Chronic Effects of an Insect Growth Regulator (teflubenzuron) on the Life Cycle and Population Growth Rate of Folsomia candida.

Current standard toxicity tests on nontarget soil invertebrates mainly focus on the endpoints survival and reproduction. Such results are likely insufficient to predict effects at higher organizational levels, for example, the population level. We assessed the effects of exposure to the pesticide teflubenzuron on the collembolan Folsomia candida, by performing a full life-cycle experiment exposing single individuals via contaminated food (uncontaminated control and 0.2, 0.32, 0.48, 0.72, 1.08, and 1.6 mg/kg dry yeast). Several life-history traits were considered by following the growth and development of newly hatched individuals over a period of 65 days. We assessed survival, body length, time to first oviposition, cumulative egg production, and hatchability of eggs. A two-stage model was applied to calculate the population growth rate (λ) combined with elasticity analysis to reveal the relative sensitivity of λ to the effects of teflubenzuron on each life-history parameter. Body length was the least sensitive life-history parameter (median effective concentration = 1.10 mg teflubenzuron/kg dry yeast) followed by time to first oviposition (0.96 mg/kg), survival (median lethal concentration = 0.87 mg/kg), cumulative egg production (0.32 mg/kg), and egg hatchability (0.27 mg/kg). Population growth decreased with increasing concentrations of teflubenzuron (λ = 1.162/day in control to 1.005/day in 0.72 mg/kg dry yeast, with populations going extinct at 1.08 and 1.6 mg/kg dry yeast). Elasticity analysis showed that changes in juvenile survival had a greater impact on the population growth rate compared with the other life-history traits. Our study provides a comprehensive overview of individual-level effects of long-term exposure to teflubenzuron and integrates these effects to assess the potential risk to collembolan populations. Environ Toxicol Chem 2024;43:1173-1183. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Lung proliferation is dependent on the duration not the timepoint of tracheal occlusion in nitrofen rats with diaphragmatic hernia.

OBJECTIVE: Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with congenital diaphragmatic hernia (CDH). Limited data are available regarding the effect of duration versus timepoint of TO. Our objective was to document the impact of TO on lung development in the near-term period in rats with nitrofen-induced CDH. METHOD: Nitrofen was administered on embryonic day (ED)9 and fetal TO was performed on ED18.5, 19, or 20 (term = ED22). Sham-operated and untouched littermates served as controls. Lungs were harvested in 0.5-day steps and only fetuses with a left-sided CDH were included in further analyses. Healthy fetuses provided a reference for normal near-term lung development. RESULTS: Duration of TO in the nitrofen rat model for CDH predicts lung growth in terms of lung-body-weight ratio as well as an increased mRNA level of the proliferation marker Ki67. Longer TO also induced a more complex airway architecture. The timepoint of TO was not predictive of lung growth. CONCLUSION: In the nitrofen rat model of CDH, a longer period of TO leads to enhanced lung growth and more refined airway architecture.

Fetal lung hypoxia and energetic cell failure in the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Congenital diaphragmatic hernia (CDH) pathogenesis is poorly understood. We hypothesize that fetal CDH lungs are chronically hypoxic because of lung hypoplasia and tissue compression, affecting the cell bioenergetics as a possible explanation for abnormal lung development. METHODS: To investigate this theory, we conducted a study using the rat nitrofen model of CDH. We evaluated the bioenergetics status using H1 Nuclear magnetic resonance and studied the expression of enzymes involved in energy production, the hypoxia-inducible factor 1α, and the glucose transporter 1. RESULTS: The nitrofen-exposed lungs have increased levels of hypoxia-inducible factor 1α and the main fetal glucose transporter, more evident in the CDH lungs. We also found imbalanced AMP:ATP and ADP:ATP ratios, and a depleted energy cellular charge. Subsequent transcription levels and protein expression of the enzymes involved in bioenergetics confirm the attempt to prevent the energy collapse with the increase in lactate dehydrogenase C, pyruvate dehydrogenase kinase 1 and 2, adenosine monophosphate deaminase, AMP-activated protein kinase, calcium/calmodulin-dependent protein kinase 2, and liver kinase B1, while decreasing ATP synthase. CONCLUSION: Our study suggests that changes in energy production could play a role in CDH pathogenesis. If confirmed in other animal models and humans, this could lead to the development of novel therapies targeting the mitochondria to improve outcomes.

The effect of tracheal occlusion in congenital diaphragmatic hernia in the nitrofen rat lung explant model.

PURPOSE: Here, we establish a tracheal occlusion (TO) model with rat lung explants in nitrofen-induced pulmonary hypoplasia in the congenital diaphragmatic hernia (CDH). METHODS: We extracted lungs from rats on an embryonic day 18. We mimicked TO in the lung explants by tying the trachea. We assessed lung weight, morphometry, and abundance of Ki-67, Active caspase-3, and Prosurfactant Protein C (proSP-C) with immunofluorescence. RESULTS: Lung weight was higher in TO + than TO - on day 1. Abundance of Ki-67 was higher in TO + than TO - (0.15 vs. 0.32, p = 0.009 for day 1, 0.07 vs. 0.17, p = 0.004 for day 2, 0.07 vs. 0.12, p = 0.044 for day 3), and Active caspase-3 was higher in TO + than TO - on day 2 and day 3 (0.04 vs. 0.03 p = 0.669 for day 1, 0.03 vs. 0.13 p < 0.001 for day 2, 0.04 vs. 0.17 p = 0.008 for day3). However, proSP-C protein abundance was lower in TO + than TO - (67.9 vs. 59.1 p = 0.033 for day 1, 73.5 vs. 51.6 p = 0.038 for day 2, 83.1 vs. 56.4 p = 0.009 for day 3). CONCLUSIONS: The TO model in lung explants mimics the outcomes of current surgical models of TO and further studies can reveal the cellular and molecular effects of TO in CDH lungs.

Yes-associated protein is dysregulated during nitrofen-induced hypoplastic lung development due to congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a birth defect associated with abnormal lung development. Yes-associated protein (YAP) is a core kinase of the Hippo pathway, which controls organ size during development. The absence of YAP protein during lung development results in hypoplastic lungs comparable to the lung phenotype in CDH (Mahoney, Dev Cell 30(2):137-150, 2014). We aimed to describe the expression of YAP during normal and nitrofen-induced abnormal lung development. METHODS: Intra-gastric administration of dams with 100 mg of nitrofen was used to induce CDH and abnormal lung development in the embryos. Immunofluorescence was performed to visualize the localization of YAP and p-YAP during lung development (E15, E18, E21). Western Blotting was used to determine the abundance of YAP and p-YAP in E21 control and nitrofen-induced hypoplastic CDH lungs. RESULTS: Immunofluorescence demonstrated cytoplasmic localization of YAP protein in airway epithelial and mesenchymal cells of nitrofen-induced hypoplastic lungs compared to nuclear localization in control lungs. Western Blotting showed a decrease (p = 0.0188) in abundance of YAP (active form) and increase in p-YAP (inactive form) in hypoplastic lungs compared to control lungs. CONCLUSION: Our results demonstrate that YAP protein is mostly phosphorylated, inactive, and expressed in the cytoplasm at the later stages of nitrofen-induced hypoplastic lung development indicating that the alteration in regulation of YAP can be associated with the pathogenesis of abnormal lung development in experimental CDH.

A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats.

BACKGROUND: Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. METHODS: Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. RESULTS: DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. CONCLUSIONS: This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. IMPACT: There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.

Bioisosteric Modification of To042: Synthesis and Evaluation of Promising Use-Dependent Inhibitors of Voltage-Gated Sodium Channels.

Three analogues of To042, a tocainide-related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use-dependent behavior. Patch-clamp experiments on hNav1.4 expressed in HEK293 cells showed that N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine, the aryloxyalkyl bioisostere of To042, exerted a higher use-dependent block than To042 thus being able to preferentially block the channels in over-excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P-glycoprotein (P-gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisostere of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be adopted in both the unbound and bound state of the ligand. Overall, N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine exhibits an interesting toxico-pharmacological profile and deserves further investigation.

Vitamin D improves pulmonary function in a rat model for congenital diaphragmatic hernia.

A congenital diaphragmatic hernia (CDH) is an anomaly caused by defects in the diaphragm; the resulting limited thorax cavity in turn restricts lung growth (pulmonary hypoplasia). This condition is related to pulmonary hypertension. Despite advances in neonatal CDH therapy, the mortality for severe pulmonary hypoplasia remains high. Therefore, it is essential to establish prenatal therapeutic interventions. Vitamin D was reported to have beneficial effects on adult pulmonary hypertension. This study aims to evaluate the efficacy of prenatal vitamin D administration for CDH. First, serum 25-hydroxyvitamin D [25(OH)D] levels in umbilical cord blood were evaluated among CDH newborns. Second, Sprague Dawley rat CDH models were exposed to nitrofen on embryo day 9 (E9). Randomly selected rats in the nitrofen-treated group were infused with calcitriol from E9 to E21. Samples from CDH pups diagnosed after birth were used for lung weight measurements, blood gas analysis, and immunohistochemical analysis. Third, microarray analysis was performed to examine the effect of vitamin D on gene expression profiles in CDH pulmonary arterial tissues. Serum 25(OH)D levels in the umbilical cord blood of newborns who did not survive were significantly lower than those who were successfully discharged. Prenatal vitamin D showed no significant effect on CDH incidence or lung weight but attenuated alveolarization and pulmonary artery remodeling accompanied the improved blood gas parameters. Vitamin D inhibited several gene expression pathways in the pulmonary arteries of CDH rats. Our results suggest that prenatal vitamin D administration attenuates pulmonary vascular remodeling by influencing several gene pathways in CDH.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. METHODS: The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and ß-Catenin were quantified by Western blot. RESULTS: Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho ß-Catenin, and BMP4. CONCLUSIONS: These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Design, Synthesis, and Herbicidal Activity of Novel Diphenyl Ether Derivatives Containing Fast Degrading Tetrahydrophthalimide.

To seek new protoporphyrinogen oxidase (PPO) inhibitors with better biological activity, a series of novel diphenyl ether derivatives containing tetrahydrophthalimide were designed based on the principle of substructure splicing and bioisomerization. PPO inhibition experiments exhibited that 6c is the most potential compound, with the half-maximal inhibitory concentration (IC50) value of 0.00667 mg/L, showing 7 times higher activity than Oxyfluorfen (IC50 = 0.0426 mg/L) against maize PPO and similar herbicidal activities to Oxyfluorfen in weeding experiments in greenhouses and field weeding experiments. In view of the inspected bioactivities, the structure-activity relationship (SAR) of this series of compounds was also discussed. Crop selection experiments demonstrate that compound 6c is safe for soybeans, maize, rice, peanuts, and cotton at a dose of 300 g ai/ha. Accumulation analysis experiments showed that the accumulation of 6c in some crops (soybeans, peanuts, and cotton) was significantly lower than Oxyfluorfen. Current work suggests that compound 6c may be developed as a new herbicide candidate in fields.

MiR-130a-5p/Foxa2 axis modulates fetal lung development in congenital diaphragmatic hernia by activating the Shh/Gli1 signaling pathway.

AIMS: Congenital diaphragmatic hernia (CDH) is a lethal birth defect characterized by congenital lung malformation, and the severity of pulmonary hypoplasia directly affects the prognosis of infants with CDH. Using a nitrofen-induced CDH rat model, we previously reported that Foxa2 expression was downregulated in CDH lungs by proteomics analysis. Here, we investigate the role of miR-130a-5p/Foxa2 axis in lung development of the nitrofen-induced CDH and evaluate its potential role in vivo prenatal therapy. MAIN METHODS: Nitrofen was orally administrated on embryonic day (E) 8.5 to establish a rat CDH model, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5 and E21.5. The binding sites of miR-130a-5p on Foxa2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. The expression levels of miR-130a-5p and Foxa2 were detected using qRT-PCR, ISH, IHC and western blotting. The role of miR-130a-5p/Foxa2 axis in CDH-associated lung development was investigated in ex vivo lung explants. KEY FINDINGS: We found that Foxa2 was downregulated in CDH lung tissues, and Foxa2 upregulating improved CDH branching morphogenesis in ex vivo lung explants. Meanwhile, we also showed that miR-130a-5p was significantly upregulated in CDH lungs and thus inversely correlated with Foxa2. Increasing miR-130a-5p abundance with mimics decreases Foxa2-driven Shh/Gli1 signaling and inhibits branching morphogenesis in ex vivo lung explants. SIGNIFICANCE: This study was the first to show that the miR-130a-5p/Foxa2 axis played a crucial role in CDH-associated pulmonary hypoplasia. These findings may provide relevant insights into the prenatal diagnosis and prenatal therapy of CDH.

The Analysis of Bifenox and Dichlobenil Toxicity in Selected Microorganisms and Human Cancer Cells.

Bifenox and Dichlobenil belong to the commonly used in Poland in agriculture group of herbicides and their residues are often detected in the environment. They are poorly known regarding their possible carcinogenic and antibacterial effect at the cellular level. Therefore, we decided to study their activity in bacterial strains Aliivibrio fisheri,E. coli, P. aeruginosa, and C. albicans (yeast) and human cancer ZR-75-1 cells. Compounds under study exhibit stimulatory effect on analyzed bacterial strains. The study performed on mammalian cells better reflects the influence of environmental pollutants on human organism, therefore we evaluated the effect of herbicides on ZR-75-1 cells. Cells viability, apoptosis and selected oxidative stress parameters in ZR-75-1 cells were investigated. Both analyzed substances exhibit stimulatory effects on analyzed parameters, however they do not stimulate apoptosis which correlate positively with the induction of oxidative stress. Bifenox and Dichlobenil enhance oxidative stress parameters by the generation of high levels of ROS, which can lead to their adaptation and resistance to the standard treatment regimen.

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs.

BACKGROUND: Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs. OBJECTIVE: We used the nitrofen/bisdiamine mouse model to induce congenital diaphragmatic hernia and accurately assess the status of alveolar epithelial cell differentiation in relation to the common bipotent progenitors. STUDY DESIGN: Pregnant Swiss mice were gavage-fed with nitrofen/bisdiamine or vehicle at embryonic day 8.5. The administered dose was optimized by assessing the survival, congenital diaphragmatic hernia and facial abnormality rates of the exposed mouse pups. NanoCT was performed on embryonic day 11.5 and 16.5 to assess the embryonic and early canalicular stages of lung development. At embryonic day 17.5 corresponding to late canalicular stage, congenital diaphragmatic hernia lungs were characterized by measuring the lung weight/body weight ratio, morphometry, epithelial cell marker gene expression levels and alveolar cell type quantification. RESULTS: Nitrofen/bisdiamine associated congenital diaphragmatic hernia lungs showed delayed development, hypoplasia with morphologic immaturity and thickened alveolar walls. Expression levels of distal epithelial progenitor marker Id2 increased, alveolar type 1 cell markers Pdpn and Hopx decreased, while type 2 cell markers pro-SPC and Muc1 remained constant during the canalicular stage. The number of Pdpn+ type 1 alveolar cells also decreased in congenital diaphragmatic hernia lungs. CONCLUSION: The mouse nitrofen/bisdiamine model is a potential model allowing the study of congenital diaphragmatic hernia lung development from early stages using a wide array of methods. Based on this model, the alveolar epithelium showed a decrease in the number of alveolar type 1 cell in congenital diaphragmatic hernia lungs while type 2 cell population remains unchanged.

Determination by chromatography and cytotoxotoxic and oxidative effects of pyriproxyfen and pyridalyl.

Pyriproxyfen (PPF) is a larvicide, used to combat the proliferation of Aedes aegypti larvae. The objective of this study was to analyze the compounds of pyriproxyfen and pyridalyl (PYL) in a commercial larvicide to analyze the cytotoxic and oxidative effects of PPF and PYL. The toxic potential of PPF and PYL were assessed based on lethal concentration (LC50) in Artemia salina, cytotoxicity based on the mitotic index and the chromosomal alterations in Allium cepa and the oxidative damage in Saccharomyces cerevisiae. The PPF and PYL compounds were identified by HPLC-PDA based on their retention times and spectral data. The wavelengths λmax (258 nm) and (271 nm) of the UV spectrum of PYL and PPF and the retention times (RT) (3.38 min) and (4.03 min), respectively. The toxicological potentials of PPF and PYL were significant at concentrations (1, 10, 100 and 1000 ppm), with an LC50 of 48 h (0.5 ppm). PPF and PYL pointed out a cytotoxic effect in A. cepa at all concentrations (0.0001, 0.001, 0.01, 0.1, 1.0, 100 and 1000 ppm), genotoxic effect at concentrations only (0.0001; 0.1; 1; 100 and 1000 ppm), and mutagenic for concentrations (0.1, 100 and 1000 ppm). In relation S. cerevisiae, PPF e PYL prompted oxidative damage at concentrations (100 and 1000 ppm) in all strains (SODWT, Sod1, Sod2, Sod1Sod2, Cat1 and Sod1Cat1). Therefore, the PPF and PYL identificated in commercial larvicide by HPLC-PDA produced cytotoxic and oxidative effects that could cause health and ecosystem risks.

Oxidative stress potential of the herbicides bifenox and metribuzin in the microalgae Chlamydomonas reinhardtii.

The widespread presence of herbicides in the aquatic environment has raised awareness about the need to develop further in depth ecotoxicological risk assessments, more specifically on potential effects on photosynthetic organisms as microalgae. The majority of the information available regarding the toxicity of herbicides towards microalgae is related to traditional toxicological and regulatory-relevant endpoints such as growth inhibition, leaving a significant gap on knowledge regarding underlying interactions and damage to biological targets. In this context, this study aimed to supplement the general toxicity information of bifenox and metribuzin in the microalgae Chlamydomonas reinhardtii using a battery of selected high-throughput methods. This multiple-endpoint approach included the measurement of formation of reactive oxygen species (ROS), alterations in reduced glutathione (GSH) content, formation of lipid peroxidation (LPO), photosystem II (PSII) performance and loss of photosynthetic pigments after 24 h exposure. Results obtained showed that both herbicides caused a concentration-dependent increase in ROS formation, with bifenox showing higher but less reactive ROS. This increase in ROS production by bifenox and metribuzin was followed by alterations in the antioxidant capacity of algae, oxidative damage in the form of LPO and alterations in pigment content. Furthermore, both herbicides impacted the photosynthetic activity of algae, as seen by alterations in the maximum and effective quantum efficiency of PSII, PSII photochemistry and energy dissipation pathways, impact in the water-splitting apparatus and reduction in the electron transport rate. The inhibitory effect of metribuzin on photosynthetic processes/components was larger than that seen for bifenox. The impact of bifenox and metribuzin in the photosynthetic processes of C. reinhardtii seems to be in close association with the formation of ROS and consequent oxidative stress and damage in algal cells. Overall, this study showed that the high-throughput methods developed could successfully characterise both potential Modes of Action and adverse effects of bifenox and metribuzin in C. reinhardtii.

Pulmonary hypertension secondary to congenital diaphragmatic hernia: factors and pathways involved in pulmonary vascular remodeling.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is characterized by pulmonary hypoplasia and pulmonary hypertension (PHTN). PHTN secondary to CDH is a result of vascular remodeling, a structural alteration in the pulmonary vessel wall that occurs in the fetus. Factors involved in vascular remodeling have been reported in several studies, but their interactions remain unclear. To help understand PHTN pathophysiology and design novel preventative and treatment strategies, we have conducted a systematic review of the literature and comprehensively analyzed all factors and pathways involved in the pathogenesis of pulmonary vascular remodeling secondary to CDH in the nitrofen model. Moreover, we have linked the dysregulated factors with pathways involved in human CDH. Of the 358 full-text articles screened, 75 studies reported factors that play a critical role in vascular remodeling secondary to CDH. Overall, the impairment of epithelial homeostasis present in pulmonary hypoplasia results in altered signaling to endothelial cells, leading to endothelial dysfunction. This causes an impairment of the crosstalk between endothelial cells and pulmonary artery smooth muscle cells, resulting in increased smooth muscle cell proliferation, resistance to apoptosis, and vasoconstriction, which clinically translate into PHTN.

Endoplasmic reticulum stress response is activated in pulmonary hypoplasia secondary to congenital diaphragmatic hernia, but is decreased by administration of amniotic fluid stem cells.

PURPOSE: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired epithelial homeostasis. Recently, amniotic fluid stem cells (AFSCs) have been shown to promote growth in hypoplastic lungs of rat fetuses with CDH. Herein, we investigated whether CDH hypoplastic lungs mount an endoplasmic reticulum (ER) stress response and whether AFSCs could re-establish pulmonary epithelial homeostasis. METHODS: Primary epithelial cells were isolated from fetal rat lungs at E14.5 from control and nitrofen-exposed dams at E9.5. Nitrofen-exposed epithelial cells were grown in medium alone or co-cultured with AFSCs. Epithelial cell cultures were compared for apoptosis (TUNEL), cytotoxicity (LIVE/DEAD assay), proliferation (5'EdU), and ER stress (CHOP, Bcl-2) using one-way ANOVA (Dunn's post-test). RESULTS: Compared to control, nitrofen-exposed epithelial cells had increased cytotoxicity and apoptosis, reduced proliferation, and activated ER stress. AFSCs restored apoptosis, proliferation, and ER stress back to control levels, and significantly reduced cytotoxicity. CONCLUSIONS: This study shows for the first time that ER stress-induced apoptosis is activated in the pulmonary epithelium of hypoplastic lungs from fetuses with CDH. AFSC treatment restores epithelial cellular homeostasis by attenuating the ER stress response and apoptosis, by increasing proliferation and migration ability, and by reducing cytotoxicity.