Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals
Abstract:Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two… Show more
“…8d). This observation in neurons agrees well with our earlier findings in HepG2 cells, where this compound was identified as being mis-classified and indeed being an uncoupler (van der Stel et al 2020 ). The uncoupling action was also found here for neuronal mitochondria at a cyazofamid concentration of 50 µM (Suppl.…”
Section: Resultssupporting
confidence: 93%
“…These data are fully concordant with our data from liver and kidney cells, where capsaicin showed only faint mitochondrial effects, and had an IC50 of > 200 µM for inhibition of cI (Fig. 8 a) (van der Stel et al 2020 ). Fig.…”
Section: Resultssupporting
confidence: 92%
“…To select a typical cI gene set, we chose deguelin as gold standard. This compound showed strong responses in all KE assays, is a well-characterized, typical cI inhibitor (Delp et al 2019 ; van der Stel et al 2020 ) and behaves less extreme than rotenone. We selected all 13 genes that were differentially regulated by deguelin at its EC 10 V (13 µM), and at the next lower concentration (3.25 µM), to make sure that no unspecific indicators of cell death were included.…”
Section: Resultsmentioning
confidence: 98%
“…In summary, the SH-SY5Y MMP assay proved to be most suitable to test mitochondrial dysfunction. We observed earlier that different cell types can give different MMP data and each test system requires a lot of method adaptations to yield robust results (van der Stel et al 2020 ). The LUHMES assay did not work under conditions that were optimal for SH-SY5Y cells.…”
Section: Resultsmentioning
confidence: 99%
“…Figure 1). In the context of the European Research project EU-ToxRisk (Daneshian et al 2016 ; Escher et al 2019 ; Graepel et al 2019 ), a set of 21 compounds was used to establish assays for mitochondrial respiratory chain functionality in neurons (Delp et al 2019 ) and for testing in different non-neuronal cell systems (representing liver and kidney) (van der Stel et al 2020 ). It could not be clarified in these studies, how the different modes of mitochondrial respiratory chain inhibition related to (or predicted) dopaminergic neurotoxicity, and how different assays of AOP:3 key events were correlated.…”
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.
“…8d). This observation in neurons agrees well with our earlier findings in HepG2 cells, where this compound was identified as being mis-classified and indeed being an uncoupler (van der Stel et al 2020 ). The uncoupling action was also found here for neuronal mitochondria at a cyazofamid concentration of 50 µM (Suppl.…”
Section: Resultssupporting
confidence: 93%
“…These data are fully concordant with our data from liver and kidney cells, where capsaicin showed only faint mitochondrial effects, and had an IC50 of > 200 µM for inhibition of cI (Fig. 8 a) (van der Stel et al 2020 ). Fig.…”
Section: Resultssupporting
confidence: 92%
“…To select a typical cI gene set, we chose deguelin as gold standard. This compound showed strong responses in all KE assays, is a well-characterized, typical cI inhibitor (Delp et al 2019 ; van der Stel et al 2020 ) and behaves less extreme than rotenone. We selected all 13 genes that were differentially regulated by deguelin at its EC 10 V (13 µM), and at the next lower concentration (3.25 µM), to make sure that no unspecific indicators of cell death were included.…”
Section: Resultsmentioning
confidence: 98%
“…In summary, the SH-SY5Y MMP assay proved to be most suitable to test mitochondrial dysfunction. We observed earlier that different cell types can give different MMP data and each test system requires a lot of method adaptations to yield robust results (van der Stel et al 2020 ). The LUHMES assay did not work under conditions that were optimal for SH-SY5Y cells.…”
Section: Resultsmentioning
confidence: 99%
“…Figure 1). In the context of the European Research project EU-ToxRisk (Daneshian et al 2016 ; Escher et al 2019 ; Graepel et al 2019 ), a set of 21 compounds was used to establish assays for mitochondrial respiratory chain functionality in neurons (Delp et al 2019 ) and for testing in different non-neuronal cell systems (representing liver and kidney) (van der Stel et al 2020 ). It could not be clarified in these studies, how the different modes of mitochondrial respiratory chain inhibition related to (or predicted) dopaminergic neurotoxicity, and how different assays of AOP:3 key events were correlated.…”
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.
Cooking oil fumes (COFs) are main pollutants in kitchen and indoor air, which threaten human health.Exposure to COFs can cause respiratory diseases and impair pulmonary function. To investigate the toxicity of COFs on human bronchial epithelial cells (Beas-2B) and explore the underlying mechanisms, MTT assay was conducted to detect the viability of Beas-2B. Intracellular reactive oxygen species (ROS) levels and nitric oxide (NO) levels were determined with DCFH-DA assay and DAF-FM assay. The expression of genes involved in in ammation were measured with quantitative real-time PCR (qRT-PCR).The phosphorylation and the expression of proteins related to Mitogen-activated protein kinase (MAPK), NF-κB signaling pathways were measured with western blot. Our results revealed that COFs decreased Beas-2B cells viability, increased the ROS levels and NO levels in cells and induced apoptosis in Beas-2B cells. The results of qRT-PCR and western blot showed that the expression of NLRP3, p65, iNOS, IL-1β, and the factors related to oxidative stress and in ammation increased, NF-κB signaling pathway and MAPK signaling pathway were activated. This study provided some useful information to alleviate the toxicity of COFs and revealed the possible mechanism for the damage on respiratory system induced by COFs. et al. 2015;Wang et al. 2018).COFs pollution produced by cooking usually includes ketones, aldehydes, hydrocarbons, fatty acids, alcohols, aromatic compounds, esters, heterocycles, and lactones is a serious health risk in china due to the cooking manners (Ding et al. 2020;Lee and Gany 2013). In China alone, the number of people exposed to COFs in 2018 has reached to 240 million and most of whom were middle-aged women . Women exposed to COFs are at higher risk for chronic bronchitis, for pregnant women, such exposure can also affect the fetus and increase the risks of adverse birth outcomes (Hou et al. 2018;Vavalà et al. 2014;Zhu et al. 2019). Studies have shown that exposure to COFs increased the risk of respiratory diseases, such as airway infection, chronic obstructive pulmonary disease, tuberculosis and
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