As a toxin of Ageratina adenophora (A. adenophora), euptox A (9-oxo-10, 11-dehydroageraphorone) is known to cause hepatotoxicity in animals. In this study, we examined the effects of euptox A on mouse liver cells and its underlying mechanisms for the first time. We found that euptox A induced liver cell cycle arrest and apoptosis in a dose-dependent manner mainly by mitochondria -related pathways, with the affected cells characterized by the appearance of DNA fragmentation, membrane blebbing, and chromatin condensation. The results showed that euptox A similarly induced hepatocyte G0 /GI arrest and apoptosis mainly by ROS accumulation and mitochondria-mediated and caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome C and AIF, activation of caspase-3/-9, Bax, as well as suppression of Bcl-2. This paper will provide new insights into the mechanisms involved in liver toxicity caused by euptox A in mice.
The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) have been approved for non-small cell lung cancer. Although EGFR TKIs are less toxic than traditional cytotoxic therapies, they cause many severe idiosyncratic drug reactions. Reactive metabolites can cause cellular damage with the release of danger-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. We tested the ability of afatinib, dacomitinib, erlotinib, gefitinib, and osimertinib to induce the release of DAMPs that activate inflammasomes. Human hepatocarcinoma functional liver cell-4 (FLC-4) cells were used for bioactivation of drugs, and the detection of inflammasome activation was performed with the human macrophage cell line, THP-1 cells. Gefitinib is known to be oxidized to a reactive iminoquinone metabolite. We found that the supernatant from the incubation of gefitinib with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells with gefitinib, the heat shock protein (HSP) 40, 70 and 90 were significantly increased. In addition, activated THP-1 cells secreted high mobility group box 1 (HMGB1) protein. These results support the hypothesis that the reactive iminoquinone metabolite can cause the release of DAMPs from hepatocytes, which in turn, can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by gefitinib, which in some patients, can cause immune-related adverse events.
Trichloroethylene (TCE) as a common organic solvent in industrial production can cause occupational medicamentosa-like dermatitis (OMDT) in some exposed workers. In addition to systemic skin damage, OMDT is also accompanied by severe kidney injury. Our previous studies show that complement (C) plays an important role in immune kidney injury caused by TCE. Specifically, C3 is mainly deposited on glomeruli. Recent studies have found that intracellular complement can be activated by cathepsin L (CTSL) and exert a series of biological effects. The purpose of this study was to explore where C3 on glomeruli comes from and what role it plays. A BALB/c mouse model of skin sensitization induced by TCE in the presence or absence of CTSL inhibitor (CTSLi,10 mg/kg). In TCE sensitization-positive mice, C3 was mainly expressed on podocytes and the expression of CTSL significantly increased in podocytes. Kidney function test and related indicators showed abnormal glomerular filtration and transmission electron microscopy revealed ultrastructure damage to podocytes. These lesions were alleviated in TCE/CTSLi positive mice. These results provide the first evidence that in TCE-induced immune kidney injury, intracellular complement in podocytes can be over-activated by CTSL and aggravates podocytes injury, thereby damaging glomerular filtration function. Intracellular complement activation and cathepsin L in podocytes may be a potential target for treating immune kidney injury induced by TCE.
Coumarin is a dietary-derived substance that is extensively metabolized by human liver to excretable 7-hydroxycoumarin. Although coumarin under daily dietary consumption is generally regarded as nontoxic, the substance is of toxicological and clinical interest because of its potential association with hepatotoxicity, which is especially evident in rats. In this study, the pharmacokinetics of coumarin were modeled after virtual oral administration in humans. The adjusted monitoring equivalents of coumarin, along with the biotransformation of coumarin to o-hydroxyphenylacetic acid (via 3,4-epoxidation) based on reported plasma concentrations from rat studies, were scaled to human coumarin equivalents using known species allometric scaling factors. Using rat and human liver preparations, data on the rapid in vitro metabolic clearance for humans (~50-fold faster than in rats) were obtained for in vitro–in vivo extrapolation. For human physiologically based pharmacokinetic (PBPK) modeling, the metabolic ratios to o-hydroxyphenylacetic acid and 7-hydroxycoumarin were set at minor (0.1) and major (0.9) levels for the total disappearance of coumarin. The resulting modeled plasma concentration curves in humans generated by simple PBPK models were consistent with reported simulated coumarin maximum concentrations. These results provide basic information to simulate plasma levels of coumarin and its primary metabolite 7-hydroxycoumarin or its secondary activated metabolite o-hydroxyphenylacetic acid (via 3,4-epoxidation) resulting from dietary foodstuff consumption. Under the current assumptions, little toxicological impact of coumarin was evident in humans, thereby indicating the usefulness of forward dosimetry using PBPK modeling for human risk assessment.
We aimed to investigate the role of programmed cell death protein 1 (PD-1) and T lymphocytes in the proliferation, apoptosis and secretion of cells from patients and mice with Graves’ disease (GD). The levels of serum hormones, related antibodies and inflammatory cytokines in GD patients were determined by electrochemiluminescence immunoassay and ELISA. The percentages of CD4 and CD8 T-lymphocytes and PD-1 expression were examined by flow cytometry. A GD mouse model, a thyroid follicular epithelial cell, and a CD4+PD-1+, CD4+PD-1- and CD8+PD-1+, CD8+PD-1- T lymphocyte co-culture system were constructed. The viability, apoptosis-related markers, serum hormones, related antibodies and inflammatory cytokines in thyroid follicular epithelial cells were determined by CCK-8, Western blot, qTR-PCR, electrochemiluminescence immunoassay and ELISA. Elevated free thyroid hormones (FT3, FT4), thyroid hormone antibodies (TRAb, TPOAb and TGAb), inflammatory cytokines, and inhibited TSH were observed in GD patients. The percentage of CD4+ T cells was increased, while that of CD8+ T cells was reduced in GD patients. PD-1 expression level was lifted in both CD4+ and CD8+ cells from GD patients. In mouse thyroid follicular epithelial cells co-cultured with CD4+PD-1+ and CD8+PD-1+ T lymphocytes, the cell viability, TH and TRAb levels and inflammatory cytokines level were the highest, while the TSH level and apoptosis were the lowest. PD-1 positive T lymphocytes were able to promote viability and inhibit apoptosis of thyroid follicular epithelial cells, which further caused a more accelerated development of GD.
Acrylonitrile (AN), which is widely utilized in the manufacture of plastics, acrylamide, acrylic fibers, and resins, is also one of main components of cigarette smoke (CS). In this study, we examined the effects of AN on the cell viability and apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cell lines. A cell viability assay confirmed that AN decreased the cell proliferation of JEG-3 and BeWo cells in a dose-dependent manner. Additionally, Western blot assay revealed that protein expression of cyclin D and cyclin E decreased, while protein expression of p21 and p27 increased in response to AN treatment for 48 hr. The changes in reactive oxygen species (ROS) levels in JEG-3 and BeWo cells exposed to AN were also measured by a dichlorofluorescein diacetate (DCFH-DA) assay, which revealed that ROS levels increased in response to AN treatment for 48 hr. Moreover, western blot assay confirmed that AN treatment of JEG-3 and BeWo cells for 4 hr promoted the expression of phosphorylated eukaryotic initiation factor 2 alpha protein (p-eIF2α), C/EBP homologous protein (CHOP) and caspase 12, which are known to be involved in ROS-mediated endoplasmic reticulum stress (ER-stress)-related apoptosis. Overall, the protein expression of p53 and Bax (a pro-apoptosis marker) increased, while the expression of Bcl-xl (an anti-apoptotic marker) decreased and the number of apoptotic cells increased in response to AN treatment for 48 hr. Taken together, these results suggest that AN has the potential to induce apoptosis of JEG-3 and BeWo human choriocarcinoma cancer cells by activating ROS.
The purpose of this study was to evaluate the sensitization potential of 82 compounds classified as volatile and/or semi-volatile organic compounds using the direct peptide reactivity assay (DPRA), given that these chemical compounds have been detected frequently and at high concentrations in a national survey of Japanese indoor air pollution and other studies. The skin sensitization potential of 81 of these compounds was evaluable in our study; one compound co-eluted with cysteine peptide and was therefore not evaluable. Twenty-five of the evaluated compounds were classified as positive. Although all glycols and plasticizers detected frequently and at high concentrations in a national survey of Japanese indoor air pollution were negative, hexanal and nonanal, which are found in fragrances and building materials, tested positive. Monoethanolamine and 1,3-butanediol, which cause clinical contact dermatitis, and several compounds reported to have weak sensitization potential in animal studies, were classified as negative. Thus, it was considered that compounds with weak sensitization potential were evaluated as negative in the DPRA. Although the sensitization potential of the formaldehyde-releasing preservative bronopol has been attributed to the release of formaldehyde (a well-known contact allergen) by its degradation, its degradation products—bromonitromethane and 2-bromoethanol—were classified as positive, indicating that these degradation products also exhibit sensitization potential. The compounds that tested positive in this study should be comprehensively assessed through multiple toxicity and epidemiological studies.