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Stroke and perinatal asphyxia have detrimental effects on neuronal cells, causing millions of deaths worldwide each year. Since currently available therapies are insufficient, there is an urgent need for novel neuroprotective strategies to address the effects of cerebrovascular accidents. One such recent approach is based on the neuroprotective properties of estrogen receptors (ERs). However, activation of ERs by estrogens may contribute to the development of endometriosis or hormone-dependent cancers. Therefore, in this study, we utilized ospemifene, a novel selective estrogen receptor modulator (SERM) already used in dyspareunia treatment. Here, we demonstrated that posttreatment with ospemifene in primary neocortical cell cultures subjected to 18 h of hypoxia and/or ischemia followed by 6 h of reoxygenation has robust neuroprotective potential. Ospemifene partially reverses hypoxia- and ischemia-induced changes in LDH release, the degree of neurodegeneration, and metabolic activity. The mechanism of the neuroprotective actions of ospemifene involves the inhibition of apoptosis since the compound decreases caspase-3 overactivity during hypoxia and enhances mitochondrial membrane potential during ischemia. Moreover, in both models, ospemifene decreased the levels of the proapoptotic proteins BAX, FAS, FASL, and GSK3β while increasing the level of the antiapoptotic protein BCL2. Silencing of specific ERs showed that the neuroprotective actions of ospemifene are mediated mainly via ESR1 (during hypoxia and ischemia) and GPER1 (during hypoxia), which is supported by ospemifene-evoked increases in ESR1 protein levels in hypoxic and ischemic neurons. The results identify ospemifene as a promising neuroprotectant, which in the future may be used to treat injuries due to brain hypoxia/ischemia.

Molecular neurobiology 60(2), 576-595 (2023). doi:10.1007/s12035-022-03087-9, Published by Humana Press, Totowa, NJ

Newly synthesized Pathway Preferential Estrogen-1 (PaPE-1) selectively activates membrane estrogen receptors (mERs), namely, mERα and mERβ, and has been shown to evoke neuroprotection; however, its effectiveness in protecting brain tissue against hypoxia and ischemia has not been verified in a posttreatment paradigm. This is the first study showing that a 6-h delayed posttreatment with PaPE-1 inhibited hypoxia/ischemia-induced neuronal death, as indicated by neutral red uptake in mouse primary cell cultures in vitro. The effect was accompanied by substantial decreases in neurotoxicity and neurodegeneration in terms of LDH release and Fluoro-Jade C staining of damaged cells, respectively. The mechanisms of the neuroprotective action of PaPE-1 also involved apoptosis inhibition demonstrated by normalization of both mitochondrial membrane potential and expression levels of apoptosis-related genes and proteins such as Fas, Fasl, Bcl2, FAS, FASL, BCL2, BAX, and GSK3β. Furthermore, PaPE-1-evoked neuroprotection was mediated through a reduction in ROS formation and restoration of cellular metabolic activity that had become dysregulated due to hypoxia and ischemia. These data provide evidence that targeting membrane non-GPER estrogen receptors with PaPE-1 is an effective therapy that protects brain neurons from hypoxic/ischemic damage, even when applied with a 6-h delay from injury onset.

Alzheimer’s disease (AD) is a multifactorial and severe neurodegenerative disorder characterized by progressive memory decline, the presence of Aβ plaques and tau tangles, brain atrophy, and neuronal loss. Available therapies provide moderate symptomatic relief but do not alter disease progression. This study demonstrated that PaPE-1, which has been designed to selectively activate non-nuclear estrogen receptors (ERs), has anti-AD capacity, as evidenced in a cellular model of the disease. In this model, the treatment of mouse neocortical neurons with Aβ (5 and 10 μM) induced apoptosis (loss of mitochondrial membrane potential, activation of caspase-3, induction of apoptosis-related genes and proteins) accompanied by increases in levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) as well as reduced cell viability. Following 24 h of exposure, PaPE-1 inhibited Aβ-evoked effects, as shown by reduced parameters of neurotoxicity, oxidative stress, and apoptosis. Because PaPE-1 downregulated Aβ-induced Fas/FAS expression but upregulated that of Aβ-induced FasL, the role of PaPE-1 in controlling the external apoptotic pathway is controversial. However, PaPE-1 normalized Aβ-induced loss of mitochondrial membrane potential and restored the BAX/BCL2 ratio, suggesting that the anti-AD capacity of PaPE-1 particularly relies on inhibition of the mitochondrial apoptotic pathway. These data provide new evidence for an anti-AD strategy that utilizes the selective targeting of non-nuclear ERs with PaPE-1.

Nuclear- and membrane-initiated estrogen signaling cooperate to orchestrate the pleiotropic effects of estrogens. Classical estrogen receptors (ERs) act transcriptionally and govern the vast majority of hormonal effects, whereas membrane ERs (mERs) enable acute modulation of estrogenic signaling and have recently been shown to exert strong neuroprotective capacity without the negative side effects associated with nuclear ER activity. In recent years, GPER1 was the most extensively characterized mER. Despite triggering neuroprotective effects, cognitive improvements, and vascular protective effects and maintaining metabolic homeostasis, GPER1 has become the subject of controversy, particularly due to its participation in tumorigenesis. This is why interest has recently turned toward non-GPER-dependent mERs, namely, mERα and mERβ. According to available data, non-GPER-dependent mERs elicit protective effects against brain damage, synaptic plasticity impairment, memory and cognitive dysfunctions, metabolic imbalance, and vascular insufficiency. We postulate that these properties are emerging platforms for designing new therapeutics that may be used in the treatment of stroke and neurodegenerative diseases. Since mERs have the ability to interfere with noncoding RNAs and to regulate the translational status of brain tissue by affecting histones, non-GPER-dependent mERs appear to be attractive targets for modern pharmacotherapy for nervous system diseases.

Triclocarban is a highly effective and broadly used antimicrobial agent. Humans are continually exposed to triclocarban, but the safety of prenatal exposure to triclocarban in the context of neurodevelopment remains unknown. In this study, we demonstrated for the first time that mice that had been prenatally exposed to environmentally relevant doses of triclocarban had impaired estrogen receptor 1 (ESR1) signaling in the brain. These mice displayed decreased mRNA and protein expression levels of ESR1 as well as hypermethylation of the Esr1 gene in the cerebral cortex. Prenatal exposure to triclocarban also diminished the mRNA expression of Esr2, Gper1, Ahr, Arnt, Cyp19a1, Cyp1a1, and Atg7, and the protein levels of CAR, ARNT, and MAP1LC3AB in female brains and decreased the protein levels of BCL2, ARNT, and MAP1LC3AB in male brains. In addition, exposure to triclocarban caused sex-specific alterations in the methylation levels of global DNA and estrogen receptor genes. Microarray and enrichment analyses showed that, in males, triclocarban dysregulated mainly neurogenesis-related genes, whereas, in females, the compound dysregulated mainly neurotransmitter-related genes. In conclusion, our data identified triclocarban as a neurodevelopmental risk factor that particularly targets ESR1, affects apoptosis and autophagy, and in sex-specific ways disrupts the epigenetic status of brain tissue and dysregulates the postnatal expression of neurogenesis- and neurotransmitter-related genes.

Benzophenone-3 (2-hydroxy-4-methoxybenzophenone, oxybenzone, or BP-3) is one of the most frequently used UV radiation absorbents, which are commonly referred to as sunscreen filters. Its widespread use in industrial applications provides protection against the photodegradation of a wide range of products but at the same time creates the risk of human exposure to benzophenone-3 unbeknownst to the individuals exposed. Topically applied benzophenone-3 penetrates individual skin layers, enters the bloodstream, and is excreted in the urine. In addition, benzophenone-3 easily crosses the placental barrier, which creates the risk of exposure to this substance in the prenatal period. Despite the widespread use and occurrence of benzophenone-3 in the human environment, little knowledge of the mechanisms underlying the effect of benzophenone-3 on the nervous system was available until recently. Only the most recent research, including studies by our group, has enabled the identification of new molecular mechanisms through which benzophenone-3 affects embryonic neuronal cells and the developing mammalian brain. Benzophenone-3 has been shown to induce neurotoxicity and apoptotic processes and inhibit autophagy in embryonic neuronal cells. Benzophenone-3 also alters expression and impairs function of receptors necessary for the proper development and function of the nervous system. The most worrying finding seems to be that benzophenone-3 contributes to an increased risk of developmental abnormalities and/or epigenetically based degeneration of neuronal cells by changing the epigenetic status of neuronal cells.

In this study, we demonstrate for the first time that amorfrutin B, a selective modulator of peroxisome proliferator-activated receptor gamma—PPARγ, can protect brain neurons from hypoxia- and ischemia-induced degeneration when applied at 6 h post-treatment in primary cultures. The neuroprotective effect of amorfrutin B suggests that it promotes mitochondrial integrity and is capable of inhibiting reactive oxygen species—ROS activity and ROS-mediated DNA damage. PPARγ antagonist and Pparg mRNA silencing abolished the neuroprotective effect of amorfrutin B, which points to agonistic action of the compound on the respective receptor. Interestingly, amorfrutin B stimulated the methylation of the Pparg gene, both during hypoxia and ischemia. Amorfrutin&nbsp, B also increased the protein level of PPARγ during hypoxia but decreased the mRNA and protein levels of PPARγ during ischemia. Under ischemic conditions, amorfrutin B-evoked hypermethylation of the Pparg gene is in line with the decrease in the mRNA and protein expression of PPARγ. However, under hypoxic conditions, amorfrutin B-dependent hypermethylation of the Pparg gene does not explain the amorfrutin B-dependent increase in receptor protein expression, which suggests other regulatory mechanisms. Other epigenetic parameters, such as HAT and/or sirtuins activities, were affected by amorfrutin B under hypoxic and ischemic conditions. These properties position the compound among the most promising anti-stroke and wide-window therapeutics.

Purpose: The aim of the study was to establish the relationship between the shape of the calcaneal tuberosity (flat, stepped, rounded, normal) and the probability that retrocalcaneal bursitis among people who train running regularly. Methods: The study included a group of 30 runners who suffered from retrocalcaneal bursitis in the past, and 30 people who never had symptoms of this disease. The study was based on a diagnostic survey, as well as on clinical examination. The surface of the calcaneal tuberosity and the slope of the calcaneus were assessed using X-rays. The mobility of the bursa, its surface size, the thickness of the Achilles tendon and its attachment rate were established during an ultrasound examination. Results: Flat surface of the calcaneal tuberosity increases fourfold the risk of suffering from retrocalcaneal bursitis (OR = 4.3). The people whose calcaneus slope is above 25° are at increased risk of suffering from such an inflammation compared with the people whose calcaneus bone is more horizontal (OR = 2.8). The analysis shows that the thickness of the Achilles tendon (p = 0.001), the surface size of the bursa (p = 0.009), as well as the flat surface of the calcaneal tuberosity (p = 0.008) are strongly associated with the occurrence of retrocalcaneal bursitis. Conclusions: The flat shape of the calcaneal tuberosity increases the risk of bursitis. The risk of inflammation is higher when the Achilles tendon is thicker and the surface of the bursa is smaller than normal.

Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3’-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.

DDE (dichlorodiphenyldichloroethylene) is an environmental metabolite of the pesticide DDT, which is still present in the environment, and its insecticidal properties are used to fight malaria and the Zika virus disease. We showed for the first time that the neurotoxic effects of DDE involve autophagy, as demonstrated by elevated levels of Becn1, Map1lc3a/MAP1LC3A, Map1lc3b, and Nup62/NUP62 and an increase in autophagosome formation. The suggestion that the aryl hydrocarbon receptor (AHR) and the constitutive androstane receptor (CAR) are involved in the neurotoxic effect of DDE was supported by increases in the mRNA and protein expression of these receptors, as detected by qPCR, ELISA, immunofluorescence labeling and confocal microscopy. Selective antagonists of the receptors, including alpha-naphthoflavone, CH223191, and CINPA 1, inhibited p,p'-DDE- and o,p'-DDE-induced LDH release and caspase-3 activity, while specific siRNAs (Ahr and Car siRNA) reduced the levels of p,p'-DDE- and o,p'-DDE-induced autophagosome formation. Although the neurotoxic effects of DDE were isomer independent, the mechanisms of p,p'- and o,p'-DDE were isomer specific. Therefore, we identified previously unknown mechanisms of the neurotoxic actions of DDE that, in addition to inducing apoptosis, stimulate autophagy in mouse neocortical cultures and induce AHR and CAR signaling.

The UV absorber benzophenone-3 (BP-3) is the most extensively used chemical substance in various personal care products. Despite that BP-3 exposure is widespread, knowledge about the impact of BP-3 on the brain development is negligible. The present study aimed to explore the mechanisms of prenatal exposure to BP-3 in neuronal cells, with particular emphasis on autophagy and nuclear receptors signaling as well as the epigenetic and post-translational modifications occurring in response to BP-3. To observe the impact of prenatal exposure to BP-3, we administered BP-3 to pregnant mice, and next, we isolated brain tissue from pretreated embryos for primary cell neocortical culture. Our study revealed that prenatal exposure to BP-3 (used in environmentally relevant doses) impairs autophagy in terms of BECLIN-1, MAP1LC3B, autophagosomes, and autophagy-related factors; disrupts the levels of retinoid X receptors (RXRs) and peroxisome proliferator-activated receptor gamma (PPARγ); alters epigenetic status (i.e., attenuates HDAC and sirtuin activities); inhibits post-translational modifications in terms of global sumoylation; and dysregulates expression of neurogenesis- and neurotransmitter-related genes as well as miRNAs involved in pathologies of the nervous system. Our study also showed that BP-3 has good permeability through the BBB. We strongly suggest that BP-3-evoked effects may substantiate a fetal basis of the adult onset of neurological diseases, particularly schizophrenia and Alzheimer’s disease.

Current evidence indicates that benzophenone-3 (BP-3) can pass through the placental and blood–brain barriers and thus can likely affect infant neurodevelopment. Despite widespread exposure, data showing the effects of BP-3 on the developing nervous system are scarce. This study revealed for the first time that prenatal exposure to BP-3 led to apoptosis and neurotoxicity, altered the levels of estrogen receptors (ERs) and changed the epigenetic status of mouse neurons. In the present study, subcutaneous injections of pregnant mice with BP-3 at 50 mg/kg, which is an environmentally relevant dose, evoked activation of caspase-3 and lactate dehydrogenase (LDH) release as well as substantial loss of mitochondrial membrane potential in neocortical cells of their embryonic offspring. Apoptosis-focused microarray analysis of neocortical cells revealed up-regulation of 22 genes involved in apoptotic cell death. This effect was supported by increased BAX and CASP3 mRNA and protein levels, as evidenced by qPCR, ELISAs and western blots. BP-3-induced apoptosis and neurotoxicity were accompanied by decreases in the mRNA and protein expression levels of ESR1 and ESR2 (also known as ERα and ERβ), with a simultaneous increase in GPER1 (also known as GPR30) expression. In addition to the demonstration that treatment of pregnant mice with BP-3 induced apoptosis, caused neurotoxicity and altered ERs expression levels in neocortical cells of their embryonic offspring, we showed that prenatal administration of BP-3 inhibited global DNA methylation as well as reduced DNMTs activity. BP-3 also caused specific hypomethylation of the genes Gper1 and Bax, an effect that was accompanied by increased mRNA and protein expression levels. In addition, BP-3 caused hypermethylation of the genes Esr1, Esr2 and Bcl2, which could explain the reduced mRNA and protein levels of the estrogen receptors. This study demonstrated for the first time that prenatal exposure to BP-3 caused severe neuronal apoptosis that was accompanied by impaired ESR1/ESR2 expression, enhanced GPER1 expression, global DNA hypomethylation and altered methylation statuses of apoptosis-related and ERs genes. We suggest that the effects of BP-3 in embryonic neurons may be the fetal basis of the adult onset of nervous system disease.

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

Purpose Tryptamine hallucinogen 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a serotonin transporter inhibitor with high affinity for serotonin 5-HT1A and 5-HT2A/C receptors. We showed previously that 5-MeO-DIPT in a single dose increased neurotransmitter release in brain regions of rats and elicited single- and double-strand DNA breaks. Herein we investigated the effects of repeated-intermittent 5-MeO-DIPT administration in adolescence on dopamine (DA), serotonin (5-HT) and glutamate release in brain regions of adult rats. Furthermore, we examined caspase-3 activity, oxidative DNA damage, the Gpx3, Sod1, Ht1a and Ht2a mRNA expression levels, and cell viability. Methods Neurotransmitter release was measured by microdialysis in freely moving animals. Caspase-3 activity was assessed colorimetrically, and oxidative DNA damage with the comet assay, while the Gpx3, Sod1, Ht1a and Ht2a mRNA expression levels were assessed by real-time polymerase chain reaction. Cell viability was studied in SH-SY5Y and Hep G2 cells by the MTT test. Results We observed changed responses of DA, 5-HT and glutamate neurons to a challenge dose of 5-MeO-DIPT when animals were treated repeatedly in adolescence with this hallucinogen. The basal extracellular levels of DA and 5-HT were decreased in the striatum and nucleus accumbens, while glutamate level was increased in the nucleus accumbens and frontal cortex. The damage of cortical DNA, increased Gpx3 and Sod1 mRNA expression and affected caspase-3 activity were also observed. Furthermore, decreased Ht1a and Ht2a mRNA expression in the frontal cortex and marked cytotoxicity of 5-MeO-DIPT were found. Conclusions These results suggest that 5-MeO-DIPT given repeatedly during adolescence affects brain neurotransmission and shows neurotoxic potential observed in adult animals.

According to the European Drug Report (2016), the use of synthetic cathinones, such as mephedrone, among young people has rapidly increased in the last years. Studies in humans indicate that psychostimulant drug use in adolescence increases risk of drug abuse in adulthood. Mephedrone by its interaction with transporters for dopamine (DAT) and serotonin (SERT) stimulates their release to the synaptic cleft. In animal studies, high repeated doses of mephedrone given to adolescent but not adult mice or rats induced toxic changes in 5-hydroxytryptamine (5-HT) neurons. The aim of our study was to investigate the effects of mephedrone given in adolescence on brain neurotransmission and possible neuronal injury in adult rats. Adolescent male rats were given mephedrone (5 mg/kg) for 8 days. In vivo microdialysis in adult rats showed an increase in dopamine (DA), 5-HT, and glutamate release in the nucleus accumbens and frontal cortex but not in the striatum in response to challenge dose in animals pretreated with mephedrone in adolescence. The 5-HT and 5-hydroxyindoleacetic acid contents decreased in the striatum and nucleus accumbens while DA turnover rates were decreased in the striatum and nucleus accumbens. The oxidative damage of DNA assessed with the alkaline comet assay was found in the cortex of adult rats. Therefore, the administration of repeated low doses of mephedrone during adolescence does not seem to induce injury to 5-HT and DA neurons. The oxidative stress seems to be responsible for possible damage of cortical cell bodies which causes maladaptive changes in serotonergic and dopaminergic neurons. Electronic supplementary material The online version of this article (10.1007/s12640-018-9908-0) contains supplementary material, which is available to authorized users.

The text is a commentary to the book by Tomasz Baldziński and Dorota Dłużniewska-Urban Mockumentary. Próba określenia istoty zjawiska [Mockumentary. An Attempt to Define the Essence of the Phenomenon]. The authoress focuses on the phenomenon of mixing the genres, and in particular of combining the conflicting elements of fiction and documentarism in contemporary cinema.

Benzophenone-3 (BP-3) is the most widely used compound among UV filters for the prevention of photodegradation. Population studies have demonstrated that it penetrates through the skin and crosses the blood-brain barrier. However, little is known about the impact of BP-3 on the nervous system and its possible adverse effects on the developing brain. We demonstrated that the neurotoxic effects of BP-3 were accompanied by the induction of apoptosis, as evidenced by apoptosis-related caspase-3 activation and apoptotic body formation as well as the inhibition of autophagy, as determined by the downregulation of autophagy-related genes, decreased autophagosome formation, and reduced LC3B-to-LC3A ratio. In this study, we showed for the first time that the BP-3-induced apoptosis of neuronal cells is mediated via the stimulation of RXRα signaling and the attenuation of RXRβ/RXRγ signaling, as demonstrated using selective antagonist and specific siRNAs as well as by measuring the mRNA and protein expression levels of the receptors. This study also demonstrated that environmentally relevant concentrations of BP-3 were able to inhibit autophagy and disrupt the epigenetic status of neuronal cells, as evidenced by the inhibition of global DNA methylation as well as the reduction of histone deacetylases and histone acetyl transferases activity, which may increase the risks of neurodevelopmental abnormalities and/or neural degenerations.

Several lines of evidence suggest that exposures to Endocrine Disrupting Chemicals (EDCs) such as pesticides increase the risks of neuropsychiatric disorders. Despite extended residual persistence of dichlorodiphenyltrichloroethane (DDT) in the environment, the mechanisms of perinatal actions of DDT that could account for adult-onset of depression are largely unknown. This study demonstrated the isomer-specific induction of depressive-like behavior and impairment of Htr1a/serotonin signaling in one-month-old mice that were prenatally exposed to DDT. The effects were reversed by the antidepressant citalopram as evidenced in the forced swimming (FST) and tail suspension (TST) tests in the male and female mice. Prenatally administered DDT accumulated in mouse brain as determined with gas chromatography and tandem mass spectrometry, led to global DNA hypomethylation, and altered the levels of methylated DNA in specific genes. The induction of depressive-like behavior and impairment of Htr1a/serotonin signaling were accompanied by p,p'-DDT-specific decrease in the levels of estrogen receptors i.e. ESR1 and/or GPER1 depending on sex. In contrast, o,p'-DDT did not induce depressive-like effects and exhibited quite distinct pattern of biochemical alterations that was related to aryl hydrocarbon receptor (AHR), its nuclear translocator ARNT, and ESR2. Exposure to o,p'-DDT increased AHR expression in male and female brains, and reduced expression levels of ARNT and ESR2 in the female brains. The evolution of p,p'-DDT-induced depressive-like behavior was preceded by attenuation of Htr1a and Gper1/GPER1 expression as observed in the 7-day-old mouse pups. Because p,p'-DDT caused sex- and age-independent attenuation of GPER1, we suggest that impairment of GPER1 signaling plays a key role in the propagation of DDT-induced depressive-like symptoms.

Although benzophenone-3 (BP-3) has frequently been reported to play a role in endocrine disruption, there is insufficient data regarding the impact of BP-3 on the nervous system, including its possible adverse effects on the developing brain. Our study demonstrated that BP-3 caused neurotoxicity and activated apoptosis via an intrinsic pathway involving the loss of mitochondrial membrane potential and the activation of caspases-9 and -3 and kinases p38/MAPK and Gsk3β. These biochemical alterations were accompanied by ROS production, increased apoptotic body formation and impaired cell survival, and by an upregulation of the genes involved in apoptosis. The BP-3-induced effects were tissue-specific and age-dependent with the most pronounced effects observed in neocortical cells at 7 days in vitro. BP-3 changed the messenger RNA (mRNA) expression levels of Erα, Erβ, Gpr30, and Pparγ in a time-dependent manner. At 3 h of exposure, BP-3 downregulated estrogen receptor mRNAs but upregulated Pparγ mRNA. After prolonged exposures, BP-3 downregulated the receptor mRNAs except for Erβ mRNA that was upregulated. The BP-3-induced patterns of mRNA expression measured at 6 and 24 h of exposure reflected alterations in the protein levels of the receptors and paralleled their immunofluorescent labeling. Erα and Pparγ agonists diminished, but Erβ and Gpr30 agonists stimulated the BP-3-induced apoptotic and neurotoxic effects. Receptor antagonists caused the opposite effects, except for ICI 182,780. This is in line with a substantial reduction in the effects of BP-3 in cells with siRNA-silenced Erβ/Gpr30 and the maintenance of BP-3 effects in Erα- and Pparγ siRNA-transfected cells. We showed for the first time that BP-3-affected mRNA and protein expression levels of Erα, Erβ, Gpr30, and Pparγ, paralleled BP-3-induced apoptosis and neurotoxicity. Therefore, we suggest that BP-3-evoked apoptosis of neuronal cells is mediated via attenuation of Erα/Pparγ and stimulation of Erβ/Gpr30 signaling.

Dibutyl phthalate (di-n-butyl phthalate, DBP) is one of the most commonly used phthalate esters. DBP is widely used as a plasticizer in a variety of household industries and consumer products. Because phthalates are not chemically bound to products, they can easily leak out to enter the environment. DBP can pass through the placental and blood–brain barriers due to its chemical structure, but little is known about its mechanism of action in neuronal cells. This study demonstrated the toxic and apoptotic effects of DBP in mouse neocortical neurons in primary cultures. DBP stimulated caspase-3 and LDH activities as well as ROS formation in a concentration (10 nM–100 µM) and time-dependent (3–48 h) manner. DBP induced ROS formation at nanomolar concentrations, while it activated caspase-3 and LDH activities at micromolar concentrations. The biochemical effects of DBP were accompanied by decreased cell viability and induction of apoptotic bodies. Exposure to DBP reduced Erα and Pparγ mRNA expression levels, which were inversely correlated with protein expression of the receptors. Treatment with DBP enhanced Ahr mRNA expression, which was reflected by the increased AhR protein level observed at 3 h after exposure. ERα, ERβ, and PPARγ antagonists stimulated DBP-induced caspase-3 and LDH activities. AhR silencing demonstrated that DBP-induced apoptosis and neurotoxicity are mediated by AhR, which is consistent with the results from DBP-induced enhancement of AhR mRNA and protein expression. Our study showed that AhR is involved in DBP-induced apoptosis and neurotoxicity, while the ERs and PPARγ signaling pathways are impaired by the phthalate.

The text discusses some aspects of Edward Kasperski’s last book entitled Metody i metodologia [Methods and Methodology]. The author refers to the idea of methodology as a research style and points out the advantages and disadvantages of comparative method in contemporary cultural studies.

Autoimmune polyglandular syndrome, APS, is characterised by the presence of two or more chronic autoimmune organ-specific diseases in one individual. There are four types of APS; however, variable clinical course often makes the proper APS-type diagnosis difficult. In this paper, we would like to present 3 patients with APS, whose diagnosis has changed over time. The first patient was a girl with alopecia and normocytic anaemia, both of which occurred when she was 9 years old. At the age of 12, an autoimmune thyroiditis was diagnosed, and antibodies against adrenal cortex were found when she turned 17. The second patient was a 10-year-old boy admitted to the hospital because of vomiting, stomach ache and stomatitis/candidiasis. He also suffered from polydypsia and decreased appetite for several days. Type 1 diabetes and Addison's disease had been diagnosed. The third patient was a 16-year-old female with acute thrombocytopenia and anaemia accompanied by Graves’ disease. Additionally, during the course of the disease, she developed symptoms of lupus-like syndrome and central facial nerve palsy. The described cases indicate that the course of the disease as well as the diagnosis of the patient with APS may change over time. Atypical development of an autoimmune disease or atypical response for standard treatment should lead to extensive diagnosis towards new elements of the syndrome. Every organ-specific autoimmune disease might be the first prodrome of APS.

There are no studies examining the effects of 3,3′-diindolylmethane (DIM) in neuronal cells subjected to ischemia. Little is also known about the roles of apoptosis and autophagy as well as AhR and ERα signaling and HDACs in DIM action. We demonstrated for the first time the strong neuroprotective capacity of DIM in mouse primary hippocampal cell cultures exposed to ischemia at early and later stages of neuronal development. The protective effects of DIM were mediated via inhibition of ischemia-induced apoptosis and autophagy that was accompanied by a decrease in AhR/CYP1A1 signaling and an increase in HDAC activity. DIM decreased the levels of pro-apoptotic factors, i.e., Fas, Caspase-3, and p38 mitogen-activated protein kinase (MAPK). DIM also reduced the protein levels of autophagy-related Beclin-1 (BECN1) and microtubule-associated proteins 1A/1B light chain (LC3), partially reversed the ischemia-induced decrease in Nucleoporin 62 (NUP62) and inhibited autophagosome formation. In addition, DIM completely reversed the ischemia-induced decrease in histone deacetylase (HDAC) activity in hippocampal neurons. Although DIM inhibited AhR/CYP1A1 signaling, it did not influence the protein expression levels of ERα and ERα-regulated CYP19A1 which are known to be controlled by AhR. This study demonstrated for the first time, that the neuroprotective action of 3,3′-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy and depends on AhR/CYP1A1 signaling and HDAC activity, thus creating the possibility of developing new therapeutic strategies that target neuronal degeneration at specific molecular levels.

The neuroprotective potential of 3,3'-diindolylmethane (DIM), which is a selective aryl hydrocarbon receptor modulator, has recently been shown in cellular and animal models of Parkinson's disease and lipopolysaccharide-induced inflammation. However, there are no data concerning the protective capacity and mechanisms of DIM action in neuronal cells exposed to hypoxia. The aim of the present study was to investigate the neuroprotective potential of DIM against the hypoxia-induced damage in mouse hippocampal cells in primary cultures, with a particular focus on DIM interactions with the aryl hydrocarbon receptor (AhR), its nuclear translocator ARNT, and estrogen receptor β (ERβ). In the present study, 18 h of hypoxia induced apoptotic processes, in terms of the mitochondrial membrane potential, activation of caspase-3, and fragmentation of cell nuclei. These effects were accompanied by substantial lactate dehydrogenase release and neuronal cell death. The results of the present study demonstrated strong neuroprotective and anti-apoptotic actions of DIM in hippocampal cells exposed to hypoxia. In addition, DIM decreased the Ahr and Arnt mRNA expression and stimulated Erβ mRNA expression level. DIM-induced mRNA alterations were mirrored by changes in protein levels, except for ERβ, as detected by ELISA, Western blotting, and immunofluorescence labeling. We also demonstrated that DIM decreased the expression of AhR-regulated CYP1A1. Using specific siRNAs, we provided evidence that impairment of AhR and ARNT, but not ERβ plays a key role in the neuroprotective action of DIM against hypoxia-induced cell damage. This study may have implication for identifying new agents that could protect neurons against hypoxia by targeting AhR/ARNT signaling.

Raloxifene is the selective estrogen receptor modulator (SERM) currently used in clinical practice to activate estrogen receptors (ERs) in bone tissue and to antagonise ERs in breast and uterine cancers. Little is known, however, about mechanisms of action of raloxifene on hypoxia-induced neuronal cell damage. The aim of the present study was to investigate the neuroprotective potential of raloxifene against hypoxia-induced damage of mouse hippocampal cells in primary cultures, with a particular focus on raloxifene interactions with the classical nuclear ERs (ERα, ERβ) and the recently identified membrane ER G-protein-coupled receptor 30 (GPR30). In this study, 18 h of hypoxia increased hypoxia inducible factor 1 alpha (Hif1α) mRNA expression and induced apoptotic processes, such as loss of the mitochondrial membrane potential, activation of caspase-3 and fragmentation of cell nuclei based on Hoechst 33342 staining. These effects were accompanied by reduced ATPase and intracellular esterase activities as well as substantial lactate dehydrogenase (LDH) release from cells exposed to hypoxia. Our study demonstrated strong neuroprotective and anti-apoptotic caspase-3-independent actions of raloxifene in hippocampal cells exposed to hypoxia. Raloxifene also inhibited the hypoxia-induced decrease in Erα mRNA expression and attenuated the hypoxia-induced rise in Erβ and Gpr30 mRNA expression levels. Impact of raloxifene on hypoxia-affected Erα mRNA was mirrored by fluctuations in the protein level of the receptor as demonstrated by Western blot and immunofluorescent labelling. Raloxifene-induced changes in Erβ mRNA expression level were in parallel with ERβ immunofluorescent labeling. However, changes in Gpr30 mRNA level were not reflected by changes in the protein levels measured either by ELISA, Western blot or immunofluorescent staining at 24h post-treatment. Using specific siRNAs, we provided evidence for a key involvement of ERα, but not ERβ or GPR30 in neuroprotective action of raloxifene against hypoxia-induced cell damage. This study may have implications for the treatment or prevention of hypoxic brain injury and the administration of current or new generations of SERMs specific to ERα. This article is part of a Special Issue entitled "Sex steroids and brain disorders".

Non-insertional Achilles tendinopathy is one of the most common overuse injuries experienced by joggers. The pes planus evaluation is often based only on the visual method without a dynamic test. Functionally inefficiency of longitudinal or transverse arch of the foot may be a significant risk factor affecting the lower limb biomechanics and causing of pain in the Achilles tendon area. Assumptions and purpose of the study: This is study was undertaken to determine and investigate the relationship between the functionally inefficient longitudinal and transverse arch of the foot and the prevalence of non-insertional Achilles tendinopathy.The study group consisted of 11 regular joggers at different levels, who were diagnosed with non-insertional Achilles tendinopathy. The clinical evaluation involved pedobarographic analysis using the Footscan pressure plate. Information on loads applied to metatarsal area, the basis of the second and third metatarsal bones were subjected to statistical analysis.All subjects who were diagnosed with overuse injuries in the Achilles tendon area showed a functionally inefficient transverse arch of the foot. Despite their pain, the individuals subjected to the study did not cease completely their physical activity.Collapse of the natural arch of the foot can lead to biomechanical disorder in the lower limb joints. This is one of the risk factors for the occurrence of changes due to overuse injuries within the Achilles tendon.

The objective of this study was to evaluate bone resistance after in ovo administration of copper nanoparticles (NanoCu) and to determine the number of cells positive for proliferating cell nuclear antigen (PCNA) in the femoral bones of broiler chickens (n = 12 per group). The study demonstrated that femoral bones from the NanoCu group were characterised by a higher weight and volume and by significantly greater resistance to fractures compared to the Control group. NanoCu promoted the proliferation of PCNA-positive cells in the long bones of chickens. A significantly higher number of PCNA-positive cells in the bones of birds in the NanoCu group compared with the Control group (137 and 122, respectively) indicate a stimulatory effect during embryogenesis. Considering the improvement in bone resistance to fractures and the effect of NanoCu on the number of PCNA-positive cells in femoral bones, NanoCu may be an alternative agent to minimise the ever-present problem of weak bones in broiler chickens.

Although an increasing body of evidence suggests that triclocarban, a phenyl ether classified as a contaminant of emerging concern, presents a risk to development, there is limited data available on the potential interplay of triclocarban with the developing mammalian nervous system. This study was aimed to investigate the impact of environmentally pervasive chemical triclocarban on autophagy and estrogen receptor-mediated signaling pathways in mouse neurons. The study showed that triclocarban impaired autophagy and disrupted estrogen receptor signaling in mouse embryonic neurons in primary culture. Triclocarban used at environmentally relevant concentrations inhibited the mRNA and protein expression of ESR1 and GPER1 but not ESR2. The triclocarban-induced decrease in the expression of estrogen receptors was supported by the colocalization of the receptors in mouse neurons and corresponded to hypermethylation of the Esr1 and Gper1 genes. Selective antagonists increased the effects of triclocarban, which suggests that the neurotoxic effects of triclocarban, in addition to decreasing estrogen receptor expression, are mediated via inhibition of the neuroprotective capacity of the receptors. Furthermore, Becn1 and Atg7 siRNAs potentiated the caspase-3-dependent effect of triclocarban, which points to triclocarban-induced impairment of autophagy. Indeed, triclocarban dysregulated the expression of autophagy-related genes, and caused a time-dependent inhibition of the mRNA expression of Becn1, Map1lc3a, Map1lc3b, Nup62, and Atg7, which was correlated with a decrease in the protein levels of MAP1LC3B, BECN1 and autophagosomes, but not NUP62 protein level which was increased. Intriguingly, the Esr1 and Gper1 siRNAs did not affect the level of autophagosomes, suggesting that the triclocarban-induced impairment of autophagy is independent of the triclocarban-induced disruption of estrogen receptor signaling in mammalian neurons. Because our data provided evidence that triclocarban has the capacity to impair autophagy and disrupt estrogen receptor signaling in brain neurons at an early developmental stage, we postulate to categorize the compound as a neurodevelopmental risk factor.

4-para-Nonylphenol (NP) is a non-ionic surfactant that has widespread and uncontrolled distribution in the environment. Little is known, however, about its actions on neuronal cells during critical developmental periods. This study aimed to investigate the mechanisms underlying the apoptotic and toxic actions of NP on mouse embryonic neuronal cells and the possible interactions of NP with estrogen receptor (ER)- and retinoid X receptor (RXR)-mediated intracellular signaling. Treatment of mouse hippocampal neuronal cell cultures with NP (5 and 10μM) induced apoptotic and neurotoxic effects. The 2 and 7 day-old mouse hippocampal cultures were vulnerable to 5 and 10μM NP, whereas 12 day-old cultures responded only to the highest concentration of NP, thus suggesting an age-dependent action of the chemical on neuronal cells. The use of specific inhibitors did not support the involvement of calpains in NP-induced apoptosis, but indicated caspase-8- and caspase-9-dependent effects of NP. Specific ER antagonists MPP and PHTPP potentiated the NP-induced loss of mitochondrial membrane potential and increase in lactate dehydrogenase (LDH) release whereas, ER agonists PPT and DPN inhibited these effects. RXR antagonist HX531 diminished the NP-evoked loss of mitochondrial membrane potential, the activity of caspase-3 and LDH release. In addition, exposure to NP inhibited ERα- and ERβ-specific immunofluorescence but stimulated RXR-specific immunolabeling in mouse hippocampal cells. In conclusion, our study demonstrated that the apoptotic and toxic actions of NP on neuronal cells in early development is accompanied by an impairment of ER- and stimulation of RXR-mediated signaling pathways. Taking into account NP-induced alterations in mRNA expression levels of particular types of RXRs, we suggest that NP affected mainly RXRα and RXRβ, but not RXRγ signaling.

The article considers black humour as an important element of postmodern culture. The term is defi ned as a variety of humour which mocks society, taboo, illness, death, and is linked with pure nonsense and the grotesque. In the article, black humor is analyzed on the example of such Polish writers as Jerzy Pilch, Krzysztof Varga, Wojciech Stamm and Paweł Goźliński, who use it as a way to express postmodern world vision as an ambiguous and dangerous labyrinth.

Possessed, the novel written by Witold Gombrowicz, like a piece of grotesque proper, mixes various styles: high and low, neutral and disturbing, sentimental/romantic and prosaic. The author consciously emphasizes particular genre markers in some passages, leading the reader down a specifi c path, just to clash them with passages of a contradictory genre type. A good case in point is the description of the prince’s residence that complies with gothic novel conventions: it is horrifying and accentuates the singularity of the historical place. Possessed, apparently, was not only to be a product for a mass readership, but an experiment binding the features of high and low art, merging various audiences; it lives up to the expectations of the common reader of mass literature, as well as of the reader of a more sophisticated taste; on the one hand, the novel copies the mechanism of mass culture, as described by Eco48, and on the other, it is a frivolous and pleasant entertainment for a highbrow reader. As an experiment, therefore, the novel is a success because of the abovementioned recent theatrical adaptations, as well as its explicit comic effects.

Extended residual persistence of the pesticide dichlorodiphenyltrichloroethane (DDT) raises concerns about its long-term neurotoxic effects. Little is known, however, about DDT toxicity during the early stages of neural development. This study demonstrated that DDT-induced apoptosis of mouse embryonic neuronal cells is a caspase-9-, caspase-3-, and GSK-3β-dependent process, which involves p,p’ -DDT-specific impairment of classical ERs. It also provided evidence for DDT-isomer-nonspecific alterations of AhR- and GPR30-mediated intracellular signaling, including changes in the levels of the receptor and receptor-regulated mRNAs, and also changes in the protein levels of the receptors. DDT-induced stimulation of AhR-signaling and reduction of GPR30-signaling were verified using selective ligands and specific siRNAs. Co-localization of the receptors was demonstrated with confocal microscopy, and the presence of functional GPR30 was detected by electrophysiology. This study demonstrates that stimulation of AhR-signaling and impairment of GPR30-signaling play important roles in the propagation of DDT-induced apoptosis during the early stages of neural development.