A novel support vector machine-based 1-day, single-dose prediction model of genotoxic hepatocarcinogenicity in rats.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Comparative analysis of immunological changes following realgar and arsenic trioxide treatments in a murine model of myelodysplastic syndrome.

BACKGROUND: Myelodysplastic syndrome (MDS) is a prevalent hematological neoplastic disorder in clinics and its immunopathogenesis has garnered growing interest. Oral and intravenous arsenic agents have long been used to treat hematological malignancies. The main component of oral arsenic is realgar (arsenic disulfide), while arsenic trioxide is the main component of intravenous arsenic. METHODS: This study aimed to assess the effects of ATO and Realgar on the enhancement of peripheral blood, drug safety, and T cell immune status in the NUP98-HOXD13 (NHD13) mice model of MDS, specifically in the peripheral blood, spleen, and liver. RESULTS: The study findings indicate that realgar and arsenic trioxide (ATO) can improve peripheral hemogram in mice, whereas realgar promotes higher peripheral blood cell production than ATO. Furthermore, the clinical administration method and dose did not cause significant toxicity or side effects and thus can be considered safe. Coexistence and interconversion of hyperimmune function and immunosuppression in mice were also observed in this study. In addition, there were interactions between immune cells in the peripheral blood, spleen, and liver to regulate the immune balance of the body and activate immunity via T-cell activation. CONCLUSION: In summary, oral and intravenous arsenic agents are beneficial in improving peripheral hemogram and immunity in mice.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

DNA Methylation Aberrations in Dimethylarsinic Acid-Induced Bladder Carcinogenesis.

Arsenic is a known human urinary bladder carcinogen. While arsenic is known to cause aberrant DNA methylation, the mechanism of arsenic-triggered bladder carcinogenesis is not fully understood. The goal of this study was to identify aberrant DNA methylation in rat bladder urothelial carcinoma (UC) induced by dimethylarsinic acid (DMAV), a major organic metabolite of arsenic. We performed genome-wide DNA methylation and microarray gene expression analyses of DMAV-induced rat UCs and the urothelium of rats treated for 4 weeks with DMAV. We identified 40 genes that were both hypermethylated and downregulated in DMAV-induced rat UCs. Notably, four genes (CPXM1, OPCML, TBX20, and KCND3) also showed reduced expression in the bladder urothelium after 4 weeks of exposure to DMAV. We also found that CPXM1 is aberrantly methylated and downregulated in human bladder cancers and human bladder cancer cells. Genes with aberrant DNA methylation and downregulated expression in DMAV-exposed bladder urothelium and in DMAV-induced UCs in rats, suggest that these alterations occurred in the early stages of arsenic-induced bladder carcinogenesis. Further study to evaluate the functions of these genes will advance our understanding of the role of aberrant DNA methylation in arsenic bladder carcinogenesis, and will also facilitate the identification of new therapeutic targets for arsenic-related bladder cancers.

Jujuboside B suppresses angiogenesis and tumor growth via blocking VEGFR2 signaling pathway.

Jujuboside B (JuB), one of the main active triterpenoid saponins from the traditional Chinese medicine Ziziphus jujuba, possesses a wide range of pharmacological activities. However, it is unknown whether JuB can inhibit tumor angiogenesis, a crucial step in solid tumor growth. In this study, we found that JuB significantly inhibited the proliferation, migration, and tube formation of human umbilical vein endothelial cells in a dose-dependent manner. JuB also suppressed angiogenesis in chick embryo chorioallantoic membranes and Matrigel plugs. Moreover, through angiogenesis inhibition, JuB delayed the growth of human HCT-15 colorectal cancer xenograft in mice. Western blot assay demonstrated that JuB inhibited the phosphorylation of VEGFR2 and its key downstream protein kinases, such as Akt, FAK, Src, and PLCγ1. In conclusion, the antiangiogenic potency and molecular mechanism of JuB are revealed for the first time, indicating that this triterpene saponin may be further explored as a potential drug candidate or lead compound for antiangiogenic cancer therapy.

Activation of Delta-Opioid Receptor Protects ARPE19 Cells against Oxygen-Glucose Deprivation/Reoxygenation-Induced Necroptosis and Apoptosis by Inhibiting the Release of TNF-α.

Purpose: Retinal ischemia-reperfusion injury (RIRI) is the basis of the pathology that leads to many retinal diseases and induces necroptosis and apoptosis. Tumor necrosis factor-α (TNF-α) is critically involved in necroptosis and apoptosis. Delta-opioid receptor (DOR) activation inhibits TNF-α release in our previous studies, it might prevent necroptosis and apoptosis by inhibiting the release of TNF-α. However, the role of TNF-α and DOR in necroptosis and apoptosis of retinal pigment epithelial (RPE) cells remains largely unknown. Here, we explored the mechanisms of TNF-α and DOR in necroptosis and apoptosis using an oxygen-glucose deprivation/reoxygenation (OGD/R) model of adult retinal pigment epithelial cell line-19 (ARPE19) cells. Materials and Methods: ARPE19 cells were exposed to OGD/R conditions to mimic RIRI in vitro. Cell viability was quantified using the Cell Counting Kit-8 (CCK-8) assay. Morphological changes were observed by inverted microscopy. TNF-α protein levels in cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). The DOR agonist TAN-67 and antagonist naltrindole (NTI) were used to pretreat cells for 1 or 2 hours before OGD24/R36 administration. Calcein acetoxymethylester/propidium iodide (Calcein-AM/PI) and Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to detect necroptotic and apoptotic ARPE19 cells, respectively. The protein expression of DOR, p-RIP1 (RIP1), p-RIP3 (RIP3), p-MLKL (MLKL), and cleaved Caspase3 (Caspase3) was measured by western blotting. Results: OGD severely damaged ARPE19 cells. Prolonged reoxygenation significantly increased TNF-α level and decreased DOR expression in ARPE19 cells. Pretreatment with the DOR agonist TAN-67 (10 µM) significantly improved ARPE19 cell viability after OGD24/R36 by reducing the number of necroptotic and apoptotic cells. Furthermore, DOR activation significantly inhibited TNF-α release and suppressed the expression of proteins related to necroptosis and apoptosis, including p-RIP1, p-RIP3, p-MLKL, and cleaved Caspase3, after OGD24/R36. This effect was reversed by the DOR antagonist NTI. Conclusion: These results strongly suggest that DOR activation inhibits necroptosis and apoptosis by decreasing TNF-α release, leading to the prevention of OGD/R-induced injury in ARPE19 cells. This study provides an innovative idea for clinical treatment strategies for retinal damage and vision loss due to RIRI.

Electroacupuncture ameliorates inflammatory response induced by retinal ischemia-reperfusion injury and protects the retina through the DOR-BDNF/Trkb pathway.

Objectives: Retinal ischemia-reperfusion injury (RIRI) is the common pathological basis of many ophthalmic diseases in the later stages, and inflammation is the primary damage mechanism of RIRI. Our study aimed to assess whether electroacupuncture (EA) has a protective effect against RIRI and to elucidate its related mechanisms. Methods: A high-intraocular pressure (HIOP) model was used to simulate RIRI in Wistar rats. EA was applied to the EA1 group [Jingming (BL1) + Shuigou (GV26)] and the EA2 group [Jingming (BL1) + Hegu (LI4)] respectively for 30 min starting immediately after the onset of reperfusion and repeated (30 min/time) at 12 h and then every 24 h until days 7 after reperfusion. The pathological changes in the retina were observed by H and E staining after HIOP. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was utilized to observe retinal cell apoptosis. The mRNA expression of IL1-ß, TNF-α, IL-4, IL-10, δ-opioid receptor (DOR), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase B (TrkB) in the retina was measured by quantitative real-time PCR. Results: HIOP caused structural disorders of the retina, decreased RGCs, and increased retinal cell apoptosis. At 1 and 3 days of RIRI, retinal apoptotic cells in the EA group were significantly reduced, while there was no distinct difference in the EA group compared with the HIOP group at 7 days of RIRI. Compared with that in the HIOP group, the expression of anti-inflammatory factors, DOR and TrkB was increased, and the expression of pro-inflammatory factors was decreased in the EA group. In contrast, HIOP had no appreciable effect on BDNF expression. Conclusion: EA at Jingming (BL1) and Shuigou (GV26) or at Jingming (BL1) and Hegu (LI4) may inhibit RIRI induced inflammation through activating the DOR-BDNF/TrkB pathway to protect the retina, especially the pair of Jingming (BL1) and Shuigou (GV26) has better inhibitory effects on inflammation.

Saikosaponin A, a Triterpene Saponin, Suppresses Angiogenesis and Tumor Growth by Blocking VEGFR2-Mediated Signaling Pathway.

Saikosaponin A (SSA), a main triterpenoid saponin component from Radix Bupleurum, has been revealed to have a variety of pharmacological activities. However, whether SSA can inhibit angiogenesis, a key step in solid tumor progression, remains unknown. In this study, we demonstrated that SSA could powerfully suppress the proliferation, migration, and tube formation of human umbilical vein endothelial cells. SSA also significantly inhibited angiogenesis in the models of the chick embryo chorioallantoic membrane and Matrigel plugs. Moreover, SSA was found to inhibit tumor growth in both orthotopic 4T1 breast cancer and subcutaneous HCT-15 colorectal tumor by the inhibition of tumor angiogenesis. Western blot assay indicated the antiangiogenic mechanism of SSA in the suppression of the protein phosphorylation of VEGFR2 and the downstream protein kinase including PLCγ1, FAK, Src, and Akt. In summary, SSA can suppress angiogenesis and tumor growth by blocking the VEGFR2-mediated signaling pathway.

A modified four vessel occlusion model of global cerebral ischemia in rats.

BACKGROUND: To develop and evaluate a modified four vessel occlusion (4VO) model of global cerebral ischemia-reperfusion (GCI/R) in rats based on the Pulsinelli and Brierley's method. NEW METHODS: Vertebral arteries (VAs) were isolated and then permanently ligated with 5-0 nylon surgical sutures under visual conditions. A total of 24 h later, GCI was induced by transient clipping of the bilateral common carotid artery for 20 min. Cognitive function and visual perception were then evaluated by behavioral and histopathological approaches. RESULTS: There was no significant difference in the survival rates between the groups. The modified 4VO group had a significantly lower body weight at each time point assessed. In the Y-maze test, the percentage of time spent and distance traveled in the III arm was significantly decreased on day 28, suggesting that cognitive function may have been impaired by the modified 4VO model. The modified 4VO procedure induced severe hippocampal damage but did not result in noticeable changes in visual perception, as indicated by the light-dark box test, and analysis of the optic tract and retinal structures. The modified 4VO procedure-induced cognitive deficits were thus likely the result of hippocampal damage, not visual perception. COMPARISON WITH EXISTING METHODS: The advantage of this model is the permanent ligation of the bilateral VAs under visual conditions rather than electrocoagulation, which is performed blind. CONCLUSIONS: This modified 4VO model can mimic the GCI/R method of the Pulsinelli and Brierley and may serve as a valuable tool for studies on GCI/R.

δ-Opioid receptor activation ameliorates lipopolysaccharide-induced inflammation and apoptosis by inhibiting the MAPK/caspase-3 pathway in BV2 microglial cells.

Delta-opioid receptor (DOR) is widely distributed in the central nervous system, and its activation protects against ischaemic/hypoxic brain injury. However, the role of DOR in microglia in ischaemic stroke has not yet been fully investigated. We found that DOR was expressed in both human and mouse cerebral microglia, besides, it was upregulated in activated BV2 microglial cells by immunofluorescence staining and Western blot. DOR activation by the specific agonist TAN-67 significantly enhanced BV2 microglial cell viability and reduced apoptosis, as evidenced by decreased cleaved caspase-3 levels and TdT-mediated aUTP-X nick end labelling (TUNEL) staining after LPS stimulation. Furthermore, activation of DOR significantly inhibited inducible nitric oxide synthase (iNOS) production and dose-dependently inhibited the mRNA and protein expression levels of other pro-inflammatory cytokines, including IL-1ß and IL-6, whereas it increased the expression of the anti-inflammatory cytokine IL-10 in LPS-stimulated BV2 microglial cells; these effects were correlated with diminished phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Moreover, these effects could be reversed by the DOR antagonist naltrindole. DOR activation can activate microglia to switch to the beneficial phenotype and inhibit LPS-induced inflammation and apoptosis via the mitogen-activated protein kinase (MAPK)/caspase-3 pathway in BV2 microglial cells. This study provides new insight into neuroprotection against and treatment of ischaemic stroke.