Konrad A. Szychowski

TBBPA causes neurotoxic and the apoptotic responses in cultured mouse hippocampal neurons in vitro

BACKGROUND Tetrabromobisphenol A (TBBPA) is a brominated flame retardant widely used in a variety of commercial and household products. TBBPA can become bioaccumulated in human body fluids, and also in different brain regions. The aim of the present study was to determine the viability and apoptosis of cultured mouse hippocampal neurons in vitro after exposure to TBBPA. Additionally, we examined the involvement of ROS generation in the effect of TBBPA. METHODS Primary hippocampal neuron cultures were prepared from Swiss mouse embryos on day 17/18 of gestation. The cultures were treated with TBBPA at concentrations ranging from 1nM to 100μM for 30min or 3, 6 or 24h. To study apoptosis, the activity of caspase-3 was measured, and apoptotic body formation was evaluated. To investigate the cytotoxic effect of TBBPA, the level of lactate dehydrogenase (LDH) was measured in the culture medium. RESULTS Our results demonstrated that TBBPA concentrations ranging from 100nM to 100μM caused caspase-3 activation and apoptotic body formation. The cytotoxic effects of TBBPA were observed at concentrations ranging from 50nM to 100μM. To detect intracellular ROS, the fluorogenic dye H2DCFDA was used. We did not observe any significant increase in the level of cellular ROS in cultured cells after TBBPA treatment. However, in a cell-free model, TBBPA at concentrations ranging from 10 to 100μM interacted with H2DCFDA and enhanced the fluorescence signal. CONCLUSION We suggest that the H2DCFDA assay cannot be used to measure TBBPA-stimulated cell-mediated ROS production.

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.

Triclosan induces Fas receptor-dependent apoptosis in mouse neocortical neurons in vitro.

Triclosan (TCS) is a commonly used antimicrobial agent in personal care and sanitizing products, as well as in household items. Numerous studies have demonstrated the presence of TCS in various human tissues. Several studies have reported the accumulation of TCS in fish and human brain tissue. The aim of the present study was to investigate the effect of TCS on apoptosis in mouse neocortical neurons after 7 days of culture in vitro following 3, 6 and 24 h of exposure. To explore the mechanism underlying the effects of TCS in neurons, we studied the activation and protein expression of the Fas receptor (FasR) and caspase-8, caspase-9 and caspase-3, as well as DNA fragmentation in TCS-treated cells. 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. The cultures were treated with concentrations of TCS ranging from 1 nM to 100 μM for 3, 6 and 24 h. The level of lactate dehydrogenase (LDH) was measured in the culture medium to exclude the cytotoxic concentrations. The cytotoxic effects were only observed when the highest concentrations of TCS were used (50 and 100 μM). To study apoptosis, the activities of caspase-8, caspase-9 and caspase-3 were measured, and DNA fragmentation was evaluated. Our results are the first time to demonstrate that TCS can induce an apoptotic process in neocortical neurons in vitro. The data demonstrated that TCS caused caspase-3 activation, DNA fragmentation and apoptotic body formation. Non-cytotoxic concentrations of TCS activated the extrinsic apoptotic signaling pathway, which is dependent on FasR and caspase-8 activation. However, it is also possible that TCS may activate the intrinsic apoptotic pathway after long-term exposure. Therefore, further studies on the mechanism underlying the effects of TCS on the nervous system are needed.

Składniki tworzyw sztucznych zaburzające funkcje układu nerwowego

Development of the chemical industry leads to the development of new chemical compounds, which naturally do not exist in the environment. These chemicals are used to reduce flammability, increase plasticity, or improve solubility of other substances. Many of these compounds, which are components of plastic, the new generation of cosmetics, medical devices, food packaging and other everyday products, are easily released into the environment. Many studies have shown that a major lipophilicity characterizes substances such as phthalates, BPA, TBBPA and PCBs. This feature allows them to easily penetrate into living cells, accumulate in the tissues and the organs, and affect human and animal health. Due to the chemical structures, these compounds are able to mimic some endogenous hormones such as estradiol and to disrupt the hormone homeostasis. They can also easily pass the placental barrier and the blood-brain barrier. As numerous studies have shown, these chemicals disturb the proper functions of the nervous system from the earliest moments of life. It has been proven that these compounds affect neurogenesis as well as the synaptic transmission process. As a consequence, they interfere with the formation of the sex of the brain, as well as with the learning processes, memory and behavior. Additionally, the cytotoxic and pro-apoptotic effect may cause neurodegenerative diseases. This article presents the current state of knowledge about the effects of phthalates, BPA, TBBPA, and PCBs on the nervous system.

Tetrabromobisphenol A (TBBPA)-stimulated reactive oxygen species (ROS) production in cell-free model using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay-limitations of method.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant, applied in a variety of commercial and household products, mainly electronic ones. Since the production of reactive oxygen species (ROS) is considered one of the principal cytotoxicity mechanisms, numerous studies undertake that aspect of TBBPA’s mechanism of action. The present study verifies if the fluorogenic substrate 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) should be used to detect ROS production induced by TBBPA. To determine the ability of TBBPA alone to stimulate the conversion of H2DCFDA to its fluorescent product 2’,7’-dichlorofluorescein (DCF), we used a cell-free model. In the experiments we check different cultured media also in combination with free radical scavenger N-acetyl-l-cysteine (NAC). Additionally, experiments with stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH·) have been made. The presented data showed that TBBPA in all tested concentrations interacts with H2DCFDA in phosphate-buffered saline (PBS) buffer while in micromolar concentrations in the DMEM/F12 medium with and without serum. The addition of NAC inhibited the interaction of TBBPA with H2DCFDA. Experiments with DPPH· showed that, in the presence of NAC, TBBPA acts like a free radical. TBBPA has similar properties to free radical and is susceptible to free radical scavenging properties of NAC. Our results indicated that H2DCFDA assay cannot be used to evaluate cellular ROS production in TBBPA studies. The study connected with TBBPA-stimulated ROS production in cell culture models using the H2DCFDA assay should be revised using a different method. However, due to the free radical-like nature of TBBPA, it can be very difficult. Therefore, further investigation of the nature of TBBPA as a compound with similar properties to free radical is required.

Comparative study of eco- and cytotoxicity during biotransformation of anthraquinone dye Alizarin Blue Black B in optimized cultures of microscopic fungi

The aim of this study was to select optimal conditions (C and N sources, initial pH and temperature) for biodecolorization of 0.03% anthraquinone dye Alizarin Blue Black B (ABBB) by microscopic fungi: Haematonectria haematococca BwIII43, K37 and Trichoderma harzianum BsIII33. The phenolic compounds, phytotoxicity (Lepidium sativum L.), biotoxicity (Microtox), cytotoxicity and yeast viability assay were performed to determine the extent of ABBB detoxification. Biodecolorization and detoxification of 0.03% ABBB in H. haematococca BwIII43 and T. harzianum BsIII33 cultures was correlated with extracellular oxidoreductases activity. In turn, secondary products, toxic to human fibroblasts and respiring sod1 Saccharomyces cerevisiae cells, were formed in H. haematococca K37 strain cultures, despite efficient decolorization.

Characterization of active compounds of different garlic (Allium sativum L.) cultivars

Abstract Garlic (Allium sativum L.) has a reputation as a therapeutic agent for many different diseases such as microbial infections, hypertension, hypercholesterolaemia, diabetes, atherosclerosis and cancer. Health benefits of garlic depend on its content of biologically-active compounds, which differs between cultivars and geographical regions. The aim of this study was to evaluate and compare the biological activity of aqueous extracts from nine garlic varieties from different countries (Poland, Spain, China, Portugal, Burma, Thailand and Uzbekistan). Antioxidant properties were evaluated through free radical scavenging (DPPH•, ABTS•+) and ion chelation (Fe2+, Cu2+) activities. The cytotoxicity of garlic extracts was evaluated in vitro using Neutral Red Uptake assay in normal human skin fibroblasts. The obtained results revealed that garlic extracts contained the highest amount of syringic and p-hydroxybenzoic acids derivatives. The lowest IC50 values for DPPH•, ABTS•+ scavenging and Cu2+ chelating ability were determined in Chinese garlic extracts (4.63, 0.43 and 14.90 μg/mL, respectively). Extracts from Spanish cultivar Morado and Chinese garlic were highly cytotoxic to human skin fibroblasts as they reduced cellular proliferation by 70–90%. We showed diverse contents of proteins and phenolic components in garlic bulbs from different varieties. The obtained results could help to choose the cultivars of garlic which contain significant amounts of active compounds, have important antioxidant properties and display low antiproliferative effect and/or low cytotoxicity against normal human skin fibroblast BJ.

The Action of Di-(2-Ethylhexyl) Phthalate (DEHP) in Mouse Cerebral Cells Involves an Impairment in Aryl Hydrocarbon Receptor (AhR) Signaling

Di-(2-ethylhexyl) phthalate (DEHP) is used as a plasticizer in various plastic compounds, such as polyvinyl chloride (PVC), and products including baby toys, packaging films and sheets, medical tubing, and blood storage bags. Epidemiological data suggest that phthalates increase the risk of the nervous system disorders; however, the impact of DEHP on the brain cells and the mechanisms of its action have not been clarified. The aim of the present study was to investigate the effects of DEHP on production of reactive oxygen species (ROS) and aryl hydrocarbon receptor (AhR), as well as Cyp1a1 and Cyp1b1 mRNA and protein expression in primary mouse cortical neurons and glial cells in the in vitro mono-cultures. Our experiments showed that DEHP stimulated ROS production in both types of mouse neocortical cells. Moreover, the results strongly support involvement of the AhR/Cyp1A1 signaling pathway in the action of DEHP in neurons and glial cells. However, the effects of DEHP acting on the AhR signaling pathways in these two types of neocortical cells were different. In neurons, AhR mRNA expression did not change, but AhR protein expression decreased in response to DEHP. A similar trend was observed for Cyp1a1 and Cyp1b1 mRNA and protein expression. Failure to induce Cyp1a1 in neurons was confirmed by EROD assay. In primary glial cells, a decrease in AhR protein level was accompanied by a decrease in AhR mRNA expression. In glial cells, mRNA and protein expression of Cyp1a1 as well as Cyp1a1-related EROD activity were significantly increased. As for Cyp1b1, both in neurons and glial cells Cyp1b1 mRNA expression did not significantly change, whereas Cyp1b1 protein level were decreased. We postulate that developmental exposure to DEHP which dysregulates AhR/Cyp1a1 may disrupt defense processes in brain neocortical cells that could increase their susceptibility to environmental toxins.