Agnieszka Wnuk

Apoptotic and neurotoxic actions of 4-para-nonylphenol are accompanied by activation of retinoid X receptor and impairment of classical estrogen receptor signaling

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.

Triclosan activates aryl hydrocarbon receptor (AhR)-dependent apoptosis and affects Cyp1a1 and Cyp1b1 expression in mouse neocortical neurons.

Triclosan (TCS) is an antimicrobial agent that is used extensively in personal care and in sanitizing products, such as soaps, toothpastes, and hair products. A number of studies have revealed the presence of TCS in human tissues, such as fat, liver and brain, in addition to blood and breast milk. The aim of the present study was to investigate the impact of TCS on AhR and Cyp1a1/Cyp1b1 signaling in mouse neocortical neurons in primary cultures. In addition to the use of selective ligands and siRNAs, expression levels of mRNA and proteins as well as caspase-3 activity, reactive oxygen species (ROS) formation, and lactate dehydrogenase (LDH) release have been measured. We also studied the involvement of the AhR in TCS-induced LDH release and caspase-3 activation as well as the effect of TCS on ROS generation. Cultures of neocortical neurons were prepared from Swiss mouse embryos on day 15/16 of gestation. The cells were cultured in phenol red-free Neurobasal medium with B27 and glutamine, and the neurons were exposed to 1 and 10µM TCS. Our experiments showed that the expression of AhR and Cyp1a1 mRNA decreased in cells exposed to 10µM TCS for 3 or 6h. In the case of Cyp1b1, mRNA expression remained unchanged compared with the control group following 3h of exposure to TCS, but after 6h, the mRNA expression of Cyp1b1 was decreased. Our results confirmed that the AhR is involved in the TCS mechanism of action, and our data demonstrated that after the cells were transfected with AhR siRNA, the cytotoxic and pro-apoptotic properties of TCS were decreased. The decrease in Cyp1a1 mRNA and protein expression levels accompanied by a decrease in its activity. The stimulation of Cyp1a1 activity produced by the application of an AhR agonist (βNF) was attenuated by TCS, whereas the addition of AhR antagonist (αNF) reversed the inhibitory effects of TCS. In our experiments, TCS diminished Cyp1b1 mRNA and enhanced its protein expression. In case of Cyp1a1 we observed paradoxical effect of TCS action, which caused the decrease in activity and protein expression of Cyp1a1 and the increase in protein level of AhR. Therefore, we determined the effects of TCS on the production of ROS. Our results revealed that TCS increased the production of ROS and that this effect of TCS was reversed by 10µM N-acetyl-L-cysteine (NAC), the ROS scavenger. To confirm an involvement of ROS in TCS-induced neurotoxicity we measured AhR, Cyp1a1, and Cyp1b1 mRNA expression levels in cells co-treated with TCS and NAC. In the presence of NAC, TCS enhanced mRNA expression of the cytochromes and AhR at 3 and 6h, respectively. We postulate that TCS exhibits primary and secondary effects. The primary effects such as impairment of Cyp1a1 signaling are mediated by TCS-induced ROS production, whereas secondary effects of TCS are due to transcriptional activity of AhR and estrogenic properties of TCS.

RXRα, PXR and CAR xenobiotic receptors mediate the apoptotic and neurotoxic actions of nonylphenol in mouse hippocampal cells.

In the present study, we investigated the role of the retinoid X receptor (RXR), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the apoptotic and toxic effects of nonylphenol in mouse primary neuronal cell cultures. Our study demonstrated that nonylphenol activated caspase-3 and induced lactate dehydrogenase (LDH) release in hippocampal cells, which was accompanied by an increase in the mRNA expression and protein levels of RXRα, PXR and CAR. Nonylphenol stimulated Rxra, Pxr, and Car mRNA expression. These effects were followed by increase in the protein levels of particular receptors. Immunofluorescence labeling revealed the cellular distribution of RXRα, PXR and CAR in hippocampal neurons in response to nonylphenol, shortening of neurites and cytoplasmic shrinking, as indicated by MAP2 staining. It also showed NP-induced translocation of receptor-specific immunofluorescence from cytoplasm to the nucleus. The use of specific siRNAs demonstrated that Rxra-, Pxr-, and Car-siRNA-transfected cells were less vulnerable to nonylphenol-induced activation of caspase-3 and LDH, thus confirming the key involvement of RXRα/PXR/CAR signaling pathways in the apoptotic and neurotoxic actions of nonylphenol. These new data give prospects for the targeting xenobiotic nuclear receptors to protect the developing nervous system against endocrine disrupting chemicals.

The Crucial Involvement of Retinoid X Receptors in DDE Neurotoxicity

Dichlorodiphenyldichloroethylene (DDE) is a primary environmental and metabolic degradation product of the pesticide dichlorodiphenyltrichloroethane (DDT). It is one of the most toxic compounds belonging to organochlorines. DDE has never been commercially produced; however, the parent pesticide DDT is still used in some developing countries for disease-vector control of malaria. DDT and DDE remain in the environment because these chemicals are resistant to degradation and bioaccumulate in the food chain. Little is known, however, about DDE toxicity during the early stages of neural development. The results of the present study demonstrate that DDE induced a caspase-3-dependent apoptosis and caused the global DNA hypomethylation in mouse embryonic neuronal cells. This study also provided evidence for DDE-isomer-non-specific alterations of retinoid X receptor α (RXRα)- and retinoid X receptor β (RXRβ)-mediated intracellular signaling, including changes in the levels of the receptor mRNAs and changes in the protein levels of the receptors. DDE-induced stimulation of RXRα and RXRβ was verified using selective antagonist and specific siRNAs. Co-localization of RXRα and RXRβ was demonstrated using confocal microscopy. The apoptotic action of DDE was supported at the cellular level through Hoechst 33342 and calcein AM staining experiments. In conclusion, the results of the present study demonstrated that the stimulation of RXRα- and RXRβ-mediated intracellular signaling plays an important role in the propagation of DDE-induced apoptosis during early stages of neural development.

Benzophenone-3 Impairs Autophagy, Alters Epigenetic Status, and Disrupts Retinoid X Receptor Signaling in Apoptotic Neuronal Cells

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.

Steroid and Xenobiotic Receptor Signalling in Apoptosis and Autophagy of the Nervous System

Apoptosis and autophagy are involved in neural development and in the response of the nervous system to a variety of insults. Apoptosis is responsible for cell elimination, whereas autophagy can eliminate the cells or keep them alive, even in conditions lacking trophic factors. Therefore, both processes may function synergistically or antagonistically. Steroid and xenobiotic receptors are regulators of apoptosis and autophagy; however, their actions in various pathologies are complex. In general, the estrogen (ER), progesterone (PR), and mineralocorticoid (MR) receptors mediate anti-apoptotic signalling, whereas the androgen (AR) and glucocorticoid (GR) receptors participate in pro-apoptotic pathways. ER-mediated neuroprotection is attributed to estrogen and selective ER modulators in apoptosis- and autophagy-related neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, stroke, multiple sclerosis, and retinopathies. PR activation appeared particularly effective in treating traumatic brain and spinal cord injuries and ischemic stroke. Except for in the retina, activated GR is engaged in neuronal cell death, whereas MR signalling appeared to be associated with neuroprotection. In addition to steroid receptors, the aryl hydrocarbon receptor (AHR) mediates the induction and propagation of apoptosis, whereas the peroxisome proliferator-activated receptors (PPARs) inhibit this programmed cell death. Most of the retinoid X receptor-related xenobiotic receptors stimulate apoptotic processes that accompany neural pathologies. Among the possible therapeutic strategies based on targeting apoptosis via steroid and xenobiotic receptors, the most promising are the selective modulators of the ER, AR, AHR, PPARγ agonists, flavonoids, and miRNAs. The prospective therapies to overcome neuronal cell death by targeting autophagy via steroid and xenobiotic receptors are much less recognized.

Prenatal exposure to benzophenone-3 (BP-3) induces apoptosis, disrupts estrogen receptor expression and alters the epigenetic status of mouse neurons

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 Disrupts the Epigenetic Status of Neuronal Cells and Induces AHR/CAR-Mediated Apoptosis

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.

Effects of exposure to 5-MeO-DIPT during adolescence on brain neurotransmission and neurotoxicity in adult rats

PurposeTryptamine 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.MethodsNeurotransmitter 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.ResultsWe 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.ConclusionsThese results suggest that 5-MeO-DIPT given repeatedly during adolescence affects brain neurotransmission and shows neurotoxic potential observed in adult animals.