The risk assessment of endocrine-disrupting chemicals (EDCs) greatly relies on in vitro screening. A 3-dimensional (3D) in vitro prostate model that can reflect physiologically-relevant prostate epithelial and stromal crosstalk can significantly advance the current androgen assessment. This study built a prostate epithelial and stromal co-culture microtissue model with BHPrE and BHPrS cells in scaffold-free hydrogels. The optimal 3D co-culture condition was defined, and responses of the microtissue to androgen (dihydrotestosterone, DHT) and anti-androgen (flutamide) exposure were characterized using molecular and image profiling techniques. The co-culture prostate microtissue maintained a stable structure for up to seven days and presented molecular and morphological features of the early developmental stage of the human prostate. The cytokeratin 5/6 (CK5/6) and cytokeratin 18 (CK18) immunohistochemical staining indicated epithelial heterogeneity and differentiation in these microtissues. The prostate-related gene expression profiling did not efficiently differentiate androgen and anti-androgen exposure. However, a cluster of distinctive 3D image features was identified and could be applied in the androgenic and anti-androgenic effect prediction. Overall, the current study established a co-culture prostate model that provided an alternative strategy for (anti-)androgenic EDC safety assessment and highlighted the potential and advantage of utilizing image features to predict endpoints in chemical screening.

4-Vinylcyclohexene diepoxide (VCD) is a hazardous industrial material which is widely used in the production of fragrances, rubber tires, antioxidants, pesticides, flame retardants and plasticizers. Previous studies have shown that exposure to VCD damages the female reproductive system, but the effects and mechanisms of VCD exposure on human granulosa cells are not reported. In this study, we used a human granulosa cell line (SVOG) to explore the effects of VCD exposure and found that VCD exposure had toxic effects on SVOG cells in vitro. VCD exposure led to excessive accumulation of intracellular ROS, caused DNA damage in cells, altered the expression of some key genes related with apoptosis and oxidative stress, and ultimately inhibited the proliferative capacity of granulosa cells, resulting in increased apoptosis. Overall, our findings provide solid evidence showing that VCD exposure produces severe damage to human granulosa cells, which is helpful for understanding the reproductive toxicity of VCD and etiology of infertility.

Salix babylonica L. is a species of the willow tree. Insulinoma is a tumor originating from pancreatic beta cells. This study aims to research the effect of different fractions of Salix babylonica L. leaf extract on INS-1 cells for treating pancreatic tumors. Cell death occurred at lower doses in the EtOAc fraction. The cells are functional in the BuOH fraction but not in EtOAc and H₂O fractions. The EtOAc fraction has a higher percentage of necrosis and ROS. INS1, INS2, and AKT gene expressions in the H₂O fraction, GLUT2, IR, HSP70 gene expressions, and WNT4 protein levels increased in the BuOH fraction. HSP90 gene expression, Beta-actin, GAPDH, insulin, HSP70, HSP90, HSF1, Beta-Catenin, and WNT7A protein levels were decreased, while IR immunolabelling intensity increased in both fractions. Ca⁺², K⁺, Na⁺, and CA-19-9 in the cell, Ca⁺² and K⁺ in secretion increased. The secondary metabolites in the EtOAc fraction cause more damage in INS-1 cells. Since the water fraction also causes the cells to die in high doses, cell function is damaged. The secondary metabolites in the BuOH fraction kill INS-1 cells with less damage. This makes the BuOH fraction of Salix babylonica L. more valuable.

The aims were to characterize the content of elements and polycyclic aromatic hydrocarbons (PAHs) in size-separated particulate matter (PM) sampled in a road tunnel, estimate the contribution of PAHs to the toxic potential, and measure the pro-inflammatory potential of PM samples and extracts with increasing polarity. Several elements/metals previously associated with cytokine responses were found. Based on PAHs levels and published PAHs potency, the calculated mutagenic and carcinogenic activities of size-separated samples were somewhat lower for coarse than fine and ultrafine PM. The AhR-activity of the corresponding PM extracts measured in an AhR-luciferase reporter model (human hepatocytes) were more similar. The highest AhR-activity was found in the neutral (parent and alkylated PAHs) and polar (oxy-PAHs) fractions, while the semi-polar fractions (mono-nitrated-PAHs) had only weak activity. The neutral and polar aromatic fractions from coarse and fine PM were also found to induce higher pro-inflammatory responses and CYP1A1 expression in human bronchial epithelial cells (HBEC3-KT) than the semi-polar fractions. Fine PM induced higher pro-inflammatory responses than coarse PM. AhR-inhibition reduced cytokine responses induced by parent PM and extracts of both size fractions. Contributors to the toxic potentials include PAHs and oxy-PAHs, but substantial contributions from other organic compounds and/or metals are likely.

Aflatoxins have been recognized as the most harmful mycotoxins leading to various toxic effects. The present study aims to determine the inhibition behavior of aflatoxins on the activity of the important phase II metabolizing enzymes, UDP-glucuronosyltransferases (UGTs), based on in vitro incubation system of recombinant human UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU). 100 μM AFB1 and AFG1 exhibited extensive inhibition towards UGT isoforms especially UGT1A7 and UGT1A8, with the inhibition ratios to be 71.38%, 72.95% and 72.79% for AFB1 to UGT1A7, AFB1 to UGT1A8 and AFG1 to UGT1A8, respectively. Molecular docking results showed that hydrogen bonds and hydrophobic contacts of the particular structure consisting of double furan ring with double bond contributed to the interaction of aflatoxins and UGTs. Kinetics analysis, including inhibition types and kinetics parameters (K_i), and in vitro-in vivo extrapolation (IVIVE) indicated that there might be a medium possibility of inhibition on UGTs by aflatoxins in vivo. In conclusion, the present study indicated that aflatoxins could possibly disturb endogenous metabolism by inhibiting the activity of UGTs so as to exhibit toxic effects.

Polo-like kinase 1 (PLK1) is crucial in regulating cell division and has been shown to have an oncogenic function in several cancers. Since PLK1 overexpression is closely related to tumorigenesis and has been correlated with poor clinical outcomes, specific inhibition of PLK1 in cancer cells is a promising approach for developing new anticancer drugs. In this context, the aim of the present study was to evaluated the potential cytotoxic effects of GSK461364A, a competitive inhibitor for PLK1, in gastric cancer cell line SNU-1 cells and explored its cytotoxic mechanism. The cells were exposed to GSK461364A at different concentrations ranging from 1 to 40 μM for 24 h, and it showed considerable cytotoxicity with an IC₅₀ value of 4.34 μM. The treatment of SNU-1 cells with GSK461364A results in cell cycle arrest at the G2/M phase, decreased mitochondrial membrane potential, and increased apoptosis as indicated by Annexin V binding assay. In addition, GSK461364A treatment significantly increased the total oxidant (TOS) level, a signal of oxidative stress, and increased cleaved PARP and 8-oxo-dG levels as an indicator of DNA damage. ELISA experiments evaluating Bax, BCL-2, and cleaved caspase 3 also confirmed the apoptotic effect of GSK461364A. Current findings suggest that GSK461364A may be a chemotherapeutic agent in patients with gastric cancer. Nevertheless, more research is needed to evaluate GSK461364A as a cancer treatment drug.

Telomere length may be maintained by telomerase nucleoprotein complex and shelterin complex, namely TRF1, TRF2, TIN2, TPP1, POT1 and RAP1 proteins and modulated by TERRA expression. Telomere loss is observed during progression of chronic myeloid leukemia (CML) from the chronic phase (CML-CP) to the blastic phase (CML-BP). The introduction of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), has changed outcome for majority of patients, however, a number of patients treated with TKIs may develop drug resistance. The molecular mechanisms underlying this phenomenon are not fully understood and require further investigation. In the present study, we demonstrate that IM-resistant BCR::ABL1 gene-positive CML K-562 and MEG-A2 cells are characterized by decreased telomere length, lowered protein levels of TRF2 and RAP1 and increased expression of TERRA in comparison to corresponding IM-sensitive CML cells and BCR::ABL1 gene-negative HL-60 cells. Furthermore, enhanced activity of glycolytic pathway was observed in IM-resistant CML cells. A negative correlation between a telomere length and advanced glycation end products (AGE) was also revealed in CD34⁺ cells isolated from CML patients. In conclusion, we suggest that affected expression of shelterin complex proteins, namely TRF2 and RAP1, TERRA levels, and glucose consumption rate may promote telomere dysfunction in IM-resistant CML cells.

Triphenyl phosphate (TPhP) is one of the most commonly found organophosphorus flame retardants (OPFRs) in the environment and the general population. Continuous daily exposure to TPhP may adversely impact male reproductive health. However, few researches were conducted to investigate the direct effects of TPhP on the progress of sperm growth and development. In this study, mouse spermatocyte GC-2spd (GC-2) cells were selected as an in vitro model, the impact of oxidative stress, mitochondrial impairment, DNA damage, cell apoptosis and the related molecular mechanisms were investigated using high content screening (HCS) system. Our study indicated that cell viability was decreased significantly in a dose-dependent manner after TPhP treatment with the half lethal concentration (LC₅₀) at 105.8, 61.61 and 53.23 μM for 24, 48 and 72 h. A concentration-related apoptosis occurrence was observed in GC-2 cells after TPhP exposure for 48 h. In addition, the elevated intracellular reactive oxygen species (ROS) and the total antioxidant capacity (T-AOC) also observed after exposing to 6, 30 and 60 μM of TPhP. Furthermore, based on the enhancement of pH2AX protein and alteration of nuclear morphology or DNA content, DNA damage might be induced by higher concentration of TPhP treatment. Simultaneously, alteration of mitochondrial structure, enhancement of mitochondrial membrane potential (MMP), reduction of cellular adenosine triphosphate (ATP) content, altered expression of Bcl-2 family proteins, release of cytochrome c and increase of caspase-3 and caspase-9 activity demonstrated that caspase-3 dependent mitochondrial pathway might play a key role in the process of GC-2 cell apoptosis. Taken together, these results showed that TPhP was a mitochondrial toxicant and apoptotic inducer, which might trigger alike responses in human spermatogenic cells. Therefore, the potential reproductive toxicity of TPhP should not be ignored.

Flutamide is a non-steroidal anti-androgen agent, which is mainly used for the treatment of prostate cancer. Flutamide is known to cause severe adverse events, which includes idiosyncratic liver injury. However, details of the mechanism of these adverse reactions have not been elucidated. We investigated whether flutamide induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested bicalutamide, enzalutamide, apalutamide, and darolutamide for their ability to activate inflammasomes in differentiated THP-1 cells. The supernatant from the incubation of flutamide and bicalutamide with human hepatocarcinoma functional liver cell-4 (FLC-4) cells increased caspase-1 activity and production of IL-1ß by differentiated THP-1 cells. In the supernatant of FLC-4 cells with flutamide and bicalutamide, the heat shock protein (HSP) 40 or 60 was significantly increased. Addition of a carboxylesterase or a CYP inhibitor to the FLC-4 cells prevented release of HSPs from the FLC-4 cells. These results suggested that the reactive metabolites of flutamide and bicalutamide can cause the release of DAMPs from hepatocytes and activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by flutamide or bicalutamide, which in some patients, can cause immune-related adverse events.