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This study systemically investigated the enantioselective bioaccumulation and degradation of etoxazole (ETZ) in earthworms along with the transcriptome and oxidative stress responses to ETZ enantiomer exposure. Based on the M-shaped bioaccumulation trends for ETZ enantiomers, R -ETZ was found to be preferentially bioaccumulated in earthworms. Sublethal toxicity analysis showed that S -ETZ induced greater changes in protein content, malondialdehyde content, detoxifying metabolic enzyme activity, and oxidative stress in earthworms, compared to those induced by R -ETZ. Integrated biomarker response analysis suggested that S -ETZ induced higher sublethal toxicity in earthworms than R -ETZ. Finally, transcriptomic analysis indicated that 845 and 314 genes were differentially expressed after R -ETZ and S -ETZ exposure, respectively, when compared to the nonexposed control group. Enrichment analysis indicated that these differentially expressed genes were primarily enriched in the digestion and absorption of proteins, lysosome, peroxisome, and peroxisome proliferator-activated receptor signaling pathways. These results suggest that earthworms exhibit distinct enantioselective responses to S -ETZ and R -ETZ. This study elucidates the enantioselective bioaccumulation, degradation, transcriptome, and oxidative stress characteristics of ETZ enantiomers in earthworms at the enantiomer level, offering a theoretical foundation to improve the risk assessment of ETZ in the soil-earthworm microsomes.

Chitin nanocrystals (ChNCs), known for their high aspect ratio, surface charge, and mobility, are promising bio-based nanomaterials for drug delivery. However, their potential as pesticide carriers in agriculture remains underexplored. Etoxazole, a diphenyl oxalate acaricide, effectively inhibits egg hatching and the normal molting process in mites but suffers from rapid degradation and short persistence in field applications. This study introduces a novel formulation, Eto@ChNC, prepared by complexing TEMPO-oxidized ChNCs with etoxazole via a one-pot method. Eto@ChNC was evaluated for controlling Tetranychus urticae, demonstrating significantly enhanced rapid action and prolonged efficacy compared to traditional formulations. The formulation increased the synergistic effects on mite eggs and deutonymphs by 41.74 % and 67.85 %, respectively, extending effectiveness by two days. The improved performance was attributed to the enhanced wetting ability of Eto@ChNC on leaf surfaces and its superior inhibition of the epidermal chitin content in T. urticae, facilitating greater etoxazole penetration. Transcriptome sequencing revealed numerous differentially expressed genes related to chitin metabolism, elucidating the molecular mechanisms underlying the increased efficacy. Safety assessments confirmed that Eto@ChNC did not elevate toxicity to earthworms or predatory mites and promoted the growth of wheat and cowpea, underscoring its environmental safety. These findings highlight Eto@ChNC as a significant advancement in bio-based acaricide formulations, offering promising applications in mite management., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Endocrine-disrupting chemicals (EDCs) are exogenous chemical compounds that interfere with the normal function of the endocrine system and are linked to direct and inherited adverse effects in both humans and wildlife. Legacy EDCs such as polychlorinated biphenyls (PCBs) are no longer used yet remain detectable in biological specimens around the world; concurrently, we are exposed to newer EDCs like the fungicide vinclozolin (VIN). This combination of individuals' direct environmental chemical exposures and any heritable changes caused by their ancestors' chemical exposures leads to a layered pattern of both direct and ancestrally inherited exposures that might have cumulative effects over generations., Objectives: We assessed consequences of both direct and ancestral exposure to EDCs over six generations, examining anxiety-like behaviors in maternal and paternal lines of female rats. We used the "two hits, three generations apart" multigenerational exposure model to explore how two distinct EDCs-the weakly estrogenic PCB mixture Aroclor 1221 (A1221) and the antiandrogenic VIN-interact on behavior across generations. We also explored serum hormones as a potential mechanism., Methods: Rats were prenatally exposed to A1221, VIN, or vehicle (DMSO) in the F1 generation, and a second exposure (same or different) was administered to the F4 generation. Anxiety-like behavior was measured in the Open Field test, Light:Dark box, and Elevated Plus Maze in the F1, F3, F4, and F6 generations. Serum concentrations of estradiol and corticosterone were analyzed., Results: Behavioral effects were not detectable in the F1 generation but emerged and became more robust across generations. Rats with ancestral VIN exposure demonstrated less anxiety-like behavior in the F3 paternal line in comparison with controls. Rats exposed to ancestral then prenatal A1221/VIN and VIN/A1221 had more anxiety-like behavior in the F4 maternal line, and those with two ancestral hits of VIN/VIN had more anxiety in the F6 paternal line, in comparison with controls., Discussion: Our findings suggest that anxiety-like behavioral phenotypes can manifest in rats following germline exposure to EDCs and that subsequent exposures across generations can intensify these effects in a lineage-dependent manner. https://doi.org/10.1289/EHP15621.

Background: Increasing evidence supports an association of endocrine-disrupting chemical (EDC) exposures with adverse biological effects in humans and wildlife. Recent studies reveal that health consequences of environmental exposures may persist or emerge across generations. This creates a dual conundrum: that we are exposed to contemporary environmental chemicals overlaid upon the inheritance of our ancestors' exposure profiles. Even when legacy EDCs are phased out, they may remain relevant due to persistence in the environment together with intergenerational inheritance of their adverse biological effects. Thus, we all possess a body burden of legacy contaminants, and we are also increasingly exposed to new generations of EDCs., Objectives: We assessed the effects of direct and ancestral exposures to EDCs across six generations on anxiety-like behaviors in male rats using our "two hits, three generations apart" multigenerational EDC exposure experimental model. We investigated two classes of EDCs with distinct hormonal actions and historical use-the weakly estrogenic polychlorinated biphenyl (PCB) mixture Aroclor 1221 (A1221) and the anti-androgenic fungicide vinclozolin (VIN)-in both the maternal and paternal line. We also determined if a hormonal mechanism drives these effects across generations., Methods: Rats were gestationally exposed to A1221, VIN, or vehicle [dimethyl sulfoxide (DMSO)] in the F1 generation. Three generations later, the F4 generation was given the same or a different exposure. Anxiety-like behavior was measured in the open field test, light:dark box, and elevated plus maze across generations. Serum was collected at the end of the experiment, and concentrations of estradiol and corticosterone were analyzed., Results: Although direct exposure did not affect behavior in F1 males, ancestral exposure to VIN decreased anxiety-like behavior in the F3 paternal line compared to vehicle. In the F4 paternal line, ancestral A1221 followed by direct exposure to VIN increased anxiety-like behavior compared to controls. In the F6 maternal line, relative to vehicle, the double ancestral hits of A1221/VIN decreased anxiety-like behavior. Serum hormones weakly predicted behavioral changes in the F4 paternal line and were modestly affected in the F4 and F6 maternal lines., Discussion: Our data suggest that anxiety-like behavioral phenotypes emerge transgenerationally in male rats in response to EDC exposure and that multiple hits of either the same or a different EDC can increase the impact in a lineage-specific manner. https://doi.org/10.1289/EHP15684.

Isoxazoline insecticides have shown broad-spectrum insecticidal activity against a variety of insect pests. However, the high toxicity of isoxazoline compounds towards honeybees restricts their application in crop protection. To mitigate this issue, a series of isoxazoline derivatives containing 2-phenyloxazoline were designed and synthesized. Bioassays revealed that several compounds exhibited promising insecticidal activities against Plutella xylostella, with G28 showing particularly excellent insecticidal activity, reflected by an LC 50 value of 0.675 mg/L, which is comparable to that of fluxametamide (LC 50  = 0.593 mg/L). Furthermore, G28 also exhibited effective insecticidal activity against Solenopsis invicta. Importantly, bee toxicity experiments indicated that G28 had significantly lower acute oral toxicity (LD 50  = 2.866 μg/adult) compared to fluxametamide (LD 50  = 1.083 μg/adult) and fluralaner (LD 50  = 0.022 μg/adult), positioning it as a promising candidate with reduced toxicity to bees. Theoretical simulation further elucidated the reasons for the selective differences in the ability of isoxazoline to achieve higher insecticidal activity while maintaining lower bee toxicity. This research suggests that isoxazoline compounds containing 2-phenyloxazoline group hold potential as new insecticide candidates and offers insights into the development of novel isoxazoline insecticides with both high efficacy and environmental safety., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Etoxazole is among the systemic pesticides with acaricidal and insecticidal characteristics. This paper reports the first evaluation of the toxic effects of Etoxazole on Allium cepa L. Etoxazole solutions were applied to three groups formed from A. cepa bulbs at 0.125 mL/L, 0.25 mL/L and 0.5 mL/L doses, respectively. The control group was treated with tap water throughout the experimental period. The toxic effects of Etoxazole became more apparent as the dose of Etoxazole was increased. The growth-limiting effect was most pronounced in the highest dose group with approximately 29%, 70% and 58.5% reductions in germination percentage, root elongation and weight gain, respectively. The genotoxic effect of Etoxazole was most severe in the 0.5 mL/L dose group. In this group, the mitotic index decreased by 30% compared to the control group, while the micronucleus frequency increased to 45.3 ± 3.74. The most observed aberrations were fragment, vagrant chromosome, sticky chromosome, unequal distribution of chromatin, bridge, reverse polarization and nucleus with vacuoles. The malondialdehyde level showed a gradual increase with increasing Etoxazole doses and reached 2.7 times that of the control group in the 0.5 mL/L Etoxazole applied group. Catalase and Superoxide dismutase activities increased in the groups exposed to 0.125 mL/L and 0.25 mL/L Etoxazole with dose dependence and decreased abruptly in the group treated with 0.5 mL/L Etoxazole. Etoxazole triggered meristematic cell damages, such as epidermis cell damage, thickening of cortex cell walls, flattened cell nucleus and indistinct transmission tissue. Considering the versatile toxicity induced by Etoxazole, we announce that this chemical has the potential to cause serious damage to non-target organisms. It should be noted that the higher the dose of exposure, the more severe the level of damage. This study will be an important reminder to limit the indiscriminate use of this highly risky agrochemical., (© 2022. The Author(s).)

Endocrine-disrupting chemicals (EDCs) are different natural and synthetic chemicals that may interfere with several mechanisms of the endocrine system producing adverse developmental, metabolic, reproductive, and neurological effects in both human beings and wildlife. Among pesticides, numerous chemicals have been identified as EDCs. MicroRNAs (miRNAs) can regulate gene expression, making fine adjustments in mRNA abundance and regulating proteostasis. We hypothesized that exposure to low doses of atrazine, cypermethrin, and vinclozolin may lead to effects on miRNA expression in SH-SY5Y cells. In particular, the exposure of SH-SY5Y cells to subtoxic concentrations of vinclozolin is able to downregulate miR-29b-3p expression leading to the increase in the related gene expression of ADAM12 and CDK6, which may promote a pro-oncogenic response through the activation of the PI3K/Akt/mTOR pathway and counteracting p53 activity. A better understanding of the molecular mechanisms of EDCs could provide important insight into their role in human disease.

Endocrine-disrupting substances (EDS) are common and pervasive in our environment and pose a serious risk to both human and animal health. Endocrine-disrupting compounds (EDCs) have been associated with a variety of detrimental human health effects, including respiratory issues, as a result of their ability to disrupt cell physiology. Vinclozolin ((RS)-3-(3,5-Dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione) is a common dicarboximide fungicide used to treat plant diseases. Several studies have analyzed the effects of vinclozolin exposure on the reproductive system, but less is known about its effect on other organs such as the lung. Mice were exposed for 28 days to orally administered vinclozolin at a dose of 100 mg/kg. Vinclozolin exposure induced histological alterations and collagen depositions in the lung. Additionally, vinclozolin induced inflammation and oxidative stress that led to lung apoptosis. Our study demonstrates for the first time that the toxicological effects of vinclozolin are not limited to the reproductive system but also involve other organs such as the lung.

Many man-made chemicals that are released into water bodies in agricultural landscapes have been identified as endocrine disruptors and can cause serious impacts on the growth and survival of aquatic species living in these environments. However, very little attention has been paid to their toxicological effects in cultured non-fish species, such as aquatic turtles. We exposed hatchlings of the Chinese soft-shelled turtle (Pelodiscus sinensis) to different concentrations of vinclozolin (0, 5, 50 and 500 μg/L) for 60 days to assess physiological and metabolic impacts of this fungicide. Despite no death occurrence, hatchling turtles exposed to the highest concentration of vinclozolin consumed less food, grew more slowly (resulting in smaller body size after exposure) and performed more poorly in behavioral swimming tests than controls and turtles exposed to lower concentrations. Hepatic metabolite profiles acquired via liquid chromatography-mass spectrometry (LC-MS) revealed multiple metabolic perturbations related to amino acid, lipid, and fatty acid metabolism in animals exposed to environmentally relevant concentrations. Specifically, many critical metabolites involved in energy-related metabolic pathways (such as some intermediates in the tricarboxylic acid cycle, lactate, and some amino acids) were present in livers of hatchling turtles exposed vinclozolin, though at lower concentrations, reflecting energy metabolism dysregulation induced by exposure to this fungicide. Overall, our results suggest that the changes in growth and behavioral performances caused by chronic vinclozolin exposure may be associated with internal physiological and metabolic disorders mediated at the biochemical level., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Prenatal and postnatal co-exposure to multiple chemicals at the same time may have deleterious effects on the developing nervous system. We previously showed that chemicals acting through similar mode of action (MoA) and grouped based on perturbation of brain derived neurotrophic factor (BDNF), induced greater neurotoxic effects on human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes compared to chemicals with dissimilar MoA. Here we assessed the effects of repeated dose (14 days) treatments with mixtures containing the six chemicals tested in our previous study (Bisphenol A, Chlorpyrifos, Lead(II) chloride, Methylmercury chloride, PCB138 and Valproic acid) along with 2,2'4,4'-tetrabromodiphenyl ether (BDE47), Ethanol, Vinclozolin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)), on hiPSC-derived neural stem cells undergoing differentiation toward mixed neurons/astrocytes up to 21 days. Similar MoA chemicals in mixtures caused an increase of BDNF levels and neurite outgrowth, and a decrease of synapse formation, which led to inhibition of electrical activity. Perturbations of these endpoints are described as common key events in adverse outcome pathways (AOPs) specific for DNT. When compared with mixtures tested in our previous study, adding similarly acting chemicals (BDE47 and EtOH) to the mixture resulted in a stronger downregulation of synapses. A synergistic effect on some synaptogenesis-related features (PSD95 in particular) was hypothesized upon treatment with tested mixtures, as indicated by mathematical modelling. Our findings confirm that the use of human iPSC-derived mixed neuronal/glial models applied to a battery of in vitro assays anchored to key events in DNT AOP networks, combined with mathematical modelling, is a suitable testing strategy to assess in vitro DNT induced by chemical mixtures., (Copyright © 2021. Published by Elsevier Inc.)

4,4'-Dimethylaminorex (4,4'-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±) cis (0.1-60 mg/kg) and (±) trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±) cis at 1, 10 or 60 mg/kg + (±) trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4'-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±) cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±) trans stereoisomer was ineffective whilst the stereoisomers' co-administration resulted in a worsening of the toxic (±) cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±) trans stereoisomer reduced the metabolism of the (±) cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

Vinclozolin (VCZ) is a fungicide with antiandrogen activity. Exposure to VCZ in maternal uterus may cause uterine, ovarian and testicular damage, hypospadias and prostate abnormality in the offspring. Hippo pathway, which is highly conservative and may be activated by miR132 and miR195a, can control organ size and tissue regeneration, and participate in injury and deformity. In the present study, VCZ was found to have caused penile malformation in the male offspring and also induced "small testis" when it was administered to the pregnant mice orally at a dose of 400 mg kg -1 day -1 on Days 12-18 of gestation. At 1, 3 and 7 weeks of age, VCZ could increase miR132, Mst1, Sav1, phosphorylated Yes-associated protein (pYap) and pLats, and decrease Yap in offspring penises and testes. Besides, it could also raise miR195a both in the testes of 1, 7-week and in the penises of all the three ages. In addition, we found the levels of some cyclin (Ccn) genes elevated in the testes, the expression of the androgen receptor (Ar) gene dereased and Jnks changed in the penises of offspring aged 1, 3 and 7 weeks. The results suggest that that gestational VCZ exposure could not only increase miR132 and miR195a in penises and testes of the offspring, but also activate Hippo pathway and down-regulate Ar. These may directly inhibit cell proliferation, accelerate cell death by up-regulating the expression of some Ccns, and ultimately lead to penile and testicular damage and malformations in the offspring., (Copyright © 2021. Published by Elsevier B.V.)

Background: Humanin (HN) is an extensive neuroprotective peptide. This study aims to investigate the neuroprotective effects of HN on Calyculin A (CA)-induced neurotoxicities in cortical neurons and the underlying mechanism., Methods: CA was added into the cultured cortical neurons to induce neurotoxicity. Cortical neurons were preincubated with HN which plays a protective role. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and Calcein-AM were applied to evaluate the neural insults. Caspase 3 signal and Tunnel were performed to test neural apoptosis. Western blot analysis was used to detect the expressions of phosphorylated tau. The corresponding kits were used to measure the contents of malondialdehyde (MDA) and superoxide dismutase (SOD), and the activity of PP2A, respectively., Results: HN preincubation preserved cell viability, protected the neurons, alleviated oxidative stress, and reserved PP2A activity. It also blocked tau overphosphorylation at Ser199/202, Ser396, and Thr231 sites and protected neurons against CA-induced insults., Conclusion: These results suggest that HN may serve as a potential therapeutic agent to prevent the pathological changes induced by CA via modulating the activity of PP2A and oxidative stress in neurodegenerative diseases.

Endocrine disrupting compounds (EDCs) are ubiquitous environmental pollutants that alter endocrine system function, induce birth defects, and a myriad of other negative health outcomes. Although the mechanism of toxicity of many EDCs have been studied in detail, little work has focused on understanding the mechanisms through which pregnant dams and fetuses protect themselves from EDCs, or if those protective mechanisms are sexually dimorphic in fetuses. In this study, we examined proteomic alterations in the livers of mouse dams and their male and female fetuses induced by vinclozolin, a model antiandrogenic EDC. Dam livers upregulated nine phase I and phase II detoxification pathways and pathway analysis revealed that more pathways are significantly enriched in dam livers than in fetal livers. Phase I and II detoxification proteins are also involved in steroid and steroid hormone biosynthesis and vinclozolin likely alters steroid levels in both the dam and the fetus. The response of the fetal liver proteome to vinclozolin exposure is sexually dimorphic. Female fetal livers upregulated proteins in xenobiotic metabolism pathways, whereas male fetal livers upregulated proteins in oxidative phosphorylation pathways. These results suggest that female fetuses increase protective mechanisms, whereas male fetuses increase ATP production and several disease pathways that are indicative of oxidative damage. Females fetuses upregulate proteins and protective pathways that were similar to the dams whereas males did not. If this sexually dimorphic pattern is typical, then males might generally be more sensitive to EDCs., (Copyright © 2021. Published by Elsevier Inc.)

Background: Herbicide safeners mitigate crop damage without reducing herbicide efficacy. Here, the protective effects of phenoxyacetyl oxazolidine derivatives as potential safeners were evaluated with a view toward reducing injury caused by sulfonylurea herbicide nicosulfuron to sensitive maize varieties., Results: Growth indices demonstrated that the bioactivity of compound 9 (N-phenoxyacety-2-methyl-2,4-diethyl-1,3-oxazolidine) was superior to that of R-28725 and all other compounds tested. Compound 9 induced endogenous glutathione and upregulated glutathione-S-transferase (GST) in maize. Thus, it could enhance maize tolerance to nicosulfuron. Compared with the untreated water control group, the maximum reaction rate of GST was increased by 37.62%, while the maximum velocity of GST was decreased by 61.93% after treatment with compound 9. Acetolactate synthase relative activity was significantly enhanced in the case of treatment with compound 9, indicating the excellent protective effects of compound 9 against nicosulfuron in maize., Conclusions: The present work demonstrates that phenoxyacetyl oxazolidine derivatives are potentially efficacious as herbicide safeners and merit further investigation., (© 2020 Society of Chemical Industry.)

The honey bee, Apis mellifera L., is the world's most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

Background: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed., Objectives: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats., Methods: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression., Results: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o -phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two ( λ -cyhalothrin , pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions., Discussion: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Living species including humans are continuously exposed to low levels of a myriad of endocrine active compounds that may affect their reproductive function. In contrast, experimental designs scrutinizing this question mostly consider the gestational/lactational period, select high unrealistic doses and, have rarely investigated the possible reproductive consequences in the progeny. The present study aimed at assessing comparatively a set of male reproductive endpoints according to exposure windows, gestational/lactational versus pre-pubertal to adulthood, using low doses of endocrine active substances in male rats as well as their unexposed male progeny. Animals were orally exposed to 1 mg/kg bw/d of genistein and/or vinclozolin, from conception to weaning or from prepuberty to young adulthood. A number of reproductive endpoints were assessed as well as testicular mRNA expression profiles, in the exposed rats and their unexposed progeny. Overall, the low dosage used only affected weakly most of classical reproductive endpoints. However, the gestational/lactational exposure to vinclozolin alone or combined to genistein significantly delayed the puberty onset. Contrasting with the gestational/lactational exposure, a decreased sperm production was found in the animals exposed to genistein and vinclozolin from the pre-pubertal period but also in their progeny for vinclozolin and the mixture. The expression level of several genes involved in meiosis, apoptosis and steroidogenesis was also affected differentially as a function of the exposure window in both exposed rats and unexposed offspring. We also provide further evidence that doses of endocrine active substances relevant with human exposure may affect the male reproductive phenotype and testicular transcriptome in the exposed generation as well as in the indirectly exposed offspring., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Vinclozolin is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor and is thought to be related to abnormalities of the reproductive tract. However, its mechanism of inducing abnormalities of the male reproductive tract is still unclear. The purpose of this study was to study the effect of gestational vinclozolin exposure on the development of rat fetal Leydig cells. Female pregnant Sprague-Dawley rats were exposed to vinclozolin (0, 25, 50, and 100 mg/kg body weight/day) by gavage from gestational day 14-21. Vinclozolin dose-dependently reduced serum testosterone levels at doses of 50 and 100 mg/kg and the anogenital distance at 100 mg/kg. RNA-seq, qPCR, and Western blotting showed that vinclozolin down-regulated the expression of Nr5a1, Sox9, Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3, Amh, Pdgfa, and Dhh and their encoded proteins. Vinclozolin reduced the number of NR2F2-positive stem Leydig cells at a dose of 100 mg/kg and enhanced autophagy in the testes. In conclusion, vinclozolin disrupts reproductive tract development and testis development in male fetal rats via several pathways., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A search for potent antitubercular agents prompted us to design and synthesize sulfamethaoxazole incorporated 4-thiazolidinone hybrids ( 7a - l ) by using a cyclocondensation reaction between 4-amino- N -(5-methylisoxazol-3-yl)benzenesulfonamide ( 4 ), aryl aldehyde ( 5a - l ), and mercapto acetic acid ( 6 ) resulting in good to excellent yields. All the newly synthesized 4-thiazolidinone derivatives were screened for their in vitro antitubercular activity against M. Bovis BCG and M. tuberculosis H37Ra ( MTB) strains. The compounds 7d , 7g , 7i , 7k , and 7l revealed promising antimycobacterial activity against M. Bovis and MTB strains with IC 90 values in the range of 0.058-0.22 and 0.43-5.31 µg/mL, respectively. The most active compounds were also evaluated for their cytotoxicity against MCF-7, HCT 116, and A549 cell lines and were found to be non-cytotoxic. Moreover, the synthesized compounds were also analyzed for ADME (absorption, distribution, metabolism, and excretion) properties and showed potential as good oral drug candidates.

Background: Vinclozolin (VCZ) is a widely used antifungal agent with capability to enter into the human food chain. VCZ metabolizes into seven metabolites M1-M7. Several studies have shown its effects on reprotoxicity. However, there is limited information available on the interaction of VCZ metabolites with nuclear receptors. In silico studies aimed at identifying interaction of endocrine disruptor with nuclear receptors serve a prescreening framework in risk assessment. Methods: We studied interactive potential of VCZ and its metabolites with human estrogen (ER) and androgen receptor (AR) using molecular docking method. Binding potential of VCZ and its metabolites with estrogen receptors 1GWR-α, 1QKM and androgen receptor 2AM9-β was checked by using Schrodinger Maestro 10.5. Estradiol (E2), a natural ligand of ER and AR was taken as a reference. Results: VCZ and its metabolites showed higher or similar binding efficiency on interaction with target proteins when compared with E2. VCZ and its metabolites also exhibited agonistic effect against 1GWR-α, 1QKM and 2AM9-β with strong binding potential to them. Conclusion: Some VCZ metabolites such as M4 and M5 showed higher binding potencies with 1GWR-α, 1QKM and 2AM9-β than E2. Toxicity data of VCZ is well endowed. However, endocrine disrupting potential of VCZ via nuclear receptor mediated pathway is less understood. This in silico study revealing that not only VCZ but its metabolites have potential to interact with 1GWR-α, 1QKM and 2AM9-β offers a platform for further exploration of VCZ in this direction.

Disruption of mitochondrial function selectively targets tumour cells that are dependent on oxidative phosphorylation. However, due to their high energy demands, cardiac cells are disproportionately targeted by mitochondrial toxins resulting in a loss of cardiac function. An analysis of the effects of mubritinib on cardiac cells showed that this drug did not inhibit HER2 as reported, but directly inhibits mitochondrial respiratory complex I, reducing cardiac-cell beat rate, with prolonged exposure resulting in cell death. We used a library of chemical variants of mubritinib and showed that modifying the 1 H -1,2,3-triazole altered complex I inhibition, identifying the heterocyclic 1,3-nitrogen motif as the toxicophore. The same toxicophore is present in a second anti-cancer therapeutic carboxyamidotriazole (CAI) and we demonstrate that CAI also functions through complex I inhibition, mediated by the toxicophore. Complex I inhibition is directly linked to anti-cancer cell activity, with toxicophore modification ablating the desired effects of these compounds on cancer cell proliferation and apoptosis., Competing Interests: ZS, RH, KP, SM, RH, RS, IC, TA, MS, MM, PF, JH, BK, AW No competing interests declared, (© 2020, Stephenson et al.)

An environmentally-friendly and fast analytical method for the stereoselective determination of etoxazole was developed and then applied to estimate stereoselective bioaccumulation and elimination in zebrafish using SFC-MS/MS. Optimal enantioseparation conditions were determined using a Chiralpak IG-3 column and CO 2 /MeOH mobile phase (80/20, v/v), at 3.0 mL/min within 1 min, 30°Me and 18 MPa. A modified QuEChERS method was developed for zebrafish sample pretreatment, and mean recoveries were 88.43-110.12% with relative standard deviations ranging from 0.32 to 5.34%. The enantioselectives of etoxazole enantiomers in zebrafish during uptake and depuration phases were evaluated. Significant enantioselective bioaccumulation was observed, with preferential accumulation of (-)-R-etoxazole compared to its antipode, during uptake at both low and high exposure concentrations. The toxic effects of etoxazole on zebrafish were further explored, and activities of antioxidant enzymes were determined in liver of zebrafish. Significant changes were observed in the SOD and GST activities and in the MDA levels, which indicated the occurrence of oxidative stress in liver of zebrafish. The toxic effects exhibited time- and dose-dependent properties. These results can facilitate the accurate risk evaluation of etoxazole and provide basic knowledge for further study of biotoxicity mechanisms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Etoxazole is an organofluorine insecticide widely used in agriculture. Exposure to insecticides is a serious environmental problem owing to their cytotoxic effects in humans and animals. Reproductive toxicity of various organofluorine insecticides have been shown in previous studies. However, few studies have evaluated the toxicity of etoxazole in mammals. We aimed to examine the toxic effects of etoxazole in porcine trophectoderm (pTr) and uterine luminal epithelial (pLE) cells. To estimate the effects of etoxazole, we conducted assays after treatment with multiple concentration of etoxazole (0, 2, 4, 6 and 9 μM) to pTr and pLE cells for 0-72 h. Etoxazole decreased the cell proliferation, viability, and migration of pTr and pLE cells. Further, etoxazole induced apoptosis via cell cycle arrest and disruption of mitochondrial membrane potential. We also found that pro-apoptotic proteins and endoplasmic reticulum (ER) stress-response proteins were activated in response to etoxazole. Finally, we observed that etoxazole altered the PI3K/AKT and MAPK signaling pathways and the mRNA expression of genes associated with implantation. Collectively, these results suggest that etoxazole disrupts normal cellular physiology and might cause early implantation failure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Epigenetic transgenerational inheritance potentially impacts disease etiology, phenotypic variation, and evolution. An increasing number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease. Previous observations have demonstrated that the agricultural fungicide vinclozolin and pesticide DDT (dichlorodiphenyltrichloroethane) induce transgenerational sperm epimutations involving DNA methylation, ncRNA, and histone modifications or retention. These two environmental toxicants were used to investigate the impacts of parent-of-origin outcross on the epigenetic transgenerational inheritance of disease. Male and female rats were collected from a paternal outcross (POC) or a maternal outcross (MOC) F4 generation control and exposure lineages for pathology and epigenetic analysis. This model allows the parental allelic transmission of disease and epimutations to be investigated. There was increased pathology incidence in the MOC F4 generation male prostate, kidney, obesity, and multiple diseases through a maternal allelic transmission. The POC F4 generation female offspring had increased pathology incidence for kidney, obesity and multiple types of diseases through the paternal allelic transmission. Some disease such as testis or ovarian pathology appear to be transmitted through the combined actions of both male and female alleles. Analysis of the F4 generation sperm epigenomes identified differential DNA methylated regions (DMRs) in a genome-wide analysis. Observations demonstrate that DDT and vinclozolin have the potential to promote the epigenetic transgenerational inheritance of disease and sperm epimutations to the outcross F4 generation in a sex specific and exposure specific manner. The parent-of-origin allelic transmission observed appears similar to the process involved with imprinted-like genes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Etoxazole, a chitin synthesis inhibitor, is widely used to control insects and mites by causing developmental defects. Despite the many advantages of pesticides, the inhibitory effects of most pesticides including etoxazole are based on biochemical reaction and their widespread application is considered as a major risk to human health and the environment because of bioaccumulation and non-target toxic effects. Though used in agricultural area, the pesticide residues run off through rivers or ocean, where diverse aquatic organisms live. Since there are no studies evaluating the risks of etoxazole exposure in embryogenesis of aquatic organisms, we investigated the adverse effects of etoxazole on development and angiogenesis in zebrafish embryos, which are considered to be an effective model for detecting ecotoxicological effects of widely used compounds, especially affecting aquatic organisms. Etoxazole induced yolk sac and heart edema, as well as loss of viability, abnormal heart rate, and developmental deficiency. Through a mechanistic approach, we also showed that etoxazole caused reactive oxygen species accumulation, inhibited the expression of cell cycle activating genes, and induced apoptosis. In addition, we investigated effects of etoxazole on cardiovascular development by demonstrating the loss of vascular structure in response to etoxazole exposure in fli1:eGFP transgenic zebrafish model. Collectively, this first assessment demonstrating the effects of etoxazole on embryogenesis and cardiovascular development provides clear evidence for the toxicity of etoxazole and contributes important data towards formulating safety guidelines on the potential hazards of etoxazole for aquatic environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Litter size and other conventional measures in rodents are common end-points in the assessment of xenobiotics for reprotoxic effects. However, since litter size may be normal despite reduced semen quality, we established and tested a mouse in vitro fertilization/in vitro culture (IVF/IVC) system to assess other aspects of reprotoxicity of xenobiotic exposure. Two pesticides, vinclozolin (V) and chlormequat (C), were added to feed in low (40 and 900 ppm, respectively) and high (300 and 2700 ppm, respectively) doses and compared to control (nil pesticide). Exposed males were used for natural mating to evaluate litter size and then used for IVF/IVC and sperm evaluation. The IVF/IVC system detected significant adverse effect of high dose of vinclozolin on blastocyst formation, which was not detected by conventional measures such as litter size or sperm motility and viability. We conclude that assessment based on IVF/IVC measures may complement litter size and other conventional end-points., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Prolonged exposure of Fenoxaprop-ethyl (FE), a post-emergence herbicide, can cause serious damage to animals through food chain. Melatonin is synthesized by the pineal gland in mammals and believed to protect cells from oxidative stress damage. In this study, we aimed to investigate the effects of FE on mouse oocyte meiosis maturation and the protective roles of melatonin on FE-exposed oocytes by in vitro maturation model. FE exposure significantly caused defects of the first polar body extrusion, which could be protected by co-culture with melatonin. Furthermore, we examined the meiotic maturation details by performing the sperm binding, actin and tubulin immunofluorescence, ROS and apoptosis detection, and histone methylation assay. Our data showed that FE exposure to oocytes led to disrupted actin filament dynamics, mis-organized spindle, and reduced the sperm binding capacity. In addition, FE-exposure increased oxidative stress level and induced oocyte apoptosis. We also found that FE exposure resulted in histone methylation changes. Treatment with melatonin could significantly improve these phenotypes in oocytes exposed to FE. In conclusion, FE exposure can cause meiotic defects by disrupting the cytoskeletal integrality and inducing excessive ROS accumulation to initiate apoptosis in oocytes, while melatonin can reduce all these damages, suggesting that melatonin has protective effects on oocytes exposed to FE during meiotic maturation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To observe the role of Hippo signaling pathway in mice hypospadias induced by vinclozolin., Methods: After 8 weeks of ICR mice were conceived, they were divided randomly into the treatment and the control groups(10 for each group). Vinclozolin was orally administraed to the pregnant mice in the treatment group with 400 mg/(kg·d) during gestation days 12-18, while those of the control group were treated with equal volume of soybean oil. About 60 days after birth, 20 penises of the mouse offspring were taken randomly from both of the two groups separately. And furthermore, the relative expression abundance of Hippo signaling pathway key genes(Mst1, Lats1, Taz, Yap), androgen receptor gene Ar of all the samples were measured by the real-time quantity reverse transcript polymerase chain reaction(qPCR). The Yes-related protein(Yap) and its phosphorylation in the downstream of Hippo signaling pathway were measured by Western blot., Results: Compared with the male mice of the control group, the anal genital distances of the treatment group were significantly shortened((9. 2501±2. 5504) vs. (16. 1253±1. 3562) mm, P<0. 05), the quality of the penises was significantly less than((0. 0293±0. 0075) vs. (0. 4731±0. 004) g, P<0. 05), the length of the penises was shorter((5. 3875±0. 4524) vs. (12. 4688±1. 2290) mm, P<0. 05), and the diameters of the penises were also less than the control((1. 5513±0. 1158) vs. (2. 6013±0. 1469) mm, P<0. 05). All the genes in the penises of the control group were expressed. The Yap and Ar in the treatment group's penises were not expressed, the expression abundance of Mst1 and Lats1 was higher than that of the control(6. 6097±1. 3188 vs. 0. 3517±0. 1524, 5. 7071±2. 7210 vs. 0. 1235±0. 0658, P<0. 05), and the expression abundance of Taz was lower than that of the control(0. 3937±0. 1519 vs. 3. 2329±0. 4339, P<0. 05). The expression of Yap was decreased in the treatment group(0. 3348±0. 0639 vs. 0. 8023±0. 4275, P<0. 05), and the phosphorylation level of Yap was higher than that of the control group(3. 9940±0. 2177 vs. 0. 3128±0. 0867, P<0. 05)., Conclusion: In the model of mice hypospadias induced by vinclozolin, the Ar was not expressed, the Hippo signaling pathway was activated and the phosphorylation of Yap was increased. This pathway may play an important role in the model.

This is the first systemic assessment of the stereoselectivity of etoxazole enantiomers. Etoxazole's stereoselective bioactivity was assessed against target organisms ( Tetranychus urticae eggs and Tetranychus cinnabarinus eggs), and its acute toxicity was assessed toward nontarget aquatic organisms ( Daphnia magna and Danio rerio). Additionally, stereoselective elimination was investigated in three species of fruits (grape and strawberry grown in a greenhouse and apple grown in an open field) and in field soil. The ovicidal activity of (+)-( S)-etoxazole against Tetranychus urticae and Tetranychus cinnabarinus eggs was about 16 and 24 times higher, respectively, than that of (-)-( R)-etoxazole. Inconsistent order of etoxazole isomer toxicity was found toward different aquatic organisms: (+)-( S)-etoxazole showed nearly 8.7 times higher acute toxicity than (-)-( R)-etoxazole toward Daphnia magna, whereas (-)-( R)-etoxazole was ∼4.5 times more toxic to Danio rerio than (+)-( S)-etoxazole. Stereoselective degradation of etoxazole enantiomers showed significant variation in various fruits and field soil. The (+)-( S)-etoxazole was preferentially dissipated in grape and strawberry fruits grown under greenhouse condition, whereas (-)-( R)-etoxazole degraded faster than its antipode in apple fruits and soils under open-field condition. Overall, the stereoselectivity of etoxazole enantiomers should be fully considered in comprehensive environmental health risk in future work.

The fungicide vinclozolin (Vz) is an endocrine disruptor with known anti-androgenic activity in vertebrates. However, there is a lack of information about the Vz mode of action in invertebrates, although some studies have shown that this compound can produce alterations in different species. Transcriptional activity was analyzed in the freshwater snail Physella acuta in order to elucidate putative cellular processes altered by this chemical during a response. In order to identify potential molecular biomarkers, a de novo transcriptome was generated for this species that constitutes a valuable source for future studies. This data, together with some already available data, permitted the identification of several genes related to detoxification mechanisms (Cyp2u1, Cyp3a7, Cyp4f22, GSTo1, GSTt2, and MRP1), stress response (Hsp20.4, Hsp17, Hsp16.6, and Cu,Zn-SOD), the hormonal system (Estrogen Receptor and Hsp90), apoptosis (Casp3), and copper homeostasis (ATOX1). Using quantitative Real-Time polymerase chain reaction, mRNA levels of these genes were examined in snails exposed to 20 or 200 µg/L Vz for 24 h. The results showed an overall weak response, with downregulation of Hsp20.4 and no statistically significant change for the other genes. These findings suggest that P. acuta can manage the concentrations of Vz found in the environment with no relevant activation of the pathways analyzed, although additional studies are needed for longer exposure times and including other metabolic pathways. The new genes described open the range of processes that can be studied at the molecular level in toxicity tests., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Fenoxaprop-ethyl (FE) is an active ingredient of commercially available herbicide formulations. Its overuse has caused much damage to the environment, livestock breeding, agricultural crops and humans. However, little is known about the effects of FE exposure on female reproductive health and the mechanisms underlying those effects. In this study, we investigated the toxic effects of FE on oocyte quality and their underlying mechanisms in mice fed a diet containing FE., Results: Ovary weight and numbers of oocytes were reduced in FE-treated mice. Moreover, oocyte quality was seriously impaired, as shown by the reduced rate of first polar body extrusion and fertilization ability in vivo. In FE-treated mice, oocytes presented reduced actin expression and abnormal meiotic spindle morphology, which indicate that cytoskeletal integrality is disrupted. Also, FE induced mitochondrial dysfunction, reflected by the accumulation of reactive oxygen species (ROS), apoptosis and autophagy, as revealed by fluorescent staining analysis and real-time polymerase chain reaction (qPCR). Finally, FE led to changes in epigenetic modifications such as histone H3K27me3 and H3K9me2 in oocytes., Conclusions: Our results indicate that FE has adverse effects on oocyte quality as assessed by maturation and fertilization potential, due to disrupted cytoskeletal integrality, and mitochondrial dysfunction leading to ROS accumulation, apoptosis and autophagy. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Background: The incidence of invasive fungal infections caused by Candida spp. has increased continuously in recent decades, especially in populations of immunocompromised patients or individuals hospitalized with serious underlying diseases. Therefore, the goal of our study was the search for new potent Candida albicans inhibitors via the development of QSAR models that could speed up this search process. A number of the most promising 1,3-oxazol-4-yltriphenylphosphonium derivatives with predicted activities were synthesized and experimentally tested. Furthermore, the toxicity of the studied compounds was determined in vitro using acetylcholinesterase enzyme as a biological marker., Methods: The classification QSAR models were created using Random Forests (WEKA-RF), k-Nearest Neighbors and Associative Neural Networks methods and different combinations of descriptors on the Online Chemical Modeling Environment (OCHEM) platform. Аntifungal properties of the investigated compounds were performed using standard disk diffusion method. The enzyme inhibitory action of the compounds was determined by modified Ellman's method using acetylcholinesterase from the electric organ of Electrophorus electricus., Results: Three classification QSAR models were developed by the WEKA-RF, k-NN and ASNN methods using the ALogPS, E-State indices and Dragon v.7 descriptors. The predictive ability of the models was tested through cross-validation, giving a balanced accuracy BA = 80-91%. All compounds demonstrated good antifungal properties against Candida spp. and slight inhibition of the acetylcholinesterase activity., Conclusion: The high percentage of coincidence between the QSAR predictions and the experimental results confirmed the high predictive power of the developed QSAR models that can be applied as tools for finding new potential inhibitors against Candida spp. Furthermore, 1,3-oxazol-4- yl(triphenyl)phosphonium salts could be considered as promising candidates for the treatment of candidiasis and the disinfection of medical equipment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Endocrine-disrupting chemicals (EDCs) can act upon a developing organism to change its endocrine health and behavior in adulthood. Beyond actions on the exposed individuals, transgenerational effects of several EDCs have been reported. This study assessed the combinatorial impact of EDC-altered maternal care and transgenerational inheritance on F3 male and female offspring. Pregnant rats were exposed to EDCs with different modes of action: the weakly estrogenic polychlorinated biphenyl (PCB) mixture Aroclor 1221, the anti-androgenic fungicide vinclozolin (VIN), or the vehicle (6% dimethylsulfoxide in sesame oil; VEH) during embryonic development. The F1 male and female offspring were bred through the paternal- or maternal-lineage with untreated partners to generate F2 offspring. This process was repeated through both maternal and paternal lineages to create the F3 generation. Maternal care of F2 dams towards their F3 offspring was altered in a lineage-dependent manner, particularly in PCB paternal-lineage animals. When F3 pups were recorded for ultrasonic vocalizations (USVs) following separation from the mother, the rate of neonatal USVs in F3 offspring were decreased in PCB paternal-lineage pups. In adulthood, anxiety-like behaviors of the F3 rats were tested, with only small effects of EDCs detected. These interactions of maternal behaviors and EDC effects across generations, especially via the paternal lineage, has implications for health and environmental responses in wildlife and humans., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Poly(2-oxazoline)s have been investigated for decades as biomaterials. Pioneering early work suggested that hydrophilic poly(2-oxazoline)s are comparable to poly(ethylene glycol) regarding their potential as biomaterials, but the ready commercial availability of the latter has led to its meteoric rise to become the gold standard of hydrophilic synthetic biomaterials. In contrast, poly(2-oxazoline)s almost fell into oblivion. However, in the last decade, this family of polymers has gained much more interest in general and as biomaterials in particular. The rich chemistry and comparably straightforward synthesis of poly(2-oxazoline)s gives many opportunities for tailoring the properties of the resulting biomaterials, allowing the chemist to explore new conjugation chemistry, and to fine-tune the molar mass, hydrophilic-lipophilic balance as well as architecture. Thus, the wide range of demands for various applications of biomaterials can be suitably addressed. This review aims to give a comprehensive and critical update of the development of poly(2-oxazoline) based biomaterials, focusing on the last 5 years, which have seen an explosive increase of interest. We believe that the research regarding this diverse family of polymers will remain strong and will keep growing, in particular after the promising first-in-human studies of a poly(2-oxazoline) drug conjugate. This review aims at researchers and students new to this polymer family and seasoned poly(2-oxazoline) experts alike and attempts to showcase how the chemical diversity of poly(2-oxazoline)s allows a relatively facile and broad access to biomaterials of all kinds., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Exposure to vinclozolin has been shown to induce the epigenetic transgenerational inheritance of increased susceptibility to disease, and to induce transgenerational changes to the epigenome. In the current study, gestating F0 generation rats were exposed to vinclozolin, and the subsequent F1, F2 and transgenerational F3 generations were evaluated for diseases and pathologies. F1 and F2 generation rats exhibited few abnormalities. However, F3 generation rats showed transgenerational increases in testis, prostate, and kidney disease, changes in the age of puberty onset in males, and an increased obesity rate in females. Overall there was an increase in the rate of animals with disease, and in the incidence of animals with multiple diseases. The objective of the current study was to analyze the sperm epigenome of F3 generation rats with specific abnormalities and compare them to rats without those abnormalities, in an effort to find epigenetic biomarkers of transgenerational disease. Unique signatures of differential DNA methylation regions (DMRs) in sperm were found that associated with testis disease, prostate disease and kidney disease. Confounding factors identified were the presence of multiple diseases in the analysis and the limited number of animals without disease. These results further our understanding of the mechanisms governing epigenetic transgenerational inheritance, and may lead in the future to the use of epigenetic biomarkers that will help predict an individual's susceptibility for specific diseases., Competing Interests: The authors have declared that no competing interests exist.

We show the potential of oligo(2-ethyl-2-oxazoline) (Ox n )-shielded graft copolymers of (2-aminoethyl)-methacrylate and N-methyl-(2-aminoethyl)-methacrylate for pDNA delivery in HEK cells. For the effect of grafting density and side chain length concerning improved transfection properties through the concept of shielding to be investigated, copolymers were synthesized via the macromonomer method using a combination of cationic ring opening polymerization and reversible addition-fragmentation chain transfer polymerization to vary the degree of grafting (DG = 10 and 30%) as well as the side chain degree of polymerization (DP = 5 and 20). Investigations of the polyplex formation, in vitro flow cytometry, and confocal laser scanning microscopy measurements on the copolymer library revealed classical shielding properties of the Ox side chains, including highly reduced cytotoxicity and a partial decrease in transfection efficiency, as also reported for polyethylene glycol shielding. In terms of the transfection efficiency, the best performing copolymers (A- g-Ox 5 (10) and M- g-Ox 5 (10)) revealed equal or better performances compared to those of the corresponding homopolymers. In particular, the graft copolymers with low DG and side chain DP transfected well with over 10-fold higher IC 50 values. In contrast, a DG of 30% resulted in a loss of transfection efficiency due to missing ability for endosomal release, and a side chain DP of 20 hampered the cellular uptake.

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

In utero exposure to vinclozolin (VIN), an antiandrogenic fungicide, is linked to multigenerational phenotypic and epigenetic effects. Mechanisms remain unclear. We assessed the role of antiandrogenic activity and DNA sequence context by comparing effects of VIN vs. M2 (metabolite with greater antiandrogenic activity) and wild-type C57BL/6 (B6) mice vs. mice carrying mutations at the previously reported VIN-responsive H19/Igf2 locus. First generation offspring from VIN-treated 8nrCG mutant dams exhibited increased body weight and decreased sperm ICR methylation. Second generation pups sired by affected males exhibited decreased neonatal body weight but only when dam was unexposed. Offspring from M2 treatments, B6 dams, 8nrCG sires or additional mutant lines were not similarly affected. Therefore, pup response to VIN over two generations detected here was an 8nrCG-specific maternal effect, independent of antiandrogenic activity. These findings demonstrate that maternal effects and crossing scheme play a major role in multigenerational response to in utero exposures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Amphibians use wetlands in urban and agricultural landscapes for breeding, growth and development. Fungicides and other pesticides used in these areas have therefore been identified as potential threats that could contribute towards amphibian population declines. However, relatively little is known about how such chemicals influence sensitive early life-stages or how short episodic exposures influence sub-lethal physiological and metabolic pathways. The present study applied untargeted metabolomics to evaluate effects in early post-hatch amphibian larvae exposed to the anti-androgenic fungicides vinclozolin and propiconazole. Recently hatched (Gosner developmental stage 25) striped marsh frog (Limnodynastes peronii) larvae were exposed for 96 h to vinclozolin at 17.5, 174.8 and 1748.6 nM and propiconazole at 5.8, 58.4 and 584.4 nM. Nuclear Magnetic Resonance (NMR) spectroscopy was performed on polar metabolites obtained from whole-body extracts. Both fungicides altered metabolite profiles compared to control animals at all concentrations tested, and there were notable differences between the two chemicals. Overall responses were consistent with altered steroidogenesis and/or cholesterol metabolism, with inconsistent responses between the two fungicides likely reflecting minor differences in the mechanisms of action of these chemicals. Broad down-regulation of the tricarboxylic acid (TCA) cycle was also observed and is indicative of oxidative stress. Interestingly, formic acid was significantly increased in larvae exposed to vinclozolin but not propiconazole, suggesting this metabolite may serve as a useful biomarker of exposure to androgen-receptor binding anti-androgenic contaminants. This study demonstrates the power of untargeted metabolomics for distinguishing between similarly acting, but distinct, pollutants and for unraveling non-endocrine responses resulting from exposure to known endocrine active contaminants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hymexazol is an efficacious and widely used fungicide. However, its environmental toxicological assessment has not been well documented. It had no report of its toxicity to fish embryo. Fish embryo acute toxicity tests are highly predictive of aquatic embryotoxicity outcome. In this study, zebrafish (Danio rerio) embryos were exposed to hymexazol at varying concentrations for the study of the developmental toxicity, melanin biosynthesis, biochemical and transcriptional endpoints. The embryotoxicity tests indicated that the 96h LC 50 value of hymexazol was 649mg/L with a 95% confidence interval range of 632-667mg/L. Hymexazol at concentrations of 417-738mg/L decreased the heart rate and increased the voluntary swing. Hymexazol inhibited normal development at concentrations above 554mg/L. the 96h EC 50 was 411mg/L. Hymexazol in a concentration range of 417-738mg/L induced cardiac edema and yolk sac edema. Exposure of hymexazol at such concentrations to zebrafish embryos for 48h decreased the pigment area density compared with the no hymexazol control. Tyrosinase activity was inhibited by hymexazol relative to the untreated control. The P53 mRNA expression level in embryos upon exposure to 480mg/L or greater of hymexazol was significantly higher than that of the control. The results indicated that hymexazol has quite low acute toxicity and low embryotoxicity to zebrafish., (Copyright © 2017. Published by Elsevier Inc.)

Vinclozolin (Vz) is a pollutant found in aquatic environments whose antiandrogenic effects in reproduction are well known in mammals. Although its reproductive effects have been less studied in invertebrates, other effects, including genotoxicity, have been described. Therefore, in this work, we studied the genotoxic effects of Vz in the freshwater benthic invertebrate Chironomus riparius. DNA damage was evaluated with the comet assay (tail area, olive moment, tail moment and % DNA in tail), and the transcriptional levels of different genes involved in DNA repair (ATM, NLK and XRCC1) and apoptosis (DECAY) were measured by RT-PCR. Fourth instar larvae of C. riparius, were exposed to Vz for 24 h at 20 and 200 μg/L. The Vz exposures affected the DNA integrity in this organism, since a dose-response relationship occurred, with DNA strand breaks significantly increased with increased dose for tail area, olive moment and tail moment parameters. Additionally, the lower concentration of Vz produced a significant induction of the transcripts of three genes under study (ATM, NLK and XRCC1) showing the activation of the cellular repair mechanism. In contrast, the expression of these genes with the highest concentration were downregulated, indicating failure of the cellular repair mechanism, which would explain the higher DNA damage. These data report for the first time the alterations of Vz on gene transcription of an insect and confirm the potential genotoxicity of this compound on freshwater invertebrates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Two of the most prevalent ovarian diseases affecting women's fertility and health are Primary Ovarian Insufficiency (POI) and Polycystic Ovarian Syndrome (PCOS). Previous studies have shown that exposure to a number of environmental toxicants can promote the epigenetic transgenerational inheritance of ovarian disease. In the current study, transgenerational changes to the transcriptome and epigenome of ovarian granulosa cells are characterized in F3 generation rats after ancestral vinclozolin or DDT exposures. In purified granulosa cells from 20-day-old F3 generation females, 164 differentially methylated regions (DMRs) (P < 1 x 10 -6 ) were found in the F3 generation vinclozolin lineage and 293 DMRs (P < 1 x 10 -6 ) in the DDT lineage, compared to controls. Long noncoding RNAs (lncRNAs) and small noncoding RNAs (sncRNAs) were found to be differentially expressed in both the vinclozolin and DDT lineage granulosa cells. There were 492 sncRNAs (P < 1 x 10 -4 ) in the vinclozolin lineage and 1,085 sncRNAs (P < 1 x 10 -4 ) in the DDT lineage. There were 123 lncRNAs and 51 lncRNAs in the vinclozolin and DDT lineages, respectively (P < 1 x 10 -4 ). Differentially expressed mRNAs were also found in the vinclozolin lineage (174 mRNAs at P < 1 x 10 -4 ) and the DDT lineage (212 mRNAs at P < 1 x 10 -4 ) granulosa cells. Comparisons with known ovarian disease associated genes were made. These transgenerational epigenetic changes appear to contribute to the dysregulation of the ovary and disease susceptibility that can occur in later life. Observations suggest that ancestral exposure to toxicants is a risk factor that must be considered in the molecular etiology of ovarian disease.

The current investigation examines whether combined exposure to three anti-androgens (flutamide, prochloraz, vinclozolin) result in interference with endocrine homeostasis when applied at very low dose levels, and whether the results of combined exposure are more pronounced than to the individual compounds. A pre-post-natal in vivo study design was chosen with more parameters than regulatory testing protocols require (additional endpoints addressing hormone levels, morphology and histopathological examinations). Dose levels were chosen to represent the lowest observed adverse effect level (LOAEL), the no observed adverse effect level (NOAEL), and the acceptable daily intake for each individual substance. Anti-androgenic changes were observable at the effect level (LOAEL) but not at lower exposures. Nipple/areola counts appeared to be a sensitive measure of effect, in addition to male sex organ weights at sexual maturation, and finally gross findings. The results indicate the absence of evidence for effects at low or very low dose levels. No (adverse) effects were seen at the NOAEL dose. A non-monotonic dose-response relationship was not evident. Combined exposure at LOAEL level resulted in enhanced responses for anogenital index, number of areolas/nipples, delayed preputial separation and reduced ventral prostate weight in comparison to the individual compounds.

Pesticides have long been considered to a risk factor of amphibian population declines. The bioaccumulation and elimination of fenoxaprop-ethyl (FE) in frogs and tadpoles were studied and the main metabolites fenoxaprop (FA) and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB) were monitored. The acute toxicity and genotoxicity of the enantiomers to tadpoles was also studied. After both oral administration and aqueous solution exposure, FE was not found in frogs, while FA was formed and accumulated in liver, kidney, brain, eggs, skin, thigh muscle and blood with preferential accumulation of R-FA. The presence of FA in frog eggs suggested maternal transfer in females and potential impacts to offsprings. The elimination of FA in frog tissues was also enantioselective with a preferential metabolism of R-FA (kidney) or S-FA (liver, eggs, skin, muscle and whole blood). FE and FA were hardly detectable in tadpoles after aqueous solution exposure, while CDHB was accumulated and eliminated as first-order kinetics with half-life of 37.1 h. Mortality of tadpoles and micronucleus rate in peripheral blood erythrocytes of tadpoles were used to evaluate the enantioselective acute toxicity and genotoxicity. Only CDHB induced significant acute toxicity to tadpole with 96-h LC 50 value of 30.4 μg/mL, and rac-FA, S-FA and CDHB showed genotoxicity., (Copyright © 2017. Published by Elsevier Ltd.)

Recent data demonstrated that activation of the muscarinic M 1 receptor by a subtype-selective positive allosteric modulator (PAM) contributes to the gastrointestinal (GI) and cardiovascular (CV) cholinergic adverse events (AEs) previously attributed to M 2 and M 3 activation. These studies were conducted using PAMs that also exhibited allosteric agonist activity, leaving open the possibility that direct activation by allosteric agonism, rather than allosteric modulation, could be responsible for the adverse effects. This article describes the design and synthesis of lactam-derived M 1 PAMs that address this hypothesis. The lead molecule from this series, compound 1 (PF-06827443), is a potent, low-clearance, orally bioavailable, and CNS-penetrant M 1 -selective PAM with minimal agonist activity. Compound 1 was tested in dose escalation studies in rats and dogs and was found to induce cholinergic AEs and convulsion at therapeutic indices similar to previous compounds with more agonist activity. These findings provide preliminary evidence that positive allosteric modulation of M 1 is sufficient to elicit cholinergic AEs.

Pesticides are used to protect crops and to eliminate pests, though non-target organisms such as mammals are also affected from their usage. Etoxazole (organoflourine pesticide) is an acaricide used to combat spider mites which are the parasites of various crops. The present study aims to investigate the effects of etoxazole on the level of MDA (malondialdehyde) and activities of CAT (catalase), GPx (glutathione peroxidase), and AChE (acetylcholinesterase) in liver and kidney tissues of Wistar rats (Rattus norvegicus var. albinos). Rats received etoxazole intraperitoneally with doses of 2.2, 11, and 22 mg/kg b.w./day for 21 days. Control rats received the same volume of the serum physiologic. Following etoxazole exposures, activities of CAT, GPx, and AChE in the liver and kidney of rats significantly decreased at all doses compared to control group. Oppositely, MDA levels in these tissues increased significantly at all doses following etoxazole exposures. The present study demonstrated that etoxazole, at all doses, had toxic effects in the liver and kidney parameters, suggesting their possible use as effective biomarkers in determining the toxic effects of etoxazole. This may suggest that these biomarkers could also be used as a tool to monitor pesticide-affected areas before severe toxic effects begin in non-target animals and humans.

The non-clinical safety profile of aleglitazar, a peroxisome proliferator activated receptor alpha/gamma agonist, and its major human metabolite M6 was studied in a complete package consisting of drug metabolism and pharmacokinetics characterization, safety pharmacology, genotoxicity, repeat dose toxicity, reproductive toxicity and carcinogenicity studies. These studies identified the following main targets similar to other PPAR agonists: red blood cell parameters, liver, heart, kidney, ovaries, testes, bone marrow, adipose tissue, and fluid accumulation. Additionally, and in the 12-month monkey study only, an increased incidence of generalized hair loss/thinning was observed in all groups including controls. In the rat carcinogenicity study there was no statistically significant increase in tumors. In the mouse carcinogenicity study, there was an increased incidence of angiomatous tumors and there were three males with gallbladder adenoma. No relevant compound-related effects were observed in safety pharmacology, genotoxicity, and a 28-day immunotoxicity rat study. Effects observed in reproductive toxicity studies were similar to those known for other PPARγ agonists. Separate studies with the human metabolite M6 did not reveal findings that would prevent human dosing. Overall, the results from the non-clinical safety studies conducted with aleglitazar and the human metabolite M6 were considered to support the clinical Phase 3 program., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the present study a series of 4-methyl-2-aryl-5-(2-aryl/benzyl thiazol-4-yl) oxazole (4a-v) have been synthesized and evaluated for their preliminary antitubercular, antimicrobial and cytotoxicity activity. Among all the synthesized compounds, 4v reported comparable activity against dormant M. tuberculosis H 37 Ra and M. bovis BCG strains with respect to standard drug rifampicin. The active compounds from the antitubercular study were further tested for anti-proliferative activity against HeLa, A549 and PANC-1 cell lines using MTT assay and showed no significant cytotoxic activity at the maximum concentration evaluated. Further, the synthesized compounds were found to have potential antibacterial activities with MIC range of 2.1-26.8 μg/mL. High potency, lower cytotoxicity and promising antimycobacterial activity suggested that these compounds could serve as good leads for further optimisation and development., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Three sweet corn genotypes, two inbred lines (IBER001 and IBER002) and their hybrid (ER00X), differ in their phenotypic responses to several P450-metabolized herbicides, used in sweet corn, namely, foramsulfuron, iodosulfuron, rimsulfuron and tembotrione. Foramsulfuron is a sulfonylurea herbicide commonly formulated with the safener isoxadifen that is used for selective post-emergence weed control in corn. Our goal was to elucidate the mechanism of these genotypes' responses to foramsulfuron and safener isoxadifen and examine the heritability of those responses. IBER001 was sensitive to foramsulfuron+isoxadifen, with an ED 50 of 3.6gaiha -1 , while IBER002 and ER00X were tolerant with ED 50 values of 808 and 700gaiha -1 , respectively. ALS enzyme extracted from each of the different genotypes was equally sensitive to foramsulfuron. Pre-treatment with malathion, a known cytochrome P450 inhibitor, increased foramsulfuron injury in IBER002 and ER00X, but had no effect on those lines when isoxadifen was applied with the herbicide. Foramsulfuron-treated IBER001 was severely injured regardless of the presence of malathion and/or isoxadifen. Pre-treatment with malathion similarly increased the phytotoxicity of iodosulfuron+safener (mefenpyr) and rimsulfuron to the tolerant genotypes, but did not increase the level of injury caused by the tembotrione+isoxadifen treatment. Segregation of F2 and backcross progenies according to their responses to foramsulfuron+isoxadifen revealed a pattern of inheritance typical of a trait controlled by a single gene inheritance, with a recessive allele conferring sensitivity. Our results support the hypothesis that foramsulfuron selectivity is associated with P450 metabolism and that isoxadifen positively affects P450 activity. The sensitive genotype that does not respond to isoxadifen is presumably homozygous for a deficient or non-functioning P450 gene., (Copyright © 2017 Elsevier Inc. All rights reserved.)