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Background: Neurogenic bladder (NB) patients exhibit varying degrees of bladder fibrosis, and the thickening and hardening of the bladder wall induced by fibrosis will further affect bladder function and cause renal failure. Our study aimed to investigate the mechanism of bladder fibrosis caused by a spinal cord injury (SCI). Methods: NB rat models were created by cutting the bilateral lumbar 6 (L6) and sacral 1 (S1) spinal nerves. RNA-seq, Western blotting, immunofluorescence, cell viability and ELISA were performed to assess the inflammation and fibrosis levels. Results: The rats showed bladder dysfunction, upper urinary tract damage and bladder fibrosis after SCI. RNA-seq results indicated that hypoxia, EMT and pyroptosis might be involved in bladder fibrosis induced by SCI. Subsequent Western blot, ELISA and cell viability assays and immunofluorescence of bladder tissue confirmed the RNA-seq findings. Hypoxic exposure increased the expression of HIF-1α and induced EMT and pyroptosis in bladder epithelial cells. Furthermore, HIF-1α knockdown rescued hypoxia-induced pyroptosis, EMT and fibrosis. Conclusion: EMT and pyroptosis were involved in the development of SCI-induced bladder fibrosis via the HIF-1α pathway. Inhibition of the HIF-1α pathway may serve as a potential target to alleviate bladder fibrosis caused by SCI.

The toxic effect of di(2-ethylhexyl) phthalate (DEHP) on prepubertal testes was examined in this study. We treated 3-week-old male mice with 4.8 mg/kg/day (milligram/kilogram/day) (no observed adverse effect level), 30 mg/kg/day (high exposure dose relative to humans), 100 mg/kg/day (level causing a reproductive system disorder), and 500 mg/kg/day (dose causing a multigenerational reproductive system disorder) of DEHP via gavage. Obvious abnormalities in the testicular organ coefficient, spermatogenic epithelium, and testosterone levels occurred in the 500 mg/kg DEHP group. Ribonucleic acid sequencing (RNA-seq) showed that differentially expressed genes (DEGs) in each group could enrich reproduction and reproductive process terms according to the gene ontology (GO) results, and coenrichment of metabolism pathway was observed by the Reactome pathway analysis. Through the analysis of common genes in the metabolism pathway, we discovered that DEHP exposure at 4.8 to 500 mg/kg or 100 mg/kg caused the same damages to the prepubertal testis. In general, we identified two key transcriptional biomarkers (fatty acid binding protein 3 (Fabp3) and carboxylesterase (Ces) 1d), which provided new insight into the gene regulatory mechanism associated with DEHP exposure and will contribute to the prediction and diagnosis of prepuberty testis injury caused by DEHP.

Di-(2-ethylhexyl) phthalate (DEHP) is a common plasticizer, which is known to be an environmental endocrine-disrupting chemical that can jeopardize the male reproductive system. Prepuberal exposure to DEHP leads to steroidogenesis disorders. However, the specific mechanism remains ambiguous. Therefore, Sprague Dawley (SD) rats underwent prepuberal DEHP exposure at a dose of 500 mg/kg per day through gavage. Additionally, the resulting testicular injury was evaluated to confirm the disturbed steroidogenesis. Changes in testicular histology, significant reduction of serum testosterone (P < 0.01) and luteinizing hormone (P < 0.001), and significantly decreased expressions of steroidogenic acute regulatory protein (P < 0.01) and 3-beta-hydroxysteroid dehydrogenase (P < 0.05) were found in DEHP-treated rats. DEHP exposure resulted in obvious intestinal damage and oxidative stress imbalance, primarily in the jejunum. Both the activation of the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway and alterations of microbiota profiles were observed in all three gut specimens, but were most notable in the jejunum. We hypothesize that the gut-microbiota-testis axis, which is mediated by the activation of the Nrf2 antioxidant pathway, could be involved in the dysfunction of prepuberal steroidogenesis induced by DEHP.

Mitochondrial DNA (mtDNA) is an attractive cellular target for anticancer agents in addition to nuclear DNA (nDNA). The cationic platinum(II) complex cis-[Pt(NP)(NH3)2Cl]NO3 (PtNP, NP = N-(2-ethylpyridine)-1,8-naphthalimide) bearing the DNA-intercalating moiety NP was designed. The structure of PtNP was fully characterized by single-crystal X-ray crystallography, NMR, and HRMS. PtNP is superior to cisplatin in both in vitro and in vivo anticancer activities with low systemic toxicity. The interaction of PtNP with CT-DNA demonstrated that PtNP could effectively bind to DNA through both covalent and noncovalent double binding modes. In addition to causing significant damage to nDNA and remarkable inhibition to DNA damage repair, PtNP also distributed in mitochondria, inducing mtDNA damage and affecting the downstream transcriptional level of mitochondrion-encoded genes. In addition, PtNP disturbed the physiological processes of mitochondria by reducing the mitochondrial membrane potential and promoting the generation of reactive oxygen species. Mechanistic studies demonstrate that PtNP induced apoptosis via mitochondrial pathways by upregulating Bax and Puma and downregulating Bcl-2 proteins, leading to the release of cytochrome c and activation of caspase-3 and caspase-9. As a dual-DNA-damage agent, PtNP is able to improve the anticancer activity by damaging both nuclear and mitochondrial DNA, thus providing a new anticancer mechanism of action for the naphthalimide monofunctional platinum(II) complexes.

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis.

Di-(2-ethylhexyl) phthalate (DEHP), one of the most commonly used endocrine-disrupting chemicals, has been shown to cause reproductive dysfunction in humans and animal models. However, very few studies have investigated the impact of DEHP at the post-transcriptional level in mouse testes, and the underlying mechanisms remain unclear. In the present research, TM3 Leydig cells were treated with 200 µM phthalic acid mono-2-ethylhexyl ester (MEHP, bio-metabolite of DEHP), and then the mRNA and lncRNA sequencing of TM3 Leydig cells was performed. Mice were exposed prepubertally to 0 or 500 mg DEHP/kg/day. RNA sequencing of mouse testes was performed to verify the RNA-seq results in vitro. The expression patterns of relevant genes and proteins were verified using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. DEHP and MEHP exposure led to testicular damage and accelerated cell aging via ROS accumulation. RNA sequencing analyses indicated that FOXO signaling and longevity regulation pathways were activated in resistance to ROS accumulation. FOXO signaling and longevity regulation pathway-related genes and proteins were also activated. By constructing a competing endogenous RNA (ceRNA) network, we observed that the ceRNA network might play a role in regulating FOXO signaling and longevity regulation pathways in response to excessive ROS accumulation and cell aging. In summary, our data here suggests that the ceRNA network may play a role in regulating FOXO signaling and longevity pathways in response to DEHP exposure in mouse testes.

Postnatal surge of gonadotrophins, Luteinizing hormone (LH) and Follicle-Stimulating hormone (FSH) known as minipuberty, is critical for gonocyte maturation into spermatogonial stem cells (SSC) in the testis. Gonadotrophins are essential for optimum fertility in men, but very little is known how they regulate germ cells during minipuberty. This study examined whether gonadotrophins play a role on gonocyte transformation in vivo. Testes from hypogonadal (hpg) mice and their wild type (WT) littermates (n = 6/group) were weighed, and processed in paraffin at postnatal days (D) 0, 3, 6 and 9. Mouse VASA homologue (germ cell marker), anti-Mullerian hormone (Sertoli cell marker) antibodies and DAPI (nuclei marker) were used for immunofluorescence followed by confocal imaging. Germ cells on or off basement membrane (BM) and Sertoli cells/tubule were counted using Image J and analyzed with GraphPad. Comparing to WT littermates, there were significantly fewer germ cells on BM/tubule (p

Di-(2-ethylhexyl) phthalate (DEHP) is an extensively used plasticizer and has been shown to cause reproductive dysfunction in humans and model animals. However, the exact mechanisms of testicular injury induced by DEHP exposure have not been fully clarified. Using gas chromatography-mass spectrometry, we found that mono-2-ethylhexyl ester (MEHP, a major biometabolite of DEHP) and DEHP concentrations were elevated in mouse serum after DEHP exposure. Using RNA-seq, we found that ferroptosis and HIF-1 signaling pathways might be involved in testicular injury due to prepubertal DEHP exposure. Subsequent Western blotting, ferrous iron and MDA measurements, and immunofluorescence of testicular sections verified the RNA-seq findings. Consistently, based on the RNA-seq findings, we found that ferroptosis and HIF-1 signaling pathways might play crucial roles in Leydig and Sertoli cell injury due to MEHP exposure in vitro. Further experiments also confirmed ferroptosis in Leydig and Sertoli cells. Using Western blotting, cellular immunofluorescence and ChIP-qPCR, we found that MEHP exposure caused HIF-1α accumulation and stabilization, resulted in HIF-1α translocation into the nucleus, and induced HIF-1α/Hmox1 binding in Leydig and Sertoli cells. To clarify whether HIF-1α plays a pivotal role in MEHP-induced ferroptosis, we knocked out Hif-1α using the CRISPR/Cas9 technique. We found that Hif-1α knockout rescued MEHP-induced ferroptosis. In summary, our findings certified that prepubertal DEHP exposure led to ferroptosis in mouse testes via the HIF-1α/HO-1 signaling pathway.

As an environmental endocrine disruptor, Di-(2-ethylhexyl) phthalate (DEHP) affects blood-testis barrier (BTB)-associated proteins expression, which compromises BTB integrity and causes infertility. Notably, DEHP-induced testicular toxicity is related to oxidative stress, but the specific mechanism remains unclear. Therefore, we sought to investigate this mechanism and determine whether vitamin C and vitamin E administration would attenuate the BTB impairment induced by DEHP in vivo and by Mono-(2-Ethylhexyl) Phthalate (MEHP) in vitro, respectively. HE staining and EM found that DEHP exposure led to spermatogenesis dysfunction and BTB disruption, respectively. The Western blot and immunofluorescence results showed that DEHP exposure caused BTB impairment through oxidative stress-mediated p38 mitogen-activated protein kinase (MAPK) signaling pathway. Furthermore, Vitamin E and vitamin C could alleviate the oxidative stress, block DEHP-induced spermatogenesis dysfunction and BTB disruption by inhibiting p38 MAPK signaling pathway. In summary, vitamin E and vitamin C are good candidates for the treatment of DEHP-induced male infertility.

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor widely used in China that is harmful to the male reproductive system. Many studies have shown that DEHP causes testicular toxicity through oxidative stress, but the specific mechanism is unknown. Because the Notch pathway is a key mechanism for regulating cell growth and proliferation, we investigated whether Notch is involved in DEHP-induced testicular toxicity and whether vitamins E and C could rescue testicular impairment in Sprague-Dawley (SD) rats. Compared with the control group, we found that DEHP exposure induced testicular toxicity through oxidative stress injury, and it decreased the testosterone level (P

Di-(2-ethylhexyl) phthalate (DEHP) is the most common phthalate that can affect the male reproductive system. DEHP exposure at the prepubertal stage could lead to the injury of immature testes, but the mechanism has not been fully clarified. In the present study, we elucidated the possible underlying mechanism of DEHP-induced prepubertal testicular injury through stereological analysis and transcriptome profiling. Compared with the control group, the DEHP-treated rats had lower body weight gain and decreased testicular weight and organ coefficient. Moreover, lower serum levels of testosterone and LH were observed in the DEHP group, in contrast to the increased FSH level. Additionally, the serum level of estradiol had no significant difference after DEHP exposure. Stereological analysis showed significant reduction in volumes of most testicular structures, especially in the seminiferous tubule and seminiferous epithelium, along with a vast decrease of spermatogenic cells and obvious structural damages with substantial pathological signs (germ cracks, cytoplasmic vacuolization, sloughing, multinucleated giant cell formation, chromatolysis desquamation and dissolution, pyknosis of nuclei) in the seminiferous tubule upon DEHP exposure at the prepubertal stage. Furthermore, transcriptome profiling identified 5548 differentially expressed genes (DEGs) upon DEHP exposure. Pathway enrichment analysis revealed several crucial signaling pathways related to retinol metabolism, oxidative phosphorylation, steroid hormone biosynthesis, and cell adhesion molecules (CAMs). In addition, seven DEGs selected from RNA-seq data were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and the results showed the same trends as the RNA-seq results. In conclusion, the above findings provide basic morphological data and lay a foundation for systematic research on transcriptome profiling in prepubertal testicular injury induced by DEHP.

To investigate the protective/ameliorative effects of vitamin E on di-2-(ethylhexyl) phthalate (DEHP)-induced reproductive toxicity, particularly in testicular toxicity in male rats, emphasizing peroxisome proliferator-activated receptor (PPAR)-dependent mechanism.Sprague-Dawley females were exposed by oral route to DEHP alone or associated with vitamin E from gestation day (GD) 12.5 to postnatal day (PND) 3 according to the following treatment regimens: vehicle control (corn oil), vitamin E (200 mg/kg)+corn oil, DEHP (500 mg/kg)+corn oil, and DEHP (500 mg/kg)+vitamin E (200 mg/kg)+corn oil. Variables including litter size, sex ratio, pup weight, post-implantation losses, and the number of viable pups were also assessed. Three male pups per litter were randomly selected and necropsied to measure paired testes weight, apoptosis, and gene expression on PND 3. To evaluate the long-term protective effects of vitamin E, three randomly selected males were necropsied to measure testis histology on PND 70.Supplementation of vitamin E (200 mg/kg) reduced malformations, increased testes weight and prevented the maternal bodyweight loss induced by DEHP. Litter size, sex ratio, and number of viable pups were unaffected, but vitamin E co-administration declined testicular cell apoptosis, decreased the PPARs expression, and protected testis histology.Vitamin E cotreatment showed protective effects against DEHP-induced testicular toxicity, including reproductive malformations, testicular weight, apoptosis and histology, and the mechanisms maybe associated with PPARs.

Di(2-ethylhexyl) phthalate (DEHP) is a widely-used plasticizer and has long been recognized as an endocrine-disrupting chemical with male reproductive toxicities. DEHP exposure at the prepubertal stage may lead to extensive testicular injury. However, the underlying mechanisms remain to be elucidated. In the present study, we gavaged male C57BL/6 mice with different concentrations of DEHP (0, 250, and 500 mg/kg-bw·d) from postnatal day 22–35, and exposed TM3 Leydig cells with 0, 100, 200, 300, and 400 μM of MEHP (bioactive metabolite of DEHP) for 12–48 h. RNA sequencing was performed both in testicular tissue and TM3 cells. The results showed that DEHP disrupts testicular development and reduces serum testosterone levels in male prepubertal mice. Bioinformatic analysis and experimental verification have revealed that DEHP/MEHP induces cell cycle arrest in TM3 cells and increases apoptosis both in vivo and in vitro. Furthermore, the p53 signaling pathway was found to be activated upon DEHP/MEHP treatment. The inhibition of p53 by pifithrin-α significantly reduced MEHP-induced injuries in TM3 cells. Cumulatively, these findings revealed the involvement of the p53 signaling pathway in DEHP-induced prepubertal testicular injury by promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

Bisphenol S (BPS) is now used as an alternative of bisphenol A (BPA), but has been implicated in male reproductive dysfunction—including diminished sperm number and quality and altered hormonal concentrations. However, the mechanisms of action subserving these effects remains unclear. In the present study, BPS at doses of 50 mg/kg bw and 100 mg/kg bw caused defects in the integrity of the blood-testis barrier (BTB) and apical ectoplasmic specialization (ES), and we also delineated an underlying molecular mechanism of action. BPS induced F-actin and α-tubulin disorganization in seminiferous tubules, which in turn led to the truncation of actin filaments and microtubules. Additionally, BPS was found to perturb the expression of the actin-binding proteins Arp3 and Eps8, which are critical for the organization of the actin filaments. mTORC1 and mTORC2 manifest opposing roles in Sertoli cell junctional function, and we demonstrated that mTORC1/rpS6/Akt/MMP9 signaling was increased and that mTORC2/rictor activity was also attenuated. In summary, we showed that BPS-induced disruption of the BTB and apical ES perturbed normal spermatogenic function that was mediated by mTORC1 and mTORC2. The imbalance in mTORC1 and mTORC2, in turn, altered the expression of actin-binding proteins, resulting in the impairment of F-actin and MT organization, and inhibited the expression of junctional proteins at the BTB and apical ES.

Application of in silico models to predict developmental toxicity has demonstrated limited success particularly when employed as a single source of information. It is acknowledged that modelling the complex outcomes related to this endpoint is a challenge; however, such models have been developed and reported in the literature. The current study explored the possibility of integrating the selected public domain models (CAESAR, SARpy and P&G model) with the selected commercial modelling suites (Multicase, Leadscope and Derek Nexus) to assess if there is an increase in overall predictive performance. The results varied according to the data sets used to assess performance which improved upon model integration relative to individual models. Moreover, because different models are based on different specific developmental toxicity effects, integration of these models increased the applicable chemical and biological spaces. It is suggested that this approach reduces uncertainty associated with in silico predictions by achieving a consensus among a battery of models. The use of tools to assess the applicability domain also improves the interpretation of the predictions. This has been verified in the case of the software VEGA, which makes freely available QSAR models with a measurement of the applicability domain.

This work provides case studies for the pharmacokinetic (PK) analog approach, where a physiologically based pharmacokinetic (PBPK) model for a target chemical (has no PK data) is evaluated using PK data from a source chemical (has existing PK data). A bottom up PBPK modeling approach (using in vitro and in silico inputs) is used to develop human oral PBPK models for caffeine and diphenhydramine. Models are evaluated using in vivo data from structural and functional PK analogs. At the end of the case studies, in vivo PK data for caffeine and diphenhydramine is introduced and both models were able to simulate plasma concentrations which agreed with the in vivo PK data. To further demonstrate that structural analogs can serve as PK analogs, in vitro metabolism and plasma protein binding was compared for a subset of structurally similar ToxCast chemicals and shown to be similar. Next steps for the PK analog approach should focus on evaluating this concept for a broader set of compounds. Using PK analogs for evaluating and establishing confidence in a PBPK model will ensure that PBPK modeling remains a viable option in animal alternative safety assessments.

Di-(2-ethylhexyl) phthalate (DEHP) is a common environmental endocrine disrupting chemical that may induce male reproductive disorders. Exposure to DEHP at a prepubertal stage could lead to prepubertal testicular injury, but the underlying mechanisms remain unclear. In this study, we exposed Sprague-Dawley rats to 0, 250, and 500 mg DEHP per kg body weight per day at the prepuberty stage from postnatal day 22 (PND 22) to PND 35 by oral gavage. Testicular injury and oxidative stress were evaluated, and the levels of 6-methyladenosine (m6A) modification and expression of modulator genes for RNA methylation were measured in testes. Furthermore, m6A modification of the important antioxidant transcription factor Nrf2 was analyzed using methylated RNA immunoprecipitation qPCR. Our results show that DEHP worsened testicular histology, decreased testosterone concentrations, downregulated expression of spermatogenesis inducers, enhanced oxidative stress, inhibited the Nrf2-mediated antioxidant pathway, and increased apoptosis in testes. Additionally, DEHP increased global levels of m6A RNA modification and altered the expression of two important RNA methylation modulator genes, FTO and YTHDC2. Moreover, m6A modification of Nrf2 mRNA increased upon DEHP exposure. Overall, these findings link oxidative stress imbalance with epigenetic effects of DEHP toxicity and provide insight into the testicular toxicity of DEHP from the new perspective of m6A modification.

Cryptorchidism is a common condition of childhood, and it is known to impair fertility potential. However, the underlying mechanisms remain unclear.This study constructed two cryptorchid rat models to investigate the roles of apoptosis and autophagy in testicular impairment induced by cryptorchidism. Pregnant rats were randomly divided into three groups. Group I: non-treated rats were used as controls. Group II: injected with drug Flutamide (Flu) 25 mg/kg/bw/d from gestation day (GD) 11-19. Group III: daily intragastric administration of 750 mg/kg/bw/d di-2-ethylhexylphosphate (DEHP) from GD 7-19. The cubs were feed normally and the testes were excised on postnatal day (PND) 30.Our results demonstrated cryptorchidism models induced noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate, decreased testosterone and severe testicular damage in histomorphology. Intriguingly, the level of apoptosis marker FAS, Cytochrome C and caspase-3 increased in Flu-induced and DEHP-induced groups. DEHP-induced treatment simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II and p62. Significant decrease of autophagy gene (LC3-II and p62) expression is found in Flu-induced rats testes.Taken together, deficient autophagy is involved in testicular spermatogenesis damage of cryptorchidism rats. And this autophagy defect is caused by deficient degradation.

In the European Union animal testing has been eliminated for cosmetic ingredients while the US Cosmetic Ingredient Review Expert Panel may request data from animal studies. The use of read-across and predictive toxicology provides a path for filling data gaps without additional animal testing. The PEG cocamines are tertiary amines with an alkyl group derived from coconut fatty acids and two PEG chains of varying length. Toxicology data gaps for the PEG cocamines can be addressed by read-across based on structure-activity relationship using the framework described by Wu et al. (2010) for identifying suitable structural analogs. Data for structural analogs supports the conclusion that the PEG cocamines are non-genotoxic and not expected to exhibit systemic or developmental/reproductive toxicity with use in cosmetics. Due to lack of reliable dermal sensitization data for suitable analogs, this endpoint was addressed using predictive software (TIMES SS) as a first step (Laboratory of Mathematical Chemistry). The prediction for PEG cocamines was the same as that for PEGs, which have been concluded to not present a significant concern for dermal sensitization. This evaluation for PEG cocamines demonstrates the utility of read-across and predictive toxicology tools to assess the safety of cosmetic ingredients.

Blood-testis barrier (BTB), constituted by tight junctions (TJs), adherens junctions and gap junctions, is important for spermatogenesis. PM2.5 is known to impair testicular functions and reproduction. However, its effects on BTB and the underlying mechanisms remain obscure.To investigate the roles of autophagy in BTB toxicity induced by PM2.5.Sprague-Dawley rats were developmentally exposed to normal saline (NS) or PM2.5 with the doses of 9 mg/kg b.w. and 24 mg/kg b.w. via intratracheal instillation for seven weeks. Success rate of mating, sperm quality, testicular morphology, expressions of BTB junction proteins and autophagy-related proteins were detected. In addition, expressions of oxidative stress markers were also analyzed.Our results demonstrated that developmental PM2.5 exposure induced noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate and severe testicular damage in histomorphology. The expressions of TJ (such as ZO-1 and occludin), gap junction (such as connexin43) were down-regulated significantly after PM2.5 treatment. Intriguingly, PM2.5 simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II and p62, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation. Moreover, the expressions of HO-1 levels remarkably increased and expression levels of Gpx and SOD were significantly decreased after PM2.5 exposure. Vitamins E and C could alleviate the PM2.5-induced oxidative stress, reverse the autophagy defect and restore the BTB impairment.Taken together, the results suggest that PM2.5 exposure destroys BTB integrity through excessive ROS-mediated autophagy. Our finding could contribute to a better understanding of PM2.5-induced male reproductive toxicity.

Structure activity relationships (SAR) and read-across are widely used animal alternative approaches for filling toxicological data gaps. A framework describing the use of expert judgment in evaluating analogs for SAR has been published and widely cited, however, reliance on expert judgment can introduce inconsistent results across experts and hinder transparency. Here we explore the use of a quantitative similarity score between an analog and a Structure of Interest (SOI) to see if these scores correlate with the expert judgement-based suitability rankings. We find these global similarity scores representing a "whole-molecule" view of similarity to be insensitive to differences in local structure which may be important for toxicity, and, therefore, cannot be substituted for expert judgement-based similarity rankings. In this paper, we suggest that the next step in the progression of SAR approaches retains the insights from expert judgment, but facilitates consistency and transparency through the development of rating "rules". This report outlines and defines analog rating rules for several compound categories. While not comprehensive, the exercises demonstrate the development of rules for categories with a large spread in molecular weight and alkyl chain length and explains the advantages that we see in this approach compared to relying solely on a computational approach or an unstructured expert judgement approach. These rules may be incorporated into analog searching work flows to define boundaries for analogs "suitable" for read-across.

Developmental and reproductive toxicity (DART) end points are important hazard end points that need to be addressed in the risk assessment of chemicals to determine whether or not they are the critical effects in the overall risk assessment. These hazard end points are difficult to predict using current in silico tools because of the diversity of mechanisms of action that elicit DART effects and the potential for narrow windows of vulnerability. DART end points have been projected to consume the majority of animals used for compliance with REACH; thus, additional nonanimal predictive tools are urgently needed. This article presents an empirically based decision tree for determining whether or not a chemical has receptor-binding properties and structural features that are consistent with chemical structures known to have toxicity for DART end points. The decision tree is based on a detailed review of 716 chemicals (664 positive, 16 negative, and 36 with insufficient data) that have DART end-point data and are grouped into defined receptor binding and chemical domains. When tested against a group of chemicals not included in the training set, the decision tree is shown to identify a high percentage of chemicals with known DART effects. It is proposed that this decision tree could be used both as a component of a screening system to identify chemicals of potential concern and as a component of weight-of-evidence decisions based on structure-activity relationships (SAR) to fill data gaps without generating additional test data. In addition, the chemical groupings generated could be used as a starting point for the development of hypotheses for in vitro testing to elucidate mode of action and ultimately in the development of refined SAR principles for DART that incorporate mode of action (adverse outcome pathways).

Blood-testis barrier (BTB) provides a suitable microenvironment for germ cells that is required for spermatogenesis. Exposure to particulate matter (PM) is recognized to occasion male reproductive impairment, but the mechanism of which remains unclear. Male Sprague-Dawley (SD) rats were used to establish animal models with PM2.5 exposure concentration of 0, 10, and 20mg/kg.b.w. once a day for four weeks. Success rate of mating, sperm quality, epididymal morphology, expressions of spermatogenesis markers, superoxide dismutases (SOD) activity and expression in testicular tissues, and expressions of BTB junction proteins were detected. In addition, in vitro experiments were also performed. After PM2.5 treatment, reactive oxygen species (ROS) production and apoptosis of Sertoli cells were analyzed. Our results indicated that after PM2.5 exposure male rats presented inferior uberty and sperm quality, with decreased expressions of spermatogenesis markers, escalated SOD activity and expression levels, and reduced expressions of tight junction, adherens junction, and gap junction proteins in testicular tissues. Meantime, PM2.5-treated Sertoli cells displayed increased SOD production and apoptosis. PM2.5 exposure engenders male reproductive function injury through breaking BTB integrity.

Propofol is currently one of the most widely used intravenous anesthetic. In the present study, we investigated the effects of propofol on neuropathogenesis in newborn rats under hypoxic conditions. Seven-day old SD rats were assigned into one of the six treatments: propofol+50% oxygen (propofol-oxygen, PO), propofol+room air (propofol-air, PA), propofol+18% oxygen (propofol-hypoxia, PH), control group: lipid emulsion solvent+50% oxygen (CO), lipid emulsion solvent+room air (CA), lipid emulsion solvent+18% oxygen (CH). The rats assigned to anesthesia or control groups received intraperitoneally (ip) propofol 50 mg/kg or identical volume of lipid emulsion solvent (5.0 ml/kg) for seven days. SaO(2) (%) and respiratory rate (RR) were monitored throughout the procedures. The rats were decapitated 24h after 7 days exposure. TUNEL staining, Nissl staining, ultrastructural changes and the expression of caspase-3 in the brain tissues were assessed. We found propofol-induced attenuation of respiration could produce lower oxygen concentrations in the blood (hypoxia) under air or mild hypoxia conditions. Interestingly, in the presence of oxygen completely rescued hypoxia to normal levels, suggesting that propofol-induced respiratory depression led to hypoxia only under air or mild hypoxic conditions (propofol/hypoxia). In addition, propofol indirectly induced apoptosis through hypoxia resulting from respiratory depression under air or hypoxic conditions, which was determined by elevated expression of caspase-3, increased TUNEL-positive cells, ultrastructural changes of neuronal cell death and loss of Nissl staining neuronal in infant SD rat brains. However, in propofol-oxygen group and all control groups, no significant apoptosis were observed. These findings indicated that propofol per se or hypoxia per se did not directly induce significant apoptosis. However, propofol-induced attenuation of respiration could produce lower oxygen concentrations in the blood under air or mild hypoxia conditions and thereby result in neuronal degeneration. So, it is important to supply with supplementary oxygen during propofol anesthesia.

A novel class of tetrazole-derived Kv1.5 blockers is disclosed. In in vitro studies, several compounds had IC50s ranging from 180 to 550 nM. In vivo studies indicated that compounds 2f and 2j increased right atrial ERP about 40% without affecting ventricular ERP.

Respiratory syncytial virus (RSV) is the most important cause of severe, lower respiratory tract infections in infants, and RSV infections have been associated with chronic wheezing and asthma during childhood. However, the mechanism of RSV-induced airway inflammation and airway hyperresponsiveness (AHR) is poorly understood. Furthermore, there are presently neither effective vaccines nor drugs available for the prevention or treatment of RSV infections. In this study, we investigated the effect of the plant extract resveratrol as a means of preventing airway inflammation and attenuating RSV-induced AHR. Our data showed that resveratrol reduced RSV lung titers and the number of infiltrating lymphocytes present in bronchoalveolar lavage fluid (BALF) and reduced inflammation. Furthermore, resveratrol attenuated airway responses to methacholine following RSV infection and significantly decreased gamma interferon (IFN-γ) levels in BALF of RSV-infected mice. Data presented in this report demonstrated that resveratrol controlled Toll-like receptor 3 (TLR3) expression, inhibited the TRIF signaling pathway, and induced M2 receptor expression following RSV infection. These data support a role for the use of resveratrol as a means of reducing IFN-γ levels associated with RSV-mediated airway inflammation and AHR, which may be mediated via TLR3 signaling.

The biological fluorescent distribution of a model antitumor monofunctional platinum(II) complex bearing a 7-nitro-2,1,3-benzoxadiazole fluorophore can be visualized in breast carcinoma MCF-7 cells, pulmonary carcinoma A549 cells, kidney epithelial 293T cells, and zebrafish larva.

A process for evaluating analogs for use in SAR (Structure–Activity Relationship) assessments was previously published ( Wu et al. 2010 ). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown.

This study investigated epigenetic (specifically, DNA methylation) changes and their impact on gene expression in testes induced by maternal exposure to Di-2-(ethylhexyl) phthalate (DEHP) in mice. Testicular dysgenesis syndrome was induced in fetuses and pups by maternal exposure to DEHP at 500 mg/kg/d, and testes were excised for analysis on gestation day (GD) 19 and postnatal days (PNDs) 3, 21, 56, and 90. High-performance liquid chromatography (HPLC) was performed to analyze DNA methylation status, and expression levels of the DNA methyltransferases were examined by quantitative real-time polymerase chain reaction (qPCR). Testis-specific gene, insulin-like hormone 3 (Insl3), and testosterone production were also detected. DEHP significantly increased DNA methylation levels on GD 19 and PND 3 ( P < .05 and P < .05) but not on PNDs 21, 56, and 90. DEHP also significantly increased the expression of DNA methyltransferases. For DNA methyltransferase 1, the difference was not significant on PND 21, and DNA methyltransferase 3a and 3b returned to normal levels on PND 56. Fetal testes were a main target for DEHP as evidenced by a reduction in Insl3 expression and testosterone production. Effects of DEHP on Insl3 expression continued until PND 21. The DEHP-induced suppression of testosterone had not recovered on PND 56. Changes in DNA methylation may play an important role in abnormal testicular function caused by environmental factors such as maternal exposure to DEHP, which may be a mechanism of DEHP-mediated testicular toxicity.

The aim of this study was to analyse epigenetic (specifically, DNA methylation) change in testes induced by maternal exposure to di-2-(ethylhexyl) phthalate (DEHP) in mice. Testicular dysgenesis syndrome was induced in foetuses by maternal exposure to DEHP. High-performance liquid chromatography was performed to analyse DNA methylation status, and expression levels of the DNA methyltransferases were examined by quantitative real-time polymerase chain reaction and western blotting. DEHP significantly had more than 10% relative increase in the global DNA methylation and also increased DNA methyltransferases' expression. Changes in DNA methylation may play an important role in abnormal testicular function caused by environmental factors such as maternal exposure to DEHP, which may be one possible mechanism of DEHP-mediated testicular toxicity.

Discussion on the role of DEHP in the critical period of gonadal development in pregnant rats (F0), studied the evolution of F1-F4 generation of inter-generational inheritance of cryptorchidism and the alteration of DNA methylation levels in testis. Pregnant SD rats were randomly divided into two groups: normal control group and DEHP experimental group. From pregnancy 7 d to 19 d, experimental group was sustained to gavage DEHP 750 mg/kg bw/day, observed the incidence of cryptorchidism in offspring and examined the pregnancy rate of female rats through mating experiments. Continuous recording the rat's weight and AGD value, after maturation (PND80) recording testis and epididymis' size and weight, detected the sperm number and quality. Subsequently, we examined the evolution morphological changes of testicular tissue for 4 generation rats by HE staining and Western Blot. Completed the MeDIP-sequencing analysis of 6 samples (F1 generation, F4 generation and Control). DEHP successfully induced cryptorchidism occurrence in offspring during pregnancy. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there was no cryptorchidism coming up in F3 and F4. Mating experiment shows conception rate 50% in F1, F2 generation was 75%, the F3 and F4 generation were 100%. HE staining showed that the seminiferous epithelium of F1 generation was atrophy and with a few spermatogenic cell, F2 generation had improved, F3 and F4 generation were tend to be normal. The DNA methyltransferase expression was up-regulated with the increase of generations by Real Time-PCR, immunohistochemistry and Western Blot. MeDIP-seq Data Analysis Results show many differentially methylated DNA sequences between F1 and F4. DEHP damage male reproductive function in rats, affect expression of DNA methyltransferase enzyme, which in turn leads to genomic imprinting methylation pattern changes and passed on to the next generation, so that the offspring of male reproductive system critical role in the development of imprinted genes imbalances, and eventually lead to producing offspring cryptorchidism. This may be an important mechanism of reproductive system damage.

Blockade of the Kv1.5 ion channel is a potentially atrial-selective avenue for the treatment of atrial fibrillation and atrial flutter. The development and biological evaluation of a series of thiazolidine-based blockers of Kv1.5 is described.

A novel class of tetrahydroindolone-derived semicarbazones has been discovered as potent Kv1.5 blockers. In in vitro studies, several compounds exhibited very good potency for blockade of Kv1.5. Compound 8i showed good selectivity for blockade of Kv1.5 vs hERG and L-type calcium channels. In an anesthetized pig model, compounds 8i and 10c increased atrial ERP about 28%, 18%, respectively, in the right atrium without affecting ventricular ERP.

Objectives To investigate the relationship between nitric oxide synthase (NOS) and oxidative stress and pathologic remodeling in the partial obstructed bladder of a rat model. Methods We surgically established partial bladder outlet obstruction (PBOO) in 2 groups of rats and allowed it to persist for 3-6 weeks. Normal and sham-operated rats served as the controls. Each group contained 6 rats for a total of 24 rats. Cystometry was used to evaluate the bladder function. The bladders were removed, and histopathologic measurements were performed to evaluate bladder hypertrophy and fibrosis and NOS immunolocalization. Biochemical measurements were used to evaluated NOS mRNA and activity and the oxidative stress level. Results The obstructed rats experienced significant increases in bladder weight, muscle hypertrophy, and deposits of collagen fibers compared with the normal and sham-operated groups. PBOO debilitated bladder contractibility, increased the residual urine volume and voiding interval, decreased the voiding volume, and caused poor bladder emptying, with an increased residual urine volume and decompensated bladder in the PBOO rats at 6 weeks. The elevation in malondialdehyde and reduction in superoxide dismutase activity in the PBOO rats suggested that oxidative stress injury occurred in the obstructed bladder. Lower inducible NOS and endothelial NOS (eNOS) mRNA expression was demonstrated through quantitative polymerase chain reaction. In particular, eNOS was significantly downregulated in the PBOO rats compared with the normal and sham-operated rats. The normal and PBOO bladder tissues did not express detectable levels of neuronal NOS mRNA or exhibit neuronal NOS immunoreactivity. The total NOS activity had decreased progressively in the PBOO groups in conjunction with the significantly decreased eNOS activity compared with that in the normal and sham-operated groups. Conclusions These findings suggest that decreases in NOS activity and expression (mainly of eNOS) concomitant with increases in reactive oxygen species generation represent the underlying pathogenic mechanism of obstructed bladder remodeling and dysfunction.