| 1. |
- Attoff, Kristina, 1985-, et al.
(författare)
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Acrylamide alters CREB and retinoic acid signaling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Acrylamide is a known neurotoxic compound that we get exposed to through food and through the environment. It can cross the placental barrier as well as the blood-brain barrier resulting in exposure of the fetus and the infant child. We used the human neuroblastoma cell line SH-SY5Y to study the effects of non-cytotoxic acrylamide exposure during 9 days of differentiation on two differentially important signaling pathways, i.e. the retinoic acid receptor (RAR) and cAMP response element-binding protein (CREB) signaling in neurons. Our results showed that exposure of non-cytotoxic concentrations of acrylamide during 9 days of differentiation induced altered expression of multiple genes that are part of the CREB and RAR activation pathways, e.g. cellular retinoic acid binding protein 1, retinol binding protein 7, CREB5 and fibroblast growth factor receptor 2. Other well-established neuronal markers such as brain-derived neurotrophic factor, syntaxin binding protein 2, transforming growth factor beta 1, the dopaminergic markers monoamine oxidase A and dopamine receptor D2 as wells as the cholinergic marker choline O-acetyltransferase were also significantly altered by acrylamide. Our results reveal that acrylamide interferes with crucial pathways involved in neuronal differentiation in vitro and raise concerns over the potential toxic outcomes in humans.
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| 2. |
- Attoff, Kristina, et al.
(författare)
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Acrylamide alters CREB and retinoic acid signalling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Acrylamide (ACR) is a known neurotoxicant which crosses the blood-brain barrier, passes the placenta and has been detected in breast milk. Hence, early-life exposure to ACR could lead to developmental neurotoxicity. The aim of this study was to elucidate if non-cytotoxic concentrations of ACR alter neuronal differentiation by studying gene expression of markers significant for neurodevelopment in the human neuroblastoma SH-SY5Y cell model. Firstly, by using RNASeq we identified two relevant pathways that are activated during 9 days of retinoic acid (RA) induced differentiation i.e. RA receptor (RAR) activation and the cAMP response element-binding protein (CREB) signalling pathways. Next, by qPCR we showed that 1 and 70 mu M ACR after 9 days exposure alter the expression of 13 out of 36 genes in the RAR activation pathway and 18 out of 47 in the CREB signalling pathway. Furthermore, the expression of established neuronal markers i.e. BDNF, STXBP2, STX3, TGFB1 and CHAT were down-regulated. Decreased protein expression of BDNF and altered ratio of phosphorylated CREB to total CREB were confirmed by western blot. Our results reveal that micromolar concentrations of ACR sustain proliferation, decrease neurite outgrowth and interfere with signalling pathways involved in neuronal differentiation in the SH-SY5Y cell model.
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| 3. |
- Cediel Ulloa, Andrea, et al.
(författare)
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Epigenetics of methylmercury
- 2023
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Ingår i: Neurotoxicology. - : Elsevier BV. - 0161-813X .- 1872-9711. ; 97, s. 34-46
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Tidskriftsartikel (refereegranskat)abstract
- Purpose of review: Methylmercury (MeHg) is neurotoxic at high levels and particularly affects the developing brain. One proposed mechanism of MeHg neurotoxicity is alteration of the epigenetic programming. In this review, we summarise the experimental and epidemiological literature on MeHg-associated epigenetic changes.Recent findings: Experimental and epidemiological studies have identified changes in DNA methylation following in utero exposure to MeHg, and some of the changes appear to be persistent. A few studies have evaluated associations between MeHg-related changes in DNA methylation and neurodevelopmental outcomes. Experimental studies reveal changes in histone modifications after MeHg exposure, but we lack epidemiological studies supporting such changes in humans. Experimental and epidemiological studies have identified microRNA-related changes associated with MeHg; however, more research is needed to conclude if these changes lead to persistent and toxic effects.Summary: MeHg appears to interfere with epigenetic processes, potentially leading to persistent changes. However, observed associations of mercury with epigenetic changes are as of yet of unknown relevance to neurodevelopmental outcomes.
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| 4. |
- Cediel Ulloa, Andrea, et al.
(författare)
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Impact of endocrine disrupting chemicals on neurodevelopment : the need for better testing strategies for endocrine disruption-induced developmental neurotoxicity
- 2022
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Ingår i: Expert Review of Endocrinology & Metabolism. - : Taylor & Francis. - 1744-6651 .- 1744-8417. ; 17:2, s. 131-141
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Brain development is highly dependent on hormonal regulation. Exposure to chemicals disrupting endocrine signaling has been associated with neurodevelopmental impairment. This raises concern about exposure to the suspected thousands of endocrine disruptors, and has resulted in efforts to improve regulation of these chemicals. Yet, the causal links between endocrine disruption and developmental neurotoxicity, which would be required for regulatory action, are still largely missing. Areas covered: In this review, we illustrate the importance of two endocrine systems, thyroid hormone and retinoic acid pathways, for neurodevelopment. We place special emphasis on TH and RA synthesis, metabolism, and how endocrine disrupting chemicals known or suspected to affect these systems are associated with developmental neurotoxicity. Expert opinion: While it is clear that neurodevelopment is dependent on proper hormonal functioning, and evidence is increasing for developmental neurotoxicity induced by endocrine disrupting chemicals, this is not grasped by current chemical testing. Thus, there is an urgent need to develop test methods detecting endocrine disruption in the context of neurodevelopment. Key to this development is further mechanistic insights on the involvement of endocrine signaling in neurodevelopment as well as increased support to develop and validate new test methods for the regulatory context.
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| 5. |
- Cediel Ulloa, Andrea, et al.
(författare)
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Methylmercury-induced DNA methylation—From epidemiological observations to experimental evidence
- 2022
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Ingår i: Frontiers in Genetics. - : Frontiers Media SA. - 1664-8021. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Methylmercury (MeHg) is a developmental neurotoxicant, and one potential mechanism of MeHg toxicity is epigenetic dysregulation. In a recent meta-analysis of epigenome-wide association studies (EWAS), associations between prenatal MeHg exposure and DNA methylation at several genomic sites were identified in blood from newborns and children. While EWASs reveal human-relevant associations, experimental studies are required to validate the relationship between exposure and DNA methylation changes, and to assess if such changes have implications for gene expression. Herein, we studied DNA methylation and gene expression of five of the top genes identified in the EWAS meta-analysis, MED31, MRPL19, GGH, GRK1, and LYSMD3, upon MeHg exposure in human SH-SY5Y cells exposed to 8 or 40 nM of MeHg during differentiation, using bisulfite-pyrosequencing and qPCR, respectively. The concentrations were selected to cover the range of MeHg concentrations in cord blood (2–8.5 μg/L) observed in the cohorts included in the EWAS. Exposure to MeHg increased DNA methylation at MED31, a transcriptional regulator essential for fetal development. The results were in concordance with the epidemiological findings where more MED31 methylation was associated with higher concentrations of MeHg. Additionally, we found a non-significant decrease in DNA methylation at GGH, which corresponds to the direction of change observed in the EWAS, and a significant correlation of GGH methylation with its expression. In conclusion, this study corroborates some of the EWAS findings and puts forward candidate genes involved in MeHg’s effects on the developing brain, thus highlighting the value of experimental validation of epidemiological association studies.
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| 6. |
- Cediel-Ulloa, Andrea, 1989-
(författare)
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Novel Endpoints To Unravel Developmental Neurotoxicity : From DNA methylation responses to methylmercury to the in vitro identification of endocrine disruptors
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The developing brain is especially sensitive to environmental stressors due to its dependence on the precise spatiotemporal regulation of multiple signals, and the long time period required for its formation. Some chemicals can interfere with molecular and cellular processes driving brain development, including epigenetic processes such as DNA methylation. Hence, identification of DNA methylation changes induced by chemical exposure may serve as early molecular markers for developmental neurotoxicity (DNT). Chemicals known as endocrine disruptors (EDCs) can produce adverse effects due to their capability to alter the endocrine system. Since brain development is highly dependent on endocrine signals, the potential adverse effects of EDCs on brain development needs to be addressed. Detection of DNT in the regulatory context has been based on in vivo testing, however, the financial costs and time intensive characteristics of these methods have resulted in a limited assessment of the DNT hazard of chemicals. In addition, in order to regulate EDCs, it is paramount to demonstrate that their adverse effects are a product of disruption of endocrine signals. Yet, at the moment, there are no approved methods which address both an endocrine mode of action and adverse neurodevelopmental outcomes. This doctoral thesis had two main aims: Firstly, to identify epigenetic changes, at the level of DNA methylation, underlying DNT induced by exposure to methylmercury (MeHg); and secondly, to develop new approach methods (NAMs) for the detection of DNT induced by endocrine disruption. Epigenetic effects were studied both in epidemiological data and experimentally in vitro. Associations between prenatal MeHg exposure and DNA methylation of GRIN2B and NR3C1 were found in children. In vitro validation of DNA methylation changes found in epigenome-wide association studies of populations exposed to MeHg, uncovered the potential involvement of the Mediator Complex Subunit 31 (MED31) in MeHg DNT. To contribute to the endocrine disruption (ED)-induced DNT field, the applicability of an in vitro model composed of murine neural progenitor cells (the C17.2 cell-line) was evaluated. We found that C17.2 neural differentiation and morphology were sensitive to retinoic acid (RAR), retinoic X (RXR), peroxisome proliferator-activated β/δ (PPARβ/δ), and glucocorticoid (GR) agonism. Furthermore, two out of 25 tested EDCs decreased neurite outgrowth and branching in the C17.2 system. These effects were recovered by co-exposure of the chemicals with antagonists of RAR, RXR, or PPARβ/δ, indicating that their DNT effect is mediated by hormonal disruption. Altogether, this thesis contributed to the development of new methodologies and endpoints for the assessment of DNT induced by MeHg and EDCs.
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| 7. |
- Cediel Ulloa, Andrea, et al.
(författare)
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Prenatal methylmercury exposure and DNA methylation in seven-year-old children in the Seychelles Child Development Study
- 2021
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Ingår i: Environment International. - : Elsevier. - 0160-4120 .- 1873-6750. ; 147
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundMethylmercury (MeHg) is present in fish and is a neurotoxicant at sufficiently high levels. One potential mechanism of MeHg toxicity early in life is epigenetic dysregulation that may affect long-term neurodevelopment. Altered DNA methylation of nervous system-related genes has been associated with adult mental health outcomes.ObjectiveTo assess associations between prenatal MeHg exposure and DNA methylation (at the cytosine of CG dinucleotides, CpGs) in three nervous system-related genes, encoding brain-derived neurotropic factor (BDNF), glutamate receptor subunit NR2B (GRIN2B), and the glucocorticoid receptor (NR3C1), in children who were exposed to MeHg in utero.MethodsWe tested 406 seven-year-old Seychellois children participating in the Seychelles Child Development Study (Nutrition Cohort 2), who were prenatally exposed to MeHg from maternal fish consumption. Total mercury in maternal hair (prenatal MeHg exposure measure) collected during pregnancy was measured using atomic absorption spectroscopy. Methylation in DNA from the children’s saliva was measured by pyrosequencing. To assess associations between prenatal MeHg exposure and CpG methylation at seven years of age, we used multivariable linear regression models adjusted for covariates.ResultsWe identified associations with prenatal MeHg exposure for DNA methylation of one GRIN2B CpG and two NR3C1 CpGs out of 12 total CpG sites. Higher prenatal MeHg was associated with higher methylation for each CpG site. For example, NR3C1 CpG3 had an expected increase of 0.03-fold for each additional 1 ppm of prenatal MeHg (B = 0.030, 95% CI 0.001, 0.059; p = 0.047). Several CpG sites associated with MeHg are located in transcription factor binding sites and the observed methylation changes are predicted to lead to lower gene expression.ConclusionsIn a population of people who consume large amounts of fish, we showed that higher prenatal MeHg exposure was associated with differential DNA methylation at seven years of age at specific CpG sites that may influence neurodevelopment and mental health.
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| 8. |
- Cediel-Ulloa, Andrea, et al.
(författare)
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The C17.2 cell line as testing system for endocrine disruption-induced developmental neurotoxicity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Hormone signaling plays an essential role during fetal life and is vital for brain development. Endocrine-disrupting chemicals (EDCs) can interfere with the hormonal milieu in this critical time period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the applicability of the neural progenitor C17. 2 cell-line, as an in vitro test method to aid in the detection of endocrine disruption induced DNT. For this, C17.2 cells were exposed during 10 days of differentiation to (ant)agonists of the thyroid hormone (Thr), glucocorticoid (Gr), retinoic acid (Rar), retinoic x (Rxr), oxysterols (Lxr), estrogen (Er), androgen (Ar), and peroxisome proliferator activated delta (Ppard) receptors, as well as to the agonist of the vitamin D (Vdr) receptor. Upon exposure and differentiation, the cells were incubated with Hoechst (nuclear staining) and subsequently stained for βIII-tubulin (neuronal marker) by immunofluorescence. Automated imaging was carried out with a 10X objective lens using an ImageXpress micro xls system (Molecular Devices) and image analysis was performed with MetaXpress® software (Molecular Devices). The C17.2 cells were responsive to the Rar and Rxr agonists which decreased neurite outgrowth, branching and neuronal differentiation as well as to the Rar antagonist which increased neurite outgrowth and branching. With this approach, we have identified that the C17.2 cells are responsive to Gr, Rar, Rxr, and Pparβ/δ, hence contributing to the development of reliable and transferable test methods for hazard assessment of EDCs.
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| 9. |
- Cediel-Ulloa, Andrea, et al.
(författare)
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The pesticides endosulfan and cypermethrin affect neuronal differentiation via retinoic and peroxisome proliferator receptor activity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Brain development is highly dependent on hormonal homeostasis, hence developmental exposure to endocrine disrupting chemicals (EDCs) is of high concern. In fact, epidemiological and in vivo studies support associations between exposure to EDCs and impaired neurodevelopment. However, the existing hazard assessment of EDCs does not consider developmental neurotoxicity (DNT) prompting an urgent requirement for innovative testing and screening tools addressing endocrine disruption (ED)-induced DNT. We have previously shown the applicability of the immortalized murine neural progenitor cells, C17.2 cells, for addressing ED-DNT. We evidenced decreased neurite outgrowth and branching when the cells were exposed to the Rar, Rxr or Pparβ/δ agonists, and concluded that this is a suitable model for the evaluation of ED-induced DNT for chemicals disrupting Rar, Rxr or Pparβ/δ signalling. In this study we further validated the C17.2 method by testing the effects of 25 EDCs on the same neuronal morphology endpoints as reported in the previous paper. Out of the tested chemicals, endosulfan and cypermethrin decreased, while benzyl butyl phthalate (BBzP) increased neurite outgrowth and branching. We proceeded to evaluate whether these effects were mediated by Rar, Rxr or Ppar β/δ agonism. The neuronal morphology effects of endosulfan and cypermethrin were rescued by co-exposures Rar and Rxr antagonists, and partially rescued by the Ppar β/δ antagonist indicating a common mechanism. With this approach, we have identified that the C17.2 cells can be used as an in vitro model to address ED-induced DNT.
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| 10. |
- Cediel Ulloa, Andrea, et al.
(författare)
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Toxicity of stainless and mild steel particles generated from gas-metal arc welding in primary human small airway epithelial cells
- 2021
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Welding fumes induce lung toxicity and are carcinogenic to humans but the molecular mechanisms have yet to be clarified. The aim of this study was to evaluate the toxicity of stainless and mild steel particles generated via gas-metal arc welding using primary human small airway epithelial cells (hSAEC) and ToxTracker reporter murine stem cells, which track activation of six cancer-related pathways. Metal content (Fe, Mn, Ni, Cr) of the particles was relatively homogenous across particle size. The particles were not cytotoxic in reporter stem cells but stainless steel particles activated the Nrf2-dependent oxidative stress pathway. In hSAEC, both particle types induced time- and dose-dependent cytotoxicity, and stainless steel particles also increased generation of reactive oxygen species. The cellular metal content was higher for hSAEC compared to the reporter stem cells exposed to the same nominal dose. This was, in part, related to differences in particle agglomeration/sedimentation in the different cell media. Overall, our study showed differences in cytotoxicity and activation of cancer-related pathways between stainless and mild steel welding particles. Moreover, our data emphasizes the need for careful assessment of the cellular dose when comparing studies using different in vitro models.
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| 11. |
- Dauter, Ulrike Maria, et al.
(författare)
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Cancer-related changes and low-to-moderate exposure to welding fumes : A longitudinal study
- 2022
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Ingår i: Scandinavian Journal of Work, Environment and Health. - : Scandinavian Journal of Work, Environment and Health. - 0355-3140 .- 1795-990X. ; 48:1, s. 21-30
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Tidskriftsartikel (refereegranskat)abstract
- Objective This study tested for an association between early cancer-related biomarkers and low-to-moderate exposure to fumes from welding mild steel.Methods Male, non-smoking participants from southern Sweden were recruited and examined (N=338, 171 welders and 167 controls); of these, 78 welders and 96 controls were examined on two occasions six years apart. Exposure to welding fumes was evaluated by measuring respirable dust, welding years, and cumulative exposure. DNA methylation of CpG sites within the cancer-related genes AHRR, F2RL3, and B3GNTL1 was measured by pyrosequencing and relative mitochondrial DNA copy number and telomere length were measured by qPCR in whole-blood samples. Multivariate models were used for longitudinal analysis.Results Median exposure to respirable dust was 0.7 mg/m3 at both timepoints, adjusted for use of personal protective equipment. Compared with controls, welders showed a significant decrease over time in DNA methylation of B3GNTL1 CpG1 and CpG4 [adjusted for age, body mass index, and smoking: β=-0.66, standard error (SE)=0.28; β=-0.48, SE=0.24, respectively]. In addition, exposure to respirable dust and cumulative exposure was associated with a decrease in methylation of F2RL3 CpG2 among all welders (adjusted β=-0.67, SE=0.23 and β=-0.03, SE=0.02, respectively). No significant associations were found for AHRR, mitochondrial DNA copy number, or telomere length.Conclusion Low-to-moderate exposure to welding fumes was associated with a small effect on selected early epigenetic biomarkers of cancer. The direction of the methylation pattern (lower methylation of specific CpG sites) indicates early lung cancer-related changes associated with mild steel welding.
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| 12. |
- Delp, Johannes, et al.
(författare)
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Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants
- 2019
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 93:6, s. 1585-1608
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Tidskriftsartikel (refereegranskat)abstract
- Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose <-> galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.
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| 13. |
- Delp, Johannes, et al.
(författare)
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Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors
- 2021
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Ingår i: Archives of Toxicology. - : Springer. - 0340-5761 .- 1432-0738. ; 95:2, s. 591-615
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Tidskriftsartikel (refereegranskat)abstract
- Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.
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| 14. |
- Hinojosa, Maria G., 1994-, et al.
(författare)
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Evaluation of mRNA markers in differentiating human SH-SY5Y cells for estimation of developmental neurotoxicity
- 2023
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Ingår i: Neurotoxicology. - : Elsevier. - 0161-813X .- 1872-9711. ; 97, s. 65-77
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Tidskriftsartikel (refereegranskat)abstract
- Current guidelines for developmental neurotoxicity (DNT) evaluation are based on animal models. These have limitations so more relevant, efficient and robust approaches for DNT assessment are needed. We have used the human SH-SY5Y neuroblastoma cell model to evaluate a panel of 93 mRNA markers that are frequent in Neuronal diseases and functional annotations and also differentially expressed during retinoic acid-induced differentiation in the cell model. Rotenone, valproic acid (VPA), acrylamide (ACR) and methylmercury chlo-ride (MeHg) were used as DNT positive compounds. Tolbutamide, D-mannitol and clofibrate were used as DNT negative compounds. To determine concentrations for exposure for gene expression analysis, we developed a pipeline for neurite outgrowth assessment by live-cell imaging. In addition, cell viability was measured by the resazurin assay. Gene expression was analyzed by RT-qPCR after 6 days of exposure during differentiation to concentrations of the DNT positive compounds that affected neurite outgrowth, but with no or minimal effect on cell viability. Methylmercury affected cell viability at lower concentrations than neurite outgrowth, hence the cells were exposed with the highest non-cytotoxic concentration. Rotenone (7.3 nM) induced 32 differentially expressed genes (DEGs), ACR (70 mu M) 8 DEGs, and VPA (75 mu M) 16 DEGs. No individual genes were significantly dysregulated by all 3 DNT positive compounds (p < 0.05), but 9 genes were differentially expressed by 2 of them. Methylmercury (0.8 nM) was used to validate the 9 DEGs. The expression of SEMA5A (encoding semaphorin 5A) and CHRNA7 (encoding nicotinic acetylcholine receptor subunit alpha 7) was downregulated by all 4 DNT positive compounds. None of the DNT negative compounds dysregulated any of the 9 DEGs in common for the DNT positive compounds. We suggest that SEMA5A or CHRNA7 should be further evaluated as biomarkers for DNT studies in vitro since they also are involved in neurodevelopmental adverse outcomes in humans.
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| 15. |
- Krebs, Alice, et al.
(författare)
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The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods
- 2020
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 94:7, s. 2435-2461
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Tidskriftsartikel (refereegranskat)abstract
- Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.
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| 16. |
- Lupu, Diana, et al.
(författare)
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Endocrine Disrupting Chemicals and Hippocampal Development : The Role of Estrogen and Androgen Signalling
- 2023
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Ingår i: Neuroendocrinology. - : S. Karger. - 0028-3835 .- 1423-0194. ; 113:12, s. 1193-1214
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Tidskriftsartikel (refereegranskat)abstract
- Hormones are important regulators of key processes during fetal brain development. Thus, the developing brain is vulnerable to the action of chemicals that can interfere with endocrine signals. Epidemiological studies have pointed toward sexually dimorphic associations between neurodevelopmental outcomes, such as cognitive abilities, in children and prenatal exposure to endocrine disrupting chemicals (EDCs). This points toward disruption of sex steroid signalling in the development of neural structures underlying cognitive functions, such as the hippocampus, an essential mediator of learning and memory processes. Indeed, during development, the hippocampus is subjected to the organizational effects of estrogens and androgens, which influence hippocampal cell proliferation, differentiation, dendritic growth and synaptogenesis in the hippocampal fields of Cornu Ammonis and the dentate gyrus. These early organizational effects correlate with a sexual dimorphism in spatial cognition and are subject to exogenous chemical perturbations. This review summarises the current knowledge about the organizational effects of estrogens and androgens on the developing hippocampus and the evidence for hippocampal-dependent learning and memory perturbations induced by developmental exposure to EDCs. We conclude that, while it is clear that sex hormone signalling plays a significant role during hippocampal development, a complete picture at the molecular and cellular levels would be needed to establish causative links between the endocrine modes of action exerted by EDCs and the adverse outcomes these chemicals can induce at the organism level.
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| 17. |
- van der Stel, Wanda, et al.
(författare)
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New Approach Methods (NAMs) Supporting Read-Across : Two Neurotoxicity AOP-based IATA Case Studies
- 2021
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Ingår i: Altex. - : ALTEX Edition. - 1868-596X .- 1868-8551. ; 38:4, s. 615-635
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Tidskriftsartikel (refereegranskat)abstract
- Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides - rotenoids and strobilurins - each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts - including transcriptomics - in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.
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