Diethylnitrosamine was purchased from Sigma-Aldrich (St. Louis, MO, USA). DEN was given to rats in drinking water (100 mg/L). The DEN solution was prepared as a fresh solution every other day and administered to rats in dark bottles. Other chemicals and reagents were of high analytical grade and were purchased from standard commercial suppliers.
Diethylnitrosamine
Manufactured by Merck Group
Sourced in United States
Diethylnitrosamine is a chemical compound used in laboratory research. It is a colorless liquid with a characteristic odor. Diethylnitrosamine is commonly used as a chemical reagent and in the synthesis of other compounds. Its core function is to serve as a versatile intermediate in organic chemistry.
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Lab products found in correlation
Oligomycin, by Merck Group (3 mentions)
Envision doublestain system, by Agilent Technologies (3 mentions)
3 3 diaminobenzidine tetrahydrochloride hydrate, by Merck Group (3 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (3 mentions)
2 acetylaminofluorene 2 aaf, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Sorafenib, by Bayer (2 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (2 mentions)
Vectastrain abc kit, by Vector Laboratories (2 mentions)
Methylene blue, by Merck Group (2 mentions)
Avidin biotin horseradish peroxidase complex abc kit, by Vector Laboratories (2 mentions)
Mouse monoclonal proliferating cell nuclear antigen antibody, by BioLegend (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Sprague dawley rat, by Charles River Laboratories (1 mentions)
Phosphorylated jnk, by Cell Signaling Technology (1 mentions)
Alt assay kit, by Abcam (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
50 protocols using diethylnitrosamine
1
Rat DEN-induced Liver Carcinogenesis
2
Diethyl Nitrosamine-Induced Liver Study
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Diethyl nitrosamine was obtained from Sigma Aldrich Ltd. A dose of 20 mg/kg body weight in 0.9% w/v NaCl and was administered intraperitonially.
3
Mycotoxin Exposure Protocol
4
Diethylnitrosamine-Induced Liver Tumor Model
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Seven week old pathogen-free male Sprague–Dawley rats (weighing 160–180 g, Charles River Laboratories, Beijing) were used in our experiments. All animals were administered diethylnitrosamine (N0756, Sigma) intraperitoneally (70 mg/kg, dissolved in saline) once a week for 10 weeks and sacrificed at 22 weeks (Zhang et al., 2012 (link); Qin et al., 2018 (link)).
5
Liver Injury Signaling Pathways
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Reagents used were tamoxifen (MP Biomedicals) and 4-hydroxytamoxifen (Sigma) LPS (From Salmonella minnesota, phenol extraction, Sigma) and diethylnitrosamine (DEN) (Sigma). Polyclonal antibodies used were TAK1, TAB1, and TAB2 described previously [14] (link), [35] (link), phosphorylated TAK1, cleaved caspase 3, phosphorylated JNK, JNK (Cell signaling), β-actin (Sigma-Aldrich), p65 and α-tubulin (Santa Cruz). An ALT assay kit (Biovision, Mountain View, CA) was used.
6
Diethylnitrosamine-Induced Liver Injury Model
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Diethylnitrosamine (DEN) was purchased from Sigma (N0258, Sigma-Aldrich, USA). Carbon tetrachloride was purchased from Hao-Sen Chemical Co., Ltd. (Shanghai, China). Ethanol (55%) was purchased from Niu Lan Shan Co., Ltd. (Beijing, China). The TRI Reagent was purchased from Sigma (T9424; Sigma-Aldrich, USA). The Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher (K1621, Thermo Fisher; U.S.A). HieffTM qPCR SYBR® Green Master Mix was purchased from Yeasen Biotech Co., Ltd. (Cat. No. 11203, Shanghai, China). Anti-pan-AKT antibody (#ab8805, Abcam, Cambridge, U.K.), anti-p-AKT (ser473) antibody (#ab126433, Abcam, Cambridge, U.K.), Anti-AFP primary antibody (cat# ab46799, Abcam, Cambridge, U.K.), β-actin antibody (cat# ab8227, Abcam, Cambridge, U.K.), anti-IL-10 primary antibody (cat#ab189392, Abcam, Cambridge, U.K.), and CTLA-4 antibody (cat#ab134090, Abcam, Cambridge, U.K.) were purchased from Abcam (UK). CD4 (cat#25229), CD8α (cat#98941), and PD-1 (cat#84651) antibodies were purchased from Cell Signaling Technology (M.A. USA).
7
Liver Cancer Induction and Metastasis
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The primary cancer group was induced by intraperitoneal diethylnitrosamine (Sigma-Aldrich, St. Louis, USA) injection in 15-day-old mice. The lack of matrillin-2 can increase the tumor proliferation in the liver [28 (link)]. The mice were investigated 4 months after the induction when liver tumors have reached the advanced multifocal status.
The metastatic group was induced by intrasplenic injection of a human melanoma HT168-M cell line (0.1 mL, ~ 5 × 105cells). This xenograft has high liver-colonizing capacity [29 (link)]. These mice were imaged only two months after the cell inoculation to mimic the early stage and the small size of metastatic lesions.
The metastatic group was induced by intrasplenic injection of a human melanoma HT168-M cell line (0.1 mL, ~ 5 × 105cells). This xenograft has high liver-colonizing capacity [29 (link)]. These mice were imaged only two months after the cell inoculation to mimic the early stage and the small size of metastatic lesions.
8
Liver Cancer Prevention with Nanoparticles
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Diethylnitrosamine (DEN) was purchased from (DEN, Sigma-Aldrich Co., St. Louis, MO, USA) and was dissolved in NaCl 0.9% (1 g in 25 ml). Sorafenib was obtained from Bayer (Berlin, Germany). Nanoparticles of Arthrospira platensis (NSP) were prepared following a previous method [17 (link)], with a size particle of 158 nm. See the supplementary materials Figures S1–S4 .
Serum alpha-fetoprotein (AFP) concentrations were estimated using kits obtained from Kamiya Biomedical Company, Washington, USA. Serum biochemical parameters were measured using ready-made kits to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (ALP), and gamma-glutamyltransferase (GGT) obtained from (Randox Laboratories Ltd., Crumlin, County Antrim, UK). Ready-made kits for reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) were purchased from Biodiagnostics, Cairo, Egypt. p53 antibodies were obtained from Biotechnologies, (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Serum alpha-fetoprotein (AFP) concentrations were estimated using kits obtained from Kamiya Biomedical Company, Washington, USA. Serum biochemical parameters were measured using ready-made kits to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (ALP), and gamma-glutamyltransferase (GGT) obtained from (Randox Laboratories Ltd., Crumlin, County Antrim, UK). Ready-made kits for reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) were purchased from Biodiagnostics, Cairo, Egypt. p53 antibodies were obtained from Biotechnologies, (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
9
Evaluating Metabolic Reprogramming Mechanisms
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Diethylnitrosamine (DEN, 73861), CCl4 (1601168), Oligomycin (O4876), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, C2920), antimycin A (A8674), rotenone (R8875), 2-deoxy-d -glucose (2-DG, D8375), PNGase F (G5166) and Swainsonine (S9263) and D-(+)-Glucose (G7021) were obtained from Sigma (St. Louis, MO, USA). SYBR green was purchased from Bio-Rad (Hercules, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-mouse immunoglobulin G were obtained from Beyotime (Haimen, Jiangsu, China). Glucose deficient DMEM medium and Trizol Reagent were obtained from Life Technologies (Carlsbad, CA, USA). Tunicamycin (B7417) was obtained from ApexBio Technology (Houston, TX, USA). UNC-2250 (S7342) was purchased from Selleck Chemicals (Houston, TX, USA). The antibodies used in this study are: MerTK (abcam; ab52968), Phospho-MerTK (abcam; ab14921), Akt (Cell Signaling Technology; 4691), Phospho-Akt (Ser473) (Cell Signaling Technology; 4060), Phospho-GSK3β (Cell Signaling Technology; 5558S), PGK1 (ZN; 501965), LDHA (ZN; 501146), PFKM (ZN; 505477), PKM2 (ZN; 505477), PDHK1 (Cell Signaling Technology; 3820S), Lamin B (Proteintech; 12987-1-AP), GAPDH (Abmart; M20028) and β-Actin (Proteintech; 60008-1-1 g).
10
Investigating Cellular Signaling Pathways
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