Toxicology in Vitro最新文献

In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK−2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.

Chloroquine (CQ) is widely used in the therapy against malarial, tumor and recently the COVID-19 pandemic, as a lysosomotropic agent to inhibit the endolysosomal trafficking in the autophagy pathway. We previously reported that CQ (20 μM, 36 h) could reprogram transcriptome, and impair multiple signaling pathways vital to porcine immature Sertoli cells (iSCs). However, whether CQ treatment could affect the metabolomic compositions of porcine iSCs remains unclear. Here, we showed that CQ (20 μM, 36 h) treatment of porcine iSCs induced significant changes of 63 metabolites (11 up and 52 down) by the metabolomics method, which were involved in different metabolic pathways. Caffeic acid and esculetin, the top two up-regulated metabolites, were validated by ELISA. The combined analysis of metabolomics and transcriptome showed caffeic acid and esculetin to be highly correlated with multiple differentially expressed genes (DEGs), including Ndrg1, S100a8, Sqstm1, S100a12, S100a9, Ill1, Lif, Ntn4 and Peg10. Furthermore, esculetin treatment (53 nM, 36 h) significantly decreased the viability and proliferation, suppressed the mitochondrial function, whereas promoted the apoptosis of porcine iSCs, similar to those by CQ treatment (20 μM, 36 h). Collectively, our results showed that CQ treatment induces metabolic changes, and its effect on porcine iSCs could be partially mediated by esculetin.

The pathogenesis of Parkinson's disease (PD) involves abnormalities in the metabolism of catecholamines. The enzyme quinone reductase 2 (NQO2) reduces quinone derivatives of catecholamines, which promotes the formation of reactive oxygen species (ROS), suggesting a role for NQO2 in the development of cellular damage typical of PD. In the present study, we investigated the relationship between 6-hydroxydophamine (6-OHDA) induced cellular damage and NQO2 activity and its levels in SH-SY5Y cell culture to establish an experimental model to evaluate the pharmacological properties of NQO2 inhibitors. Cellular damage was evaluated using the MTT and comet assays. It was shown that oxidative damage of SH-SY5Y cells upon incubation with 6-OHDA for 6, 12 and 24 h was accompanied by an increase in NQO2 activity. The increase in NQO2 protein level in SH-SY5Y cells was observed 24 h after incubation with 6-OHDA at concentrations of 50 and 100 μM. Oxidative damage of SH-SY5Y cells upon 1 h incubation with 6-OHDA is increased in the presence of the selective enzyme co-substrate 1-benzyl-1,4-dihydronicotinamide (BNAH), but is not accompanied by changes in NQO2 activity and protein levels. The data obtained demonstrate the contribution of NQO2 to the cytotoxic mechanism of 6-OHDA action.

Chlorpyrifos (CPF) is one of the organophosphorus pesticides widely used throughout the world. Epidemiological studies suggested a link between CPF exposure and neurologic disorders, while the molecular mechanisms remain inconclusive. In the present study, we investigated the impacts of chlorpyrifos-oxon (CPO), the major toxic CPF metabolite, on cell apoptosis, and explored possible mechanism associated with endoplasmic reticulum (ER) stress in SH-SY5Y cells. Results showed that CPO exposure induced dose-dependent apoptosis and expression of ER stress-related proteins in SH-SY5Y cells. Pretreatment with 4-PBA (an ER stress inhibitor) effectively inhibited the expression of GRP78, GRP94, p-IRE1α, and XBP1-s, and apoptotic events. Pretreatment with STF-083010 (an IRE1α inhibitor) partially attenuated CPO-induced apoptosis. In addition, CPO exposure significantly evoked the generation of reactive oxygen species (ROS) which could be eliminated by pretreatment of 4-PBA. Of note, buffering the ROS generation with antioxidant NAC had little impact on the expression of p-IRE1α, and only partially attenuated CPO-induced apoptosis. In contrast, co-pretreatment with NAC and STF-083010 effectively inhibited CPO-induced apoptotic events. Collectively, our results indicate that CPO exposure exerts neuronal cytotoxicity via ER stress downstream-regulated IRE1α/XBP1 signaling pathway and ROS generation-triggered apoptosis. These findings highlight the role of ER stress in CPF-induced neurotoxicity, and provide a promising target for the intervention of organophosphate-associated neurodegenerative diseases.

The general population is exposed to many chemicals which have putative, but incompletely understood, links to breast cancer. Cell Painting is a high-content imaging-based in vitro assay that allows for unbiased measurements of concentration-dependent effects of chemical exposures on cellular morphology. We used Cell Painting to measure effects of 16 human exposure relevant chemicals, along with 21 small molecules with known mechanisms of action, in non-tumorigenic mammary epithelial cells, the MCF10A cell line. Using CellProfiler image analysis software, we quantified 3042 morphological features across approximately 1.2 million cells. We used benchmark concentration modeling to identify features both conserved and different across chemicals. Benchmark concentrations were compared to exposure biomarker concentration measurements from the National Health and Nutrition Examination Survey to assess which chemicals induce morphological alterations at human-relevant concentrations. We found significant feature overlaps between chemicals, including similarities between the organochlorine pesticide DDT metabolite p,p’-DDE and an activator of Wnt signaling CHIR99201. We validated these findings by assaying the activation of Wnt, as reflected by translocation of ꞵ-catenin, following p’-p’ DDE exposure. Consistent with Wnt signaling activation, low concentration p’,p’-DDE (25 nM) significantly enhanced the nuclear translocation of ꞵ-catenin. Overall, these findings highlight the ability of Cell Painting to enhance mode-of-action studies for toxicants which are common in our environment but incompletely characterized with respect to breast cancer risk.

While many plastic additives show endocrine disrupting properties, this has not been studied for micro- and nanoplastics (MNPs) particles despite their ubiquitous presence in humans. The objective of this study was to determine the effects of various sizes and concentrations of polystyrene (PS)-MNPs (50–10,000 nm, 0.01–100 μg/mL) on estrogen- and androgen receptor (ER and AR) activity and steroidogenesis in vitro. Fluorescent (F)PS-MNPs of ≤1000 nm were internalized in VM7 and H295R cells and FPS-MNPs ≤200 nm in AR-ecoscreen cells. H295R cells displayed the highest uptake and particles were closer to the nucleus than other cell types. None of the sizes and concentrations PS-MNPs tested affected ER or AR activity. In H295R cells, PS-MNPs caused some statistically significant changes in hormone levels, though these showed no apparent concentration or size-dependent patterns. Additionally, PS-MNPs caused a decrease in estriol (E3) with a maximum of 37.5 % (100 μg/mL, 50 nm) and an increase in gene expression of oxidative stress markers GPX1 (1.26-fold) and SOD1 (1.23-fold). Taken together, our data show limited endocrine-disrupting properties of PS-MNPs in vitro. Nevertheless the importance of E3 in the placenta warrants further studies in the potential effects of MNPs during pregnancy.

Folates are essential nutrients for fetal development during pregnancy. Valproic acid (VPA), an inhibitor of histone deacetylases (HDACs), alters the expression of folate carriers in placental cells; however, the underlying mechanisms remain unclear. Here, we aimed to determine the profiles of folate carriers (folate receptor alpha [FOLR1], solute carrier [SLC]-19A1, and SLC46A1) after inhibition of HDACs, especially class I and IIa HDACs, using different inhibitors and gene knockdown tests. Quantitative polymerase chain reaction revealed that BeWo cells (a trophoblast model) expressed HDACs and folate carriers, similar to human placental villi. FOLR1 expression was upregulated by VPA, apicidin, and trichostatin A, but downregulated by MS-275 after 24 h treatment. VPA and apicidin upregulated the expression of SLC46A1. These inhibitors downregulated SLC19A1 expression. TMP269 (a class IIa inhibitor) did not affect folate carrier levels. HDAC1/2 knockdown upregulated FOLR1 and SLC46A1 levels, whereas HDAC1/3 knockdown downregulated FOLR1 levels. Our findings suggest that the pharmacological inhibition of class I HDACs alters the expression of folate carriers in BeWo cells. By contrast, HDAC inhibitors exert different regulatory effects on folate carriers. Moreover, HDAC1/2 inhibition may be a potential mechanism involved in altering FOLR1 and SLC46A1 levels.

Risperidone (RIS) is a widely used antipsychotic drug with reported alteration in immune response. The current study investigated mitochondrial disruption as the underlying mechanism of RIS-induced immunotoxicity in isolated human peripheral blood monocytes (hPBM). RIS was cytotoxic to hPBM in exposure duration and concentration-dependent patterns. Functionally, RIS was shown to increase the release of IL-6, TNF-α, and IL-8 with a decrease in test particle phagocytosis in concertation and exposure time-based patterns. It was found that RIS decreased ATP production in isolated monocytes' mitochondria, with an estimated EC50 of around 70 μM after 24 h with parallel inhibition of mitochondrial complexes I and III activities and decreased mitochondrial membrane potential and oxygen consumption rates with increased lactate production from by the treated cells in comparison to controls. Structurally, RIS in 100 μM concentration significantly increased the mitochondrial membrane fluidity with significant increase in increased unsaturated/saturated fatty acids ratios of the mitochondrial membranes of the treated cells. Interestingly, water-soluble CoQ10 formulation significantly decreased the cytotoxic effect of RIS and improved the phagocytic activity of RIS-treated cells. To conclude, the current data suggests mitochondrial disruption as the underlying mechanism of RIS-induced immunotoxicity with shown protective effect of water-soluble CoQ10 formulation.

Repeated dietary dose testing is used to assess longer term toxicity of chemicals, such as pesticides, to mammals. However, the internal pesticide concentration varies significantly as feeding rate relative to body size fluctuates over time. Toxicokinetic-toxicodynamic (TK-TD) models can estimate internal toxicant concentration over time and link this directly to observed effects on endpoints such as the growth rate of laboratory rats. Using TK-TD models it is therefore possible to predict the effects that would result from a constant internal concentration of a pesticide. This presents the possibility of comparison with data from in vitro experiments, potentially facilitating quantitative in vitro to in vivo extrapolation (QIVIVE). We used in vivo TK-TD models to identify relevant internal concentrations and then estimated the experimental conditions required to replicate these in cultured cells, using in vitro TK models. Cell population growth was measured, with a view to extrapolating through time and comparing effect sizes with in vivo predictions. However, observed cell proliferation was not significantly affected by the tested concentrations of any of the five pesticides in this study and so extrapolation was not possible. In light of this negative result, we highlight areas for future work towards QIVIVE of graded sublethal effects in mammals. The most pressing objective is improving the accuracy of in vivo TK predictions, which could be achieved with dietary dosing in TK studies.