (8 to 12 days after the AAP) or fourth successive IAP (12 to 16 days), with gradual loss in transmission efficiency and rate of viruliferous insects over time, suggesting a persistent but nonpropagative mode of transmission. Infected test plants showed mottling symptoms with mild chlorotic streaks and height reduction. This is the first report of transmission of a mastrevirus by D. maidis, facilitating the completion of Koch's postulate for MSMV.

TOCSY, hydrogen decoupling and computational calculations to an unequivocal structural elucidation of a new sesquiterpene derivative and identification of other constituents from Praxelis sanctopaulensis.

INTRODUCTION: Praxelis genus comprises 24 species, however, only two species of this genus have been chemically investigated. Here we investigated Praxelis sanctopaulensis, a native plant from Brazil, that occurs mainly in Cerrado regions. OBJECTIVE: The goal was to identify the specialised metabolites from P. sanctopaulensis, and compare with those described from Praxelis and Chromolaena species. METHODS: The phytochemical study of P. sanctopaulensis was performed through different chromatography techniques, including high-performance liquid chromatography (HPLC), gas chromatography flame ionisation detector (GC-FID), and ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). The structures of the compounds were established based on spectroscopic analysis, total correlated spectroscopy (TOCSY), hydrogen decoupling and computational calculations was used to an unequivocal structural elucidation of a new sesquiterpene. The antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), and antimicrobial assay was performed by the microdilution method. Comparison of the flavonoids described P. sanctopaulensis was carried out using principal component analysis. RESULTS: The phytochemical investigation of P. sanctopaulensis led to the isolation of a pair of diastereomers, praxilone A and praxilone B. Seven known compounds were isolated from this species, another 14 fatty acids were detected in hexane fraction, and 26 compounds were identified from ethyl acetate fraction. All these compounds are being described for the first time in this species, with the exception of viridifloric acid. The ethyl acetate fraction showed potent antioxidant activity. CONCLUSIONS: Forty-seven compounds are described from P. sanctopaulensis. The combination of different techniques of nuclear magnetic resonance (NMR) spectroscopy and computational calculations allowed the unequivocal structure elucidation of a new cadinene. The clustering analysis showed similarities between the flavonoids identified in P. sanctopaulensis and in Chromolaena species.

Neutrophil extracellular traps in inflammatory bowel diseases: Implications in pathogenesis and therapeutic targets.

Crohn's disease (CD) and ulcerative colitis (UC) are the two main forms of inflammatory bowel disease (IBD). Among the various immune cells involved in IBD, neutrophils are the first to infiltrate and appear to contribute to the impairment of the epithelial barrier, destruction of tissues by oxidative and proteolytic damage, as well as to the perpetuation of inflammation by the release of cytokines and chemokines associated with pro-inflammatory effects. In addition to basic effector mechanisms, such as phagocytosis and chemotaxis, neutrophils can also form extracellular traps (NETs), which is made up of a mesh-like structure - which contains its chromatin (DNA + histones) together with granules and enzymes, such as myeloperoxidase (MPO) and neutrophilic elastase (NE) - and that acts as a trap that can result in the death of extracellular pathogens and/or can promote tissue damage. Recent evidence indicates that NETs also play an important and significant role in the pathogenesis of IBD. Previous studies have reported increased levels of NETs in tissue and serum samples from patients with IBD, as well as in experimental colitis. In this review, we discuss current knowledge about the formation of NETs and their role in the pathophysiology of IBD, pointing out potential mechanisms by which NETs promote tissue damage, as well as their involvement in complications associated with IBD. In addition, we propose potential targets for therapy to regulate the production of NETs, making it possible to expand the current spectrum of therapies for IBD.

Sensitivity of the Nipple-Areola Complex in Reduction Mammaplasty Following Periareolar Dermis Section.

BACKGROUND: Preservation of the periareolar dermis, after de-epithelization in reduction mammaplasty, may result in reduced nipple-areola complex (NAC) mobility. Consequently, distortion in shape and difficulty correcting the areola position can occur. OBJECTIVES: The aim of this study was to evaluate NAC viability and sensitivity resulting from periareolar dermis section after de-epithelization in patients who underwent reduction mammaplasty surgery. METHODS: This study was a randomized, controlled double-blind clinical trial. A total of 41 patients (82 breasts) were randomly allocated into 2 groups and underwent reduction mammaplasty-involving the superior pedicle-with a final inverted T-shaped scar. A sectioning of the periareolar dermis was performed on the breasts in the experimental group. Semmes-Weinstein monofilaments were used to evaluate NAC sensitivity at the preoperative evaluation, and 3, 6, and 52 weeks after surgery. RESULTS: No cases of partial or total NAC necrosis were noted in either group. The comparison between the groups did not show significant differences regarding NAC sensitivity. However, a statistically significant difference was noted when sensitivity levels at the preoperative evaluation and 3 weeks after surgery were compared in the experimental group. A significant difference was noted when the scores of the sensitivity 3 weeks after surgery were compared between the control and the experiment groups. Nevertheless, after 1 year, no statistical differences were observed in the experimental group or between both groups. CONCLUSIONS: The sectioning of the periareolar dermis does not affect the viability or the pressure sensitivity of the NAC.

A Synthetic Model of the Mucosa for Oral Penetration Studies.

The main objective of this study is the evaluation of the use of a synthetic membrane, Nuclepore, as a model for permeation studies through sublingual mucosa. The permeability of two types of membranes, porcine sublingual oral mucosa and a synthetic Nuclepore membrane, to water was compared. Moreover, the water permeability of membranes modified with waterproofing formulations was measured to study their ability to protect against the penetration of viruses, toxins, etc. A relatively high correlation (R2 0.88) was obtained between the transmucosal water loss (TMWL) values of the artificial membrane and the mucosa. These results support the possible use of this synthetic membrane in the screening of the water permeability of formulations. In addition, studies of the permeation of different actives, drugs, and biocides through the two membranes were carried out, and these results were compared with their skin permeation data. The synthetic membrane does not seem to discern between compounds in terms of permeability. However, the permeation of caffeine through intact or modified membranes incorporating waterproofing formulations presents similar permeation profiles through the synthetic membrane and mucosa. The results from these assays should lend support to the use of this synthetic membrane when screening formulations to be applied in oral penetration studies.

Permeation Protection by Waterproofing Mucosal Membranes.

The permeability of the oral or nasal mucosa is higher than that of the skin. Mucosa permeability depends mainly on the thickness and keratinization degree of the tissues. Their permeability barrier is conditioned by the presence of certain lipids. This work has the main aim of reinforcing the barrier effect of oral mucosa with a series of formulations to reduce permeation. Transmembrane water loss of different formulations was evaluated, and three of them were selected to be tested on the sublingual mucosa permeation of drugs. Caffeine, ibuprofen, dexamethasone, and ivermectin were applied on porcine skin, mucosa, and modified mucosa in order to compare the effectiveness of the formulations. A similar permeation profile was obtained in the different membranes: caffeine > ibuprofen~dexamethasone > ivermectin. The most efficient formulation was a liposomal formulation composed of lipids that are present in the skin stratum corneum. Impermeability provided by this formulation was notable mainly for the low-molecular-weight compounds, decreasing their permeability coefficient by between 40 and 80%. The reinforcement of the barrier function of mucosa provides a reduction or prevention of the permeation of different actives, which could be extrapolated to toxic compounds such as viruses, contaminants, toxins, etc.

The chemistry of Vernonanthura nudiflora (Less.) H. Rob. flowers and its antimicrobial activities.

Twenty-one known specialised metabolites were isolated from the flowers of Vernonanthura nudiflora (Less.) H. Rob., the structures of the compounds were established based on 1 D and 2 D NMR spectroscopic experiments. Others 28 compounds were putatively identified using the dereplication technique by UHPLC-HRMS/MS. Twenty-three of the compounds are being reported for the first time in this species. The mixture of sesquiterpene lactones piptocarphins A and B (17 + 18), and the flavone velutin (14) were tested against several microorganisms and showed promising activity against Mycobacterium tuberculosis with MIC of 15.6 µg/mL and 31.2 µg/mL, respectively. Furthermore, 17 + 18 showed greater cytotoxicity against VERO cells (IC50 = 7.0 ± 1.73) compared to compound 14 (IC50 85.0 ± 10.6 µg/mL). These findings reveal the feasibility of using the UHPLC-ESI-HRMS/MS-based dereplication strategy in complex fractions to identify specialised metabolites, moreover to V. nudiflora flowers being a source of compounds with antimycobacterial potential.

Reproductive toxicity following in utero and lactational exposure to a human-relevant phthalate mixture in rats.

This rodent (Wistar rats) study examined reproductive effects of in utero/lactational exposure to a mixture of 6 antiandrogenic phthalates (PMix): diisobutyl phthalate, di-n-butyl phthalate, diisopentyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, and diisononyl phthalate. The PMix was defined based on exposure data from pregnant women in Brazil. Experimental groups were established by extrapolating the estimated human dose to rats (0.1 mg/kg/day), followed by up to 3 additional doses corresponding to 5, 1000, and 5000 times the starting rat dose: 0 (control), 0.1, 0.5, 100, and 500 mg/kg/day. The fetal experiment assessed gestational exposure effects on fetal gonads, whereas the postnatal experiment evaluated reproductive parameters in males and females after in utero and lactational exposure. Prenatal exposure decreased fetal testicular testosterone production at 0.5 and 500 mg/kg/day. PMix 500 also reduced mRNA expression of steroidogenesis-related genes, upregulated transcript expression of the retinoic acid-degrading enzyme Cyp26b1, and increased multinucleated gonocytes incidence in fetal testes. Postnatal assessment revealed antiandrogenic effects at the highest dose, including reduced anogenital distance, nipple retention, and decreased weight of reproductive organs. Early puberty onset (preputial separation) was observed at the lowest dose in males. In contrast, females did not show significant changes in fetal and adult endpoints. Overall, the PMix recapitulated early and late male rat phthalate syndrome phenotypes at the highest dose, but also induced some subtle changes at lower doses, which warrant confirmation and mechanistic assessments. Our data support the use of epidemiologically defined mixtures for exposure risk assessments over traditional toxicological approaches.

Study of chemical constituents, antioxidants and antimicrobial activities of Tamarindus indica L. seed.

The fruits of Tamarindus indica L. are consumed worldwide, with various parts of the plant being used for medicinal purposes. The residues (pericarp and seeds) generated during cellulose processing are of significant value as they contain bioactive compounds with diverse biological activities. The objective of this study was to evaluate the chemical constituents of the ethyl acetate fraction as possible substitutes for synthetic compounds with biological properties using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) analysis and the evaluation of the antioxidant activity (ferric reducing antioxidant power [FRAP], 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid [ABTS], and 1-diphenyl-2-picrylhydrazyl [DPPH]), total phenolic compounds (TPC), and antimicrobial activity of the hydroalcoholic extract and tamarind seed fractions were also performed. The chemical investigation of the acetate fraction using UHPLC-HRMS/MS resulted in the putative identification of 14 compounds, including flavonoids, (+)-catechin/(-)-epicatechin, procyanidin B2, procyanidin C2, isoquercetin, quercetin, luteolin, rutin, taxifolin, eriodictyol, kaempferide, hydroxybenzoic acid, protocathecuic acid, and protocathecuic acid methyl and ethyl esters derivatives. The crude hydroalcoholic extract exhibited the best results in terms of TPC: 883.87 gallic acid equivalent (GAE; mg/g) and antioxidant activity: FRAP: 183.29 GAE (mg/g), ABTS: 39.67%, and DPPH: 91.08%. The extract exhibited excellent antibacterial activity against gram-positive bacteria, specifically Staphylococcus aureus minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC; 62.5/125 g/mL) and Bacillus cereus MIC/MBC (125/250 g/mL), and gram-negative bacteria, specifically Aeromonas hydrophila MIC/MBC (125/250 µg/mL) and Pseudomonas aeruginosa MIC/MBC (250/500 g/mL). Morphological damage to cells was observed using flow cytometry and scanning electron microscopy. Tamarind seeds contain unique bioactive compounds that should be explored for their use as novel food preservatives. PRACTICAL APPLICATION: Original data were obtained regarding the Tamarindus indica L. seed extract and the ethyl acetate and hexane fractions. This research aimed to investigate the potential of these for food preservation and as alternatives to additives and synthetic compounds added to cattle feed. This paper reports novel findings regarding the chemical composition of the extract and its antioxidant activity, along with its antimicrobial activity against bacteria (gram-positive: Staphylococcus aureus, Bacillus cereus, and gram-negative: Salmonella enterica serovar Enteritidis, Escherichia coli, Pseudomonas aeruginosa, and Aeromonas hydrophila) and yeasts (Candida albicans and Saccharomyces cerevisiae).

Exposure to phthalates and female reproductive health: A literature review.

Endocrine-disrupting chemicals (EDCs) are exogenous compounds that have been known for their ability to interfere with the action of hormones and affect endocrine pathways, including the ones involved in the development and function of both male and female reproductive systems. EDCs comprise a wide class of compounds, such as pesticides, bisphenol A, phthalates and, parabens, that are present in the environment and in several daily use products. Phthalate esters, compounds commonly used as plasticizers and additives in many industrial applications, have attracted special attention because of the widespread human exposure and the potential for disruption of androgen-dependent development in males. Although phthalates are rapidly metabolized and excreted, their ubiquitous presence ensures continuous exposures throughout different life stages from conception to adult life, as documented by a number of human biomonitoring studies worldwide. Although most research efforts have been placed on the impact of phthalates on male reproductive development and functions, there is a large body of recent experimental and observational data indicating that phthalates can negatively affect female reproductive health, and in particular alter ovarian and uterine functions, potentially contributing to disorders like polycystic ovarian syndrome, endometriosis, and other common female reproductive problems. This review summarizes the most recent experimental and epidemiologic literature on the potential effects of phthalate exposures on female reproductive health and their impact on female fertility.

Chemical constituents and dereplication study of Lessingianthus brevifolius (Less.) H.Rob. (Asteraceae) by UHPLC-HRMS and molecular networking.

Chemical investigation of Lessingianthus brevifolius (Less.) H.Rob. aerial parts resulted in the isolation of the hirsutinolide-type sesquiterpene lactones piptocarphol, spicatolide D, piptocarphin D and 8α-acetoxy-10α-hydroxy-13-O-methylhirsutinolide, and also of a cadinanolide identified as 13-O-methylvernojalcanolide 8-O-acetate. Flavonoids, triterpenes and chlorogenic acids were also isolated. In addition, a dereplication study was carried out using UHPLC-HRMS and molecular networking, resulting in the identification of fifteen known compounds, being two sesquiterpene lactones and thirteen flavonoids. Some of the compounds are being described for the first time in L. brevifolius, and also in the Lessingianthus genus.

CETSA interaction proteomics define specific RNA-modification pathways as key components of fluorouracil-based cancer drug cytotoxicity.

The optimal use of many cancer drugs is hampered by a lack of detailed understanding of their mechanism of action (MoA). Here, we apply a high-resolution implementation of the proteome-wide cellular thermal shift assay (CETSA) to follow protein interaction changes induced by the antimetabolite 5-fluorouracil (5-FU) and related nucleosides. We confirm anticipated effects on the known main target, thymidylate synthase (TYMS), and enzymes in pyrimidine metabolism and DNA damage pathways. However, most interaction changes we see are for proteins previously not associated with the MoA of 5-FU, including wide-ranging effects on RNA-modification and -processing pathways. Attenuated responses of specific proteins in a resistant cell model identify key components of the 5-FU MoA, where intriguingly the abrogation of TYMS inhibition is not required for cell proliferation.

Phytoprostane and phenolic compounds from Chromolaena palmaris.

The chemical investigation of Chromolaena palmaris (Sch.Bip. ex Baker) R.M. King & H. Rob. expands the phytochemical composition knowledge of Chromolaena genus, since this is the first chemical investigation of this species. Twenty-five compounds were identified, including a phytoprostane, 17 flavonoids, 6 phenolic acids, and a caffeoyl-glucoside derivative obtained by classical chromatography and UHPLC-HRMS/MS analysis. Moreover, anti-Mycobacterium tuberculosis and antiproliferative activities of C. palmaris were evaluated. Dichloromethane fraction showed cytotoxicity towards human cancer cell lines, presenting TGI values on glioma (U251) of 27.8 µg mL-1. Furthermore, compounds 1 and 2 exhibited antimicrobial activity against Mycobacterium tuberculosis with MIC of 62.5 and 15.6 µg mL-1, respectively.

Controversies on Endocrine and Reproductive Effects of Glyphosate and Glyphosate-Based Herbicides: A Mini-Review.

Glyphosate-based herbicides (GBHs) are among the most used pesticides worldwide, presenting high potential for human exposure. Recently, a debate was raised on glyphosate risks to human health due to conflicting views over its potential carcinogenic and endocrine disruptive properties. Results from regulatory guideline studies, reports from Regulatory Agencies, and some literature studies point to a lack of endocrine disrupting properties of the active ingredient glyphosate. On the other hand, many in vivo and in vitro studies, using different experimental model systems, have demonstrated that GBHs can disrupt certain hormonal signaling pathways with impacts on the hypothalamic-pituitary-gonadal axis and other organ systems. Importantly, several studies showed that technical-grade glyphosate is less toxic than formulated GBHs, indicating that the mixture of the active ingredient and formulants can have cumulative effects on endocrine and reproductive endpoints, which requires special attention from Regulatory Agencies. In this mini-review, we discuss the controversies related to endocrine-disrupting properties of technical-grade glyphosate and GBHs emphasizing the reproductive system and its implications for human health.

The endocrine disrupting effects of sodium arsenite in the rat testis is not mediated through macrophage activation.

Arsenic (As) is an endocrine disrupting chemical that can disturb the male reproductive system. In a previous study, it was suggested that testicular macrophages could display a role in endocrine disruption induced by As exposure. This work aimed to evaluate the effects of chronic As exposure in the testis function of Wistar rats and examine the participation of macrophage activation and inflammatory response in these processes. We examined gene expression of steroidogenic machinery and immunological markers by RT-QPCR, plasma testosterone concentrations, sperm count and morphology, and histomorphometrical parameters after 60-days exposure to 1 or 5 mg.kg-1.day-1 of sodium arsenite, combined or not with 50 µg.kg-1 of LPS administered one day before euthanasia. We have demonstrated that As exposure reduced the weight of androgen-dependent organs and induced changes in spermatogenesis, in particular at the highest dose. LPS and As co-exposure promoted a decrease in testosterone synthesis, but did not increase the overexpression of markers of macrophage activation seen in LPS-only rats. Our results suggest that As does not alter the testicular macrophage function, but under immunological challenges LPS and As can display a complex interaction, which could lead to endocrine disruption.

Maize Bushy Stunt Phytoplasma Favors Its Spread by Changing Host Preference of the Insect Vector.

Plant pathogenic bacteria may influence vector behavior by inducing physiological changes in host plants, with implications for their spread. Here, we studied the effects of maize bushy stunt phytoplasma (MBSP) on the host selection behavior of the leafhopper vector, Dalbulus maidis (DeLong and Wolcott). Choice assays contrasting leaves of healthy (mock-inoculated) vs. infected maize (Zea mays L.) were conducted during the asymptomatic and symptomatic phases of plant infection, with leafhopper males or females previously exposed to infected plants (bacteriliferous insects) or not. In each assay, 40 adults were released in choice arenas where only the leaves of two plants from each treatment were offered and visible, and the insects landed on the leaves were counted 1, 2, 3, 5, 7, 9, 11 and 23 h after release. During the asymptomatic phase of plant infection, an effect was observed only on bacteriliferous females, who preferred leaves of healthy plants 5 h after release or later. The symptomatic phase triggered a pull-push effect on non-bacteriliferous females, who were first attracted to symptomatic leaves but hours later moved to healthy leaves. Non-bacteriliferous males initially preferred symptomatic leaves (up to 5 h after release) and later became equally distributed between treatments. Bacteriliferous males and females initially did not discriminate between healthy and symptomatic leaves, but only the females tended to move to healthy leaves 9 h after release. Oviposition was drastically reduced on symptomatic leaves. The changes in vector behavior induced by MBSP favor its primary spread, since bacteriliferous females prefer healthy leaves at early (asymptomatic) stages of the crop. At later stages, secondary spread may be favored because non-bacteriliferous females are initially attracted to infected (symptomatic) leaves, allowing pathogen acquisition and subsequent transmission as they move to healthy plants.

Prepubertal acrylamide exposure causes dose-response decreases in spermatic production and functionality with modulation of genes involved in the spermatogenesis in rats.

The increase in human infertility prevalence due to male reproductive disorders has been associated with extensive endocrine-disrupting chemical (EDC) exposure. Acrylamide (AA) is a compound formed spontaneously during heat processing of some foods that are mainly consumed by children and adolescents. In this study, we evaluated the prepubertal AA exposure effects on male adult reproductive physiology using a prepubertal experimental model to analyze the pubertal development, spermatogenesis hormones levels and genes expression involved in male reproductive function. This study is the first one to use the validated protocol to correlate the AA exposure with puberty development, as well as the AA-induced endocrine disrupting effects on reproductive axis. AA did not affect the age at puberty, the reproductive organ's weight and serum hormonal levels. AA reduces spermatogenesis, induces morphological and functional defects on sperm and alters transcript expression of sexual hormone receptors (Ar and Esr2), the transcript expression of Tnf, Egr2, Rhcg and Lrrc34. These findings suggest that excessive AA consumption may impair their reproductive capacity at adulthood, despite no changes in hormonal profile being observed.

Prenatal diclofenac exposure delays pubertal development and induces behavioral changes in rats.

Diclofenac is a non-steroidal anti-inflammatory drug widely used by the general population and, although generally contraindicated during pregnancy, it is also used by some pregnant women. This study investigated endocrine, reproductive and behavioral effects of diclofenac in male and female offspring rats exposed in utero from gestational days 10-20. Pregnant rats were treated with diclofenac at doses of 0.2, 1 and 5 mg/kg/day via oral gavage. Anogenital distance (AGD), number of nipples, and developmental landmarks of puberty onset - vaginal opening (VO), first estrus (FE) and preputial separation (PPS) - were evaluated in the offspring. At adulthood, behavioral and reproductive parameters were assessed. Male and female rats were tested in the elevated plus maze test to assess locomotor activity and anxiety-like behaviors, while male rats were also evaluated in the partner preference test. No significant effects were observed on AGD and number of nipples in both males and females. Diclofenac treatment induced an overall delay in developmental landmarks of puberty onset in male and female offspring, which reached statistical significance for PPS at the lowest diclofenac dose. Prenatal exposure to all tested doses abolished the preference of male rats for an estrous female, suggesting an impairment of brain masculinization. No changes were observed on male or female reproductive parameters at adulthood. Overall, our results indicate that prenatal exposure to therapeutically relevant doses of diclofenac may have an impact in the pubertal development of rats and negatively affect male partner preference behavior.