Organophosphorus Flame Retardant TDCPP Displays Genotoxic and Carcinogenic Risks in Human Liver Cells

Tris(1,3-Dichloro-2-propyl)phosphate (TDCPP) is an organophosphorus flame retardant (OPFR) widely used in a variety of consumer products (plastics, furniture, paints, foams, and electronics). Scientific evidence has affirmed the toxicological effects of TDCPP in in vitro and in vivo test models; however, its genotoxicity and carcinogenic effects in human cells are still obscure. Herein, we present genotoxic and carcinogenic properties of TDCPP in human liver cells (HepG2). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and neutral red uptake (NRU) assays demonstrated survival reduction in HepG2 cells after 3 days of exposure at higher concentrations (100–400 μM) of TDCPP. Comet assay and flow cytometric cell cycle experiments showed DNA damage and apoptosis in HepG2 cells after 3 days of TDCPP exposure. TDCPP treatment incremented the intracellular reactive oxygen species (ROS), nitric oxide (NO), Ca2+ influx, and esterase level in exposed cells. HepG2 mitochondrial membrane potential (ΔΨm) significantly declined and cytoplasmic localization of P53, caspase 3, and caspase 9 increased after TDCPP exposure. qPCR array quantification of the human cancer pathway revealed the upregulation of 11 genes and downregulation of two genes in TDCPP-exposed HepG2 cells. Overall, this is the first study to explicitly validate the fact that TDCPP bears the genotoxic, hepatotoxic, and carcinogenic potential, which may jeopardize human health.

Adaptive morphogenesis of lamb liver under Anthropobiocenosis conditions lamb liver morphogenesis

Investigated the histological features of the structure of hepar of one-day-old lambs (ODL) using a complex of morphological techniques. It was found that hepar in ODL is formed by stroma and parenchyma. The stromal elements of the organ are formed by loose lugs of hematopoietic connective tissue, which in turn forms a capsule (2.80-7.23 microns thick), as well as the septa of the lobules, which include the hepar triads and paravasal tissue of efferent blood vessels. As a result of the studies, the prevalence of the stroma in the hematopoietic foci around large afferent blood vessels, especially in the hepar hilus, was revealed. The hepar parenchyma in ODL has structural incompleteness. It is formed by hepar cells and numerous resinusoidal cells located in the subcapsular, peripheral, central zones in the lobus hepatis. Hepar cells located in the subcapsular zone (SZ) have a more optically cleared (OC) cytoplasm, and the practically cleared cytoplasm of the hepatocyte diameter (HD) decreases towards the central zone (CZ). In the hepar parenchyma of the ODL, hematopoiesis foci (HF) are established in each of the lobes, which tend to decrease from the periphery to the center of the lobes. Hemopoetic cells (HC) are located between hepatocytes, densely layering on top of each other, and there are also cells located sparsely between the liver cells (LC). The number of hepatocytes per 1 mm2 of the area of the histological specimen in the left lobe (LL) of the ODL reaches the highest value 5983.79 ± 90.40, in the right lobe (RL) of the liver it is 5358.80 ± 646.60, while in the middle it has a minimum value and reaches 5133.10 ± 205.75.

S100A6 Promotes Inflammation and Infiltration of Mononuclear/Macrophages via the p-P38 and p-JNK Pathways in Acute Liver Injury

BackgroundThe phagocytic S100 protein, which mediates inflammatory responses and recruits inflammatory cells to sites of tissue damage, has long been known to be expressed in cells of myeloid origin. S100A6 belongs to the A group of the S100 protein family of Ca2+-binding proteins. Currently, the mechanism by which S100A6 mediates the inflammatory response and recruits inflammatory cells to the tissue injury site is unknown.MethodsA mouse model of carbon tetrachloride (CCl4)-induced acute liver injury (ALI) was established, and the transcriptomes of postinjury 2d and 5d liver tissues were sequenced. Enzyme-linked immunosorbent assay was used to determine the expression of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) in the supernatant of the liver. Immunohistochemical analysis confirmed the expression of S100A6 in the liver cells. In vitro experiments proved the pro-inflammatory function of S100A6, and western blotting (WB) showed that the pathways were activated. The transwell experiment showed the infiltration of mononuclear/macrophages.ResultsWe found that S100A6 is highly expressed in liver cells during the most severe period of ALI, suggesting that it acts as an endogenous danger signal and has a pro-inflammatory function. In vitro, the mouse S100A6 recombinant protein was used to stimulate liver Kupffer cells to promote the secretion of TNF-α, IL-1β, IL-6, and IL-8. Further mechanistic experiments revealed that S100A6 acts as an endogenous danger signal to activate p-P38 and p-JNK downstream of the TLR4 and P65 pathways. Similarly, transcriptome data showed that S100A6 can activate the inflammatory response in Kuffer cells. WB revealed that S100A6 had no significant effect on cell apoptosis. To continue to explore the mechanism of monocyte/macrophage infiltration, we found that TNF-α stimulates liver cells as the main source of CCL2. TNF-α can initiate the p-P38 and p-JNK pathways of liver cells to produce CCL2, thereby recruiting the infiltration of mononuclear/macrophages. ConclusionsTaken together, S100A6 is an endogenous danger signal that mediates inflammatory responses and recruits inflammatory cells to sites of tissue damage.

In Vitro and In Vivo Protective Effects of Lentil (Lens culinaris) Extract against Oxidative Stress-Induced Hepatotoxicity

Excessive oxidative stress plays a role in hepatotoxicity and the pathogenesis of hepatic diseases. In our previous study, the phenolic extract of beluga lentil (BLE) showed the most potent in vitro antioxidant activity among extracts of four common varieties of lentils; thus, we hypothesized that BLE might protect liver cells against oxidative stress-induced cytotoxicity. BLE was evaluated for its protective effects against oxidative stress-induced hepatotoxicity in AML12 mouse hepatocytes and BALB/c mice. H2O2 treatment caused a marked decrease in cell viability; however, pretreatment with BLE (25–100 μg/mL) for 24 h significantly preserved the viability of H2O2-treated cells up to about 50% at 100 μg/mL. As expected, BLE dramatically reduced intracellular reactive oxygen species (ROS) levels in a dose-dependent manner in H2O2-treated cells. Further mechanistic studies demonstrated that BLE reduced cellular ROS levels, partly by increasing expression of antioxidant genes. Furthermore, pretreatment with BLE (400 mg/kg) for 2 weeks significantly reduced serum levels of alanine transaminase and triglyceride by about 49% and 40%, respectively, and increased the expression and activity of glutathione peroxidase in CCl4-treated BALB/c mice. These results suggest that BLE protects liver cells against oxidative stress, partly by inducing cellular antioxidant system; thus, it represents a potential source of nutraceuticals with hepatoprotective effects.

Non-Clinical investigation of Tuberculosis Drugs - Conjugated Norbornene-Based Nanocarriers Toxic Impacts on Zebrafish

INTRODUCTION: Rifampicin conjugated (R-CP), and rifampicin -isoniazid dual conjugated (RI-CP) norbornene-derived nanocarriers are newly designed for pH stimuli-responsive delivery of tuberculosis (TB) drugs. Its biosafety level is yet to be well established. OBJECTIVES: To assess the impacts of the nanocarriers on liver cells using zebrafish animal model and human liver cell line model (HepG2). METHODS: Initially, lethal dose concentration for the norbornene-derived nanocarrier systems in zebrafish was determined. The toxic effects were analysed at the sub-lethal drug concentration by histopathological study, total GSH level, gene expression and DNA damage in zebrafish liver cells. Fish erythrocyte nuclear abnormalities were also evaluated. Cell viability and oxidative stress level (ROS generation) after exposure to the nanoconjugates was determined using HepG2 cell in the in vitro study. RESULTS: In vivo studies of both R-CP and RI-CP showed 100% mortality at 96 hours for exposure concentration >100mg/l and showed toxic changes in zebrafish liver histology, GSH, and DNA damage levels. A noticeable upregulated PXR, CYP3A and cyp2p6 genes was observed in RI-CP exposure than in RIF or R-CP molecules. The in vitro study revealed a dose-dependent effect on cell viability and ROS generation for RIF, R-CP and RI-CP exposures in HepG2 cells. CONCLUSION: The current study reports that the rifampicin conjugated (R-CP) and rifampicin-isoniazid conjugated (RI-CP) norbornene derived nanocarriers exhibit enhanced toxic responses in both adult zebrafish and HepG2 cells. The pH-sensitive norbornene derived nanocarriers on conjugation with different drugs exhibited varied impacts on hepatic cells. Hence the present investigation recommends a complete metabolomics analysis and norbornene carrier-drug interaction study to be performed for each drug conjugated norbornene nanocarrier to ensure its biosafety.

HepG2 liver cells treated with fumonisin B1 in galactose supplemented media have altered expression of genes and proteins known to regulate cholesterol flux

Fumonisin B1 (FB1) contributes to mycotoxicosis in animals and has been associated with the incidence of some cancers in humans. The effect of FB1 on lipidomic profiles, sphingolipids and cholesterol levels have been demonstrated in experimental models, however, the events leading to altered cholesterol levels are unclear. This study investigates the molecular mechanisms that regulate the effect of FB1 on cholesterol homeostasis in galactose supplemented HepG2 liver cells. Galactose supplementation is a proven method utilised to circumvent the Crabtree effect exhibited by cancer cells, which forces cancer cells to activate the mitochondria. HepG2 cells were cultured in galactose supplemented media and treated with FB1 (IC50 = 25 μM) for 6 h. Cell viability was determined using the MTT assay. Metabolic status was evaluated using ATP luciferase assay, and cholesterol regulatory transcription factors (SIRT1, SREBP-1C, LXR, LDLR, PCSK9, and ABCA1) were investigated using western blotting and qPCR. FB1 in galactose supplemented HepG2 cells increased gene expression of SIRT1 (P<0.05), SREBP-1C, LXR, and LDLR; however, PCSK9 (P<0.05) was decreased. Furthermore, protein expression of SIRT1, LXR, and LDLR was elevated upon FB1 treatment, while SREBP-1C and PCSK9 were reduced. The data provides evidence that SIRT1 reduced the expression of PCSK9 and deacetylated LXR to prevent degradation of LDLR. This could result in a dysregulated cholesterol flux, which may contribute to FB1 mediated toxicity.