-
Chinese Journal of Integrative Medicine Jun 2022To screen the active components from Fuzheng Huayu Recipe (FZHY) and redesign a new recipe composed of the active components, and validate the effect of active...
OBJECTIVE
To screen the active components from Fuzheng Huayu Recipe (FZHY) and redesign a new recipe composed of the active components, and validate the effect of active components formulation from FZHY against liver fibrosis.
METHODS
Thirty-two components from FZHY were evaluated for their activities against liver fibrosis respectively, with 6 kinds of cell models in vitro, including oxidative stressed hepatocyte in L-02, hypoxia injured/proliferative hepatic sinusoidal endothelial cells in SK-HEP-1 and human hepatic sinusoidal endothelial cells (HHSEC), and activated hepatic stellate cell in LX-2. The comprehensive activity of each component against liver fibrosis was scored according to the role of original herbs in FZHY and cell functions in fibrogenesis. Totally 7 active components were selected and combined with equal proportion to form a novel active components formulation (ACF). The efficacy of ACF on liver fibrosis were evaluated on activation of LX-2 and proliferation of HHSEC in vitro and in liver fibrosis model mice induced by dimethylnitrosamine (DMN). Totally 72 mice were divided into 6 groups using a random number table, including normal, high-dose ACF control (20 µ mol/L × 7 components/kg body weight), model, low-, medium-, high-dose ACF groups (5, 10, 20 µ mol/L × 7 components/kg body weight, respectively). Hematoxylin eosin and Sirius red stainings were used to observe inflammation and fibrosis change of liver tissue; scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to observe the effect of ACF on ultrastructure of hepatic sinusoids.
RESULTS
Fifteen components from FZHY showed higher scores for their activity on against liver fibrosis. Among them, 7 components including tanshinone II A, salvianolic acid B, cordycepin, amygdalin, quercetin, protopanaxatriol, and schizandrin B were recombined with equal proportions to form ACF. ACF at 1,2, 4 µ mol/L showed strong inhibitory effects on activation of LX-2 and proliferation of HHSEC in vitro (all P<0.01). Compared with the model group, ACF attenuated liver collagen deposition, improved sinusoidal capillarization in a dose-dependent manner (all P<0.05).
CONCLUSION
ACF exerts a satisfactory effect against experimental liver fibrosis and attenuates sinusoidal capillarization, which warrant a further research and development for herbal components formulation on liver fibrosis.
Topics: Animals; Body Weight; Drugs, Chinese Herbal; Endothelial Cells; Liver; Liver Cirrhosis; Mice
PubMed: 34581939
DOI: 10.1007/s11655-021-3293-x -
Molecules (Basel, Switzerland) Nov 2020A GC-MS/MS method with EI ionization was developed and validated to detect and quantify -nitrosodimethylamine (NDMA) and seven other nitrosamines in 105 samples of...
A GC-MS/MS method with EI ionization was developed and validated to detect and quantify -nitrosodimethylamine (NDMA) and seven other nitrosamines in 105 samples of metformin tablets from 13 different manufactures. Good linearity for each compound was demonstrated over the calibration range of 0.5-9.5 ng/mL. The assay for all substances was accurate and precise. NDMA was not detected in the acquired active pharmaceutical ingredient (API); however, NDMA was detected in 64 (85.3%) and 22 (91.7%) of the finished product and prolonged finished product samples, respectively. European Medicines Agency recommends the maximum allowed limit of 0.032 ppm in the metformin products. Hence, 28 finished products and 7 pronged dosage products were found to exceed the acceptable limit of daily intake of NDMA contamination. The implications of our findings for the testing of pharmaceutical products are discussed.
Topics: Artifacts; Calibration; Dimethylnitrosamine; Drug Contamination; Drug Design; Europe; Gas Chromatography-Mass Spectrometry; Limit of Detection; Linear Models; Metformin; Pharmaceutical Preparations; Powders; Solvents; Tablets; Tandem Mass Spectrometry; Temperature
PubMed: 33202951
DOI: 10.3390/molecules25225304 -
Archives of Toxicology Oct 2023N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in...
N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.
Topics: Humans; Nitrosamines; Cytochrome P-450 Enzyme System; Carcinogens; DNA Damage; Dimethylnitrosamine; Mutagens
PubMed: 37486449
DOI: 10.1007/s00204-023-03560-x -
Journal of Hazardous Materials May 2023N-nitrosamines are formed during different industrial processes and are of significant concern due to their carcinogenic and mutagenic properties. This study reports...
N-nitrosamines are formed during different industrial processes and are of significant concern due to their carcinogenic and mutagenic properties. This study reports concentrations of N-nitrosamines in eight different industrial wastewater treatment plants in Switzerland and the variability of their abundance. Only four N-nitrosamines species, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDPA) and N-nitrosomorpholine (NMOR) were above the limit of quantification in this campaign. Remarkably high concentrations (i.e. up to 975 μg NDMA/L, 90.7 μg NDEA/L, 1.6 μg NDPA/L and 710 μg NMOR/L) of these N-nitrosamines were detected at seven of eight sites. These concentrations are two to five orders of magnitude higher than those typically detected in municipal wastewater effluents. These results suggest that industrial effluents may be a major source of N-nitrosamines. Although very high concentrations of N-nitrosamine have been detected in industrial discharges, various processes in surface water can partially mitigate their concentrations (e.g. photolysis, biodegradation and volatilization) and hence the risk to human health and aquatic ecosystems. Nevertheless, there is little information on long-term effects on aquatic organisms and therefore the discharge of N-nitrosamines to the environment should be avoided until the impact on ecosystems is assessed. During winter a less efficient mitigation of N-nitrosamines can be expected (lower biological activity, less sunlight) and therefore, emphasis should be put on this season in future risk assessment studies.
Topics: Humans; Switzerland; Ecosystem; Nitrosamines; Dimethylnitrosamine; Diethylnitrosamine
PubMed: 36867906
DOI: 10.1016/j.jhazmat.2023.131094 -
European Journal of Clinical... Jun 2020Hepatic fibrosis is a worldwide incurable disease; due to the complex and unclear mechanism, there lack the effective therapeutic targets. However, the mechanism of...
BACKGROUND
Hepatic fibrosis is a worldwide incurable disease; due to the complex and unclear mechanism, there lack the effective therapeutic targets. However, the mechanism of miR-23a-5p underling this pathological process is largely not clear. The purpose of this study was to investigate the role of miR-23a-5p in hepatic fibrosis and HSC activation.
METHODS
The content of miR-23a-5p in hepatic fibrosis induced by N-nitrosodimethylamine (NDMA) and HSC activation induced by platelet-derived growth factor (PDGF) was detected by qRT-PCR. H&E staining, Masson staining and Shear wave electrography (SWE) were used to detect the degree of hepatic fibrosis. Immunohistochemistry staining, qRT-PCR and Western blot detect the related markers of liver fibrosis or HSC activation, as well as the related pathway genes and proteins. Dual-luciferase reporter system verifies the interaction between miR-23a-5p with PTEN or miR-23a-5p with lncRNA LOC102551149 in HSC-T6. siRNA and miRNA mimic transfer to HSC-T6 to detect the function of lncRNA LOC102551149 and miR-23a-5p on HSC activation.
RESULTS
After hepatic fibrosis and HSC activation happened, the expression of miR-23a-5p was up-regulated, whereas anti-miR-23a-5p can alleviate hepatic fibrosis and HSC activation. Further research shows miR-23a-5p can target PTEN and degrade it, causing activation of PI3K/Akt/mTOR/Snail pathway. lncRNA LOC102551149 can be used as a competition endogenous RNA (ceRNA) targeting miR-23a-5p through base pairing, and siRNA LOC102551149 or exogenous miR-23a-5p can induce HSC activation through PI3K/Akt/mTOR/Snail pathway.
CONCLUSION
We demonstrate mechanism pathway of miR-23a-5p on hepatic fibrosis and HSC activation, which may develop a therapeutic target for hepatic fibrosis.
Topics: Animals; Blotting, Western; Cell Line; Dimethylnitrosamine; Gene Knockdown Techniques; Hepatic Stellate Cells; Liver Cirrhosis; MicroRNAs; PTEN Phosphohydrolase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Rats; Real-Time Polymerase Chain Reaction
PubMed: 32306379
DOI: 10.1111/eci.13243 -
Food Additives & Contaminants. Part A,... Apr 2023A simple and sensitive method based on liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry (LC-APCI-MS/MS) was developed and...
A simple and sensitive method based on liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry (LC-APCI-MS/MS) was developed and validated to determine the levels of 13N-nitrosamines and N-nitrosatable substances migrated from rubber teats into artificial saliva. The migration test from rubber teats was conducted at 40 °C and for 24 h in artificial saliva, and the migrated artificial saliva solution was analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) without further extracting steps. The sensitivity of N-nitrosamines was examined by applying atmospheric chemical ionisation and electrospray ionisation to optimise the mass spectrometric conditions, and the atmospheric chemical ionisation (APCI) mode exhibited 1.6-19 times higher sensitivity. Method validation showed acceptable linearity, precision, and accuracy, and the detection and quantification limits were 0.07-0.35 and 0.24-1.1 μg kg, respectively. The developed liquid chromatography-atmospheric chemical ionisation-tandem mass spectrometry method was applied to 39 domestic and imported rubber teats. From 39 samples, N-nitrosamines [N-nitrosodimethylamine (NDMA), N-nitrosomorpholine (NMOR), and N-nitroso n-methyl N-phenylamine (NMPhA)] were detected in 30 samples, with N-nitrosatable substances in 17 samples give rise to NDMA, NMOR, and N-nitrosodiethylamine. However, the levels were below the specific migration limit of Korean Standards and Specifications for Food Containers, Utensils, and Packages and EC Directive 93/11/EEC.
Topics: Rubber; Tandem Mass Spectrometry; Nitrogen Radioisotopes; Chromatography, Liquid; Saliva, Artificial; Nitrosamines; Dimethylnitrosamine
PubMed: 36972401
DOI: 10.1080/19440049.2023.2193276 -
Archives of Toxicology Jun 2024Dietary exposure to N-nitrosamines has recently been assessed by the European Food Safety Authority (EFSA) to result in margins of exposure that are conceived to...
Dietary exposure to N-nitrosamines has recently been assessed by the European Food Safety Authority (EFSA) to result in margins of exposure that are conceived to indicate concern with respect to human health risk. However, evidence from more than half a century of international research shows that N-nitroso compounds (NOC) can also be formed endogenously. In this commentary of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), the complex metabolic and physiological biokinetics network of nitrate, nitrite and reactive nitrogen species is discussed with emphasis on its influence on endogenous NOC formation. Pioneering approaches to monitor endogenous NOC have been based on steady-state levels of N-nitrosodimethylamine (NDMA) in human blood and on DNA adduct levels in blood cells. Further NOC have not been considered yet to a comparable extent, although their generation from endogenous or exogenous precursors is to be expected. The evidence available to date indicates that endogenous NDMA exposure could exceed dietary exposure by about 2-3 orders of magnitude. These findings require consolidation by refined toxicokinetics and DNA adduct monitoring data to achieve a credible and comprehensive human health risk assessment.
Topics: Humans; Risk Assessment; Nitrosamines; Dietary Exposure; Dimethylnitrosamine; DNA Adducts; Food Contamination; Food Safety; Animals; Nitrites; Nitrates; Reactive Nitrogen Species
PubMed: 38573336
DOI: 10.1007/s00204-024-03726-1 -
Applied Biochemistry and Biotechnology Apr 2022Dimethylnitrosamine (DMN) is an established carcinogen. It is toxic to several organs, viz., the liver, kidney, and lungs, and immune system. Several drugs have been...
Dimethylnitrosamine (DMN) is an established carcinogen. It is toxic to several organs, viz., the liver, kidney, and lungs, and immune system. Several drugs have been used in the past to modulate its toxicity using experimental animal models. The present study was designed to investigate the effect of zinc oxide nanoparticles (ZnONPs) on renal toxicity caused by DMN in laboratory rat. Since oxidative mechanisms are mainly involved in its toxicity, the proposed study focuses on the amelioration of oxidative stress response by ZnONPs, if any. The present results show that administration of ZnONPs (50 mg/kg body weight/rat) to DMN (2 μl/100 g body weight/rat)-treated rats diminuted the concentration of malonaldehyde, HO, and NO in the kidney. However, reduced glutathione (GSH) concentration increased after ZnONP treatment. Results on glutathione S-transferase and glutathione peroxidase favored its antioxidative effects. These results are supported by the recovery of oxidative DNA damage and less pronounced histopathological changes in the kidney. It is hypothesized that ZnONPs might be toxic to renal tissue; however, its strong therapeutic/antioxidative potential helps in ameliorating DMN-induced renal toxicity in rat.
Topics: Animals; Antioxidants; Body Weight; Dimethylnitrosamine; Hydrogen Peroxide; Nanoparticles; Oxidative Stress; Rats; Zinc Oxide
PubMed: 34855113
DOI: 10.1007/s12010-021-03689-4 -
Life Sciences Feb 2021To investigate the effects and mechanism of miR-322/424 in liver fibrosis.
AIMS
To investigate the effects and mechanism of miR-322/424 in liver fibrosis.
MAIN METHODS
miR-322/424 expression in liver cirrhosis patients, mouse and rat liver fibrosis was determined by qPCR. Mice liver fibrosis was established by CCl, and intervened by miR-322/424 agomir or antagomir. Liver hydroxyproline content and Sirius red staining were used to evaluate collagen deposition. CD31 expression was used to evaluate liver microvessel density. In vitro, the effects of miR-322/424 mimic or inhibitor on human hepatic sinusoidal endothelial cells (HHSECs) migration and tube formation were investigated. A dual luciferase reporter assay was performed to confirm the direct interaction between miR-322/424 and Cullin2. mRNA expression of elongin B/C, Cullin2, and RBX1 was determined by qPCR. HIF-1α protein expression was determined by Western blotting.
KEY FINDINGS
miR-322/424 level in liver cirrhosis patients, mouse liver fibrosis induced by CCl and BDL, and rat liver fibrosis induced by CCl and dimethylnitrosamine was increased. miR-322/424 agomir exacerbated CCl-induced mouse liver fibrosis, whereas the opposite effect was observed for miR-322/424 antagomir. miR-322/424 agomir significantly upregulated liver CD31 expression; opposite effects occurred with miR-322/424 antagomir. In vitro, miR-322/424 mimic significantly promoted tube formation and cell migration, and increased von Willebrand factor expression, whereas miR-322/424 inhibitor had the opposite effect. Dual-Luciferase Reporter Assay identified Cullin2 as miR-322/424 target. miR-322/424 decreased the mRNA expression of elongin B/C, Cullin2, and RBX1 and increased HIF-1α protein expression in HHSECs.
SIGNIFICANCE
miR-322/424 plays a central role in the pathogenesis of liver fibrosis by targeting Cullin2, and enhancing HIF-1α-mediated hepatic angiogenesis.
Topics: Animals; Cells, Cultured; Cullin Proteins; Disease Models, Animal; Hemangioma; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; MicroRNAs; Neovascularization, Pathologic; Rats; Rats, Wistar
PubMed: 33333053
DOI: 10.1016/j.lfs.2020.118819 -
Biomedicine & Pharmacotherapy =... Jul 2022Osteopontin (OPN) is a matricellular cytokine and a stress-induced profibrogenic molecule that promotes activation of stellate cells during the pathogenesis of hepatic...
Osteopontin (OPN) is a matricellular cytokine and a stress-induced profibrogenic molecule that promotes activation of stellate cells during the pathogenesis of hepatic fibrosis. We studied the protective effects of epigallocatechin-3-gallate (EGCG) to suppress oxidative stress, inhibit OPN expression, and prevent experimentally induced hepatic fibrosis. Liver injury was induced with intraperitoneal injections of N-nitrosodimethylamine (NDMA) in a dose of 1 mg/100 g body weight on 3 consecutive days of a week for 28 days. A group of rats received 0.2 mg EGCG/100 g body weight orally everyday during the study. The animals were sacrificed on day 28th from the beginning of exposure. Serum levels of AST, ALT, OPN, malondialdehyde, collagen type IV, and hyaluronic acid were measured. Immunohistochemistry and/or real-time PCR were performed for α-SMA, 4-HNE, OPN, collagen type I, and type III. Serial administrations of NDMA produced well developed fibrosis and early cirrhosis in rat liver. Treatment with EGCG significantly reduced serum/plasma levels of AST, ALT, OPN, malondialdehyde, collagen type IV, and hyaluronic acid and prevented deposition of collagen fibers in the hepatic tissue. Protein and/or mRNA levels demonstrated marked decrease in the expression of α-SMA, 4-HNE, OPN, collagen type I, and type III. Treatment with EGCG prevented excessive generation of reactive oxygen species, suppressed oxidative stress, significantly reduced serum and hepatic OPN levels, and markedly attenuated hepatic fibrosis. The results indicated that EGCG could be used as a potent therapeutic agent to prevent hepatic fibrogenesis and related adverse events.
Topics: Animals; Body Weight; Catechin; Collagen Type I; Collagen Type IV; Dimethylnitrosamine; Fibrosis; Hepatic Stellate Cells; Hyaluronic Acid; Liver; Liver Cirrhosis; Malondialdehyde; Osteopontin; Rats
PubMed: 35594711
DOI: 10.1016/j.biopha.2022.113111