apap nac  (MedChemExpress)

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: Protein S- glutathionylation (PSSG) was involved in acetaminophen (APAP)-induced hepatotoxicity, and the expression of glutaredoxin-1 (Glrx1) was inhibited after APAP administration. (A) The total PSSG of the liver in wild type ( WT ) mice after APAP (200 mg/kg) treatment was detected under non-reducing conditions (left panel). Ponceau staining was used as a loading control (right panel). (B) Hepatic mRNA expression of genes related to PSSG in WT mice after APAP treatment for 12 h ( n = 6). (C) mRNA expression of genes related to PSSG in mouse primary hepatocytes (MPHs). MPHs isolated from WT mice were cultured with 0 or 5 mM APAP for 24 h ( n = 6). (D) Hepatic mRNA expression of Glrx1 in mice after APAP treatment for 0, 3, 12, and 24 h ( n = 5). (E) mRNA expression of Glrx1 in MPHs treated with or without APAP for 3, 12, and 24 h ( n = 6). (F) Hepatic protein expression of Glrx1 in mice after APAP treatment for 12 h (left panel) and quantifications of relative protein expression (right panel, n = 3). (G) Immunofluorescence staining of Glrx1 (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) in the liver of WT mice after saline or 200 mg/kg APAP treatment for 24 h. Quantification are shown on the right ( n = 6). Data are shown as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test. DTT: dithiothreitol, Gstp : glutathione S -transferase P, Trx : thioredoxin.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Expressing, Staining, Control, Isolation, Cell Culture, Immunofluorescence, Saline, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: Glutaredoxin-1 (Glrx1) ablation aggravated acetaminophen (APAP)-induced liver injury. Eight-to-ten-week-old male wild type ( WT ) and Glrx1 knockout ( Glrx1 −/− ) mice were treated with saline or APAP (200 mg/kg) for 3, 6, and 12 h to induce hepatotoxicity. (A) Serum alanine aminotransferase (ALT) (left panel) and aspartate aminotransferase (AST) (right panel) levels at 3, 6, and 12 h (saline: n = 6, APAP: n = 8). (B) Representative hematoxylin and eosin staining of liver sections (3 and 12 h) and the quantifications of the necrosis areas ( n = 5). (C) Hepatic reduced glutathione (GSH) content of mice after saline or APAP treatment for 3 h ( n = 6). (D) Content of hepatic malondialdehyde (MDA) in mice after saline or APAP treatment ( n = 6). (E) Relative hepatic content of reactive oxygen species (ROS) in mice after APAP treatment ( n = 6). (F) Western blotting analysis of the protein expression of extracellular signal-regulated kinases (ERK), phosphor-ERK (p-ERK), c-Jun N-terminal kinase (JNK) and phosphor-JNK (p-JNK) (left panel) and the relative quantification of p-ERK ratio to ERK and p-JNK ratio to JNK (right panel, n = 3). (G) Relative mRNA expression ( n = 5–6) and (H) protein levels ( n = 6) of inflammatory factor interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor alpha (TNFα) in the liver after APAP treatment. Data are presented as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Knock-Out, Saline, Staining, Western Blot, Expressing, Quantitative Proteomics, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: Glutaredoxin-1 (Glrx1) ablation increased toxic metabolites of acetaminophen (APAP). (A) Schematic of APAP metabolism in the liver with three pathways. About 45%–60% of APAP are metabolized by phase II conjugating enzyme UDP-glucuronosyltransferases (UGTs) to form APAP-glucuronide (APAP-Glu); about 25%–35% are metabolized by phase II conjugating enzyme sulfotransferases (SULTs) to form APAP-sulfate (APAP-Sul); about 9% are metabolized by cytochrome P450 enzymes (Cyp450s), mainly cytochrome p450 1a2 (Cyp1a2), cytochrome p450 2e1 (Cyp2e1) and cytochrome p450 3a11 (Cyp3a11) to form N -acetyl- p -benzoquinone imine (NAPQI), and NAPQI is converted to APAP-glutathione (APAP-GSH), APAP-Cysteine (APAP-CYS) and 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC). (B) Plasma concentration-time curves of APAP (left panel), plasma area under the curve (AUC) of APAP (middle panel), and liver concentration of APAP (right panel) in the wild type ( WT ) and Glrx1 knockout ( Glrx1 −/− ) mice after APAP treated for 3 h ( n = 6). (C) Concentration-time curves of APAP-GSH (left panel), plasma AUC of APAP-GSH (middle panel), and liver concentration of APAP-GSH (right panel) in the WT and Glrx1 −/− mice after APAP treated for 3 h ( n = 6). (D) Concentration-time curves of APAP-CYS (left panel), plasma AUC of APAP-CYS (middle panel), and liver concentration of APAP-CYS (right panel) in the WT and Glrx1 −/− mice after APAP treated for 3 h ( n = 6). (E) Concentration-time curves of APAP-NAC (left panel), plasma AUC of APAP-NAC (middle panel), and liver concentration of APAP-NAC (right panel) in the WT and Glrx1 −/− mice after APAP treated for 3 h ( n = 6). Data are presented as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Clinical Proteomics, Concentration Assay, Knock-Out, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: The activity of cytochrome p450 3a11 (Cyp3a11) and its S- glutathionylation modification was increased in glutaredoxin-1 (Glrx1) deficiency mice. (A) mRNA expression of cytochrome p450 1a2 ( Cyp1a2 ) , cytochrome p450 2e1 ( Cyp2e1 ) and Cyp3a11 in the wild type ( WT ) and Glrx1 knockout ( Glrx1 −/− ) mice after APAP treatment ( n = 6). (B) Western blotting analysis of the hepatic protein expression of Cyp1a2, Cyp2e1 and Cyp3a11. (C) The relative quantification of Cyp1a2, Cyp2e1 and Cyp3a11 protein expression levels ( n = 3). (D) Relative activity of Cyp1a2, Cyp2e1 and Cyp3a11 in liver microsomes ( n = 6). (E) Hepatic protein S- glutathionylation (PSSG) in WT and Glrx1 −/− mice under non-reducing conditions (left panel). Ponceau staining used as a loading control (right panel). (F–H) The S- glutathionylation of Cyp3a11, Cyp2e1 and Cyp1a2 in the WT and Glrx1 −/− mouse liver samples were measured by immunoprecipitation with an anti-glutathione (anti-GSH) body, followed by the detection of anti-Cyp3a11 (F), anti-Cyp2e1 (G) and anti-Cyp1a2 (H) antibodies. Data are presented as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test. DTT: dithiothreitol.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Activity Assay, Modification, Expressing, Knock-Out, Western Blot, Quantitative Proteomics, Staining, Control, Immunoprecipitation, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: Liver-specific overexpression of Glrx1 ( AAV8-Glrx1 ) alleviated acetaminophen (APAP)-induced hepatotoxicity. Eight-to-ten-week-old male control mice ( AAV8 vector containing only green fluorescent protein, AAV8-GFP ) and AAV8-Glrx1 mice were treated with saline or APAP (200 mg/kg) for 3, 6, and 12 h. (A) Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (saline: n = 5, APAP: n = 8). (B) Representative hematoxylin and eosin staining and quantifications of necrosis areas of the liver sections at 3 and 12 h ( n = 5). (C) Reduced glutathione (GSH) content of mice after saline or APAP treatment for 3 h (saline: n = 5, APAP: n = 7). (D) Hepatic content of malondialdehyde (MDA) in mice treated with APAP for 3 h ( n = 6). (E) The relative hepatic level of reactive oxygen species (ROS) was measured by BBoxiProbe® O08 red probe after APAP challenge for 3 h ( n = 7). (F) Western blotting analysis of the protein levels of extracellular signal-regulated kinases (ERK), phosphor-ERK (p-ERK), c-Jun N-terminal kinase (JNK) and phosphor-JNK (p-JNK) after APAP challenge for 3 h (left panel), and the quantification of p-ERK ratio to ERK and p-JNK ratio to JNK (right panel, n = 3). (G) Relative mRNA expression of interleukin-1 beta ( Il-1β ), tumor necrosis factor alpha ( Tnfα ) ( n = 6). (H) Protein levels of IL-1β and interleukin-6 (IL-6) were measured by the enzyme-linked immunosorbent assay (ELISA) kits ( n = 6). Data are presented as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Over Expression, Control, Plasmid Preparation, Saline, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: Liver-specific overexpression of Glrx1 ( AAV8-Glrx1 ) reduced toxic metabolites of acetaminophen (APAP) and inhibited cytochrome p450 3a11 (Cyp3a11) activity. (A) Plasma concentration of APAP-glutathione (APAP-GSH) (left panel), APAP-Cysteine (APAP-CYS) (middle panel) and 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC) in control (AAV8 vector containing only green fluorescent protein, AAV8-GFP ) mice and AAV8-Glrx1 mice after APAP treatment for 3 h ( n = 6). (B) Liver concentration of APAP-GSH (left panel), APAP-CYS (middle panel) and APAP-NAC (right panel) after APAP treatment for 3 h ( n = 6). (C) Relative activity of cytochrome p450 3a11 (Cyp3a11) in liver microsomes ( n = 7). (D) The S- glutathionylation of Cyp3a11 in AAV8-GFP and AAV8-Glrx1 mouse liver samples. Data are presented as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Over Expression, Activity Assay, Clinical Proteomics, Concentration Assay, Control, Plasmid Preparation, Two Tailed Test

Journal: Journal of Pharmaceutical Analysis

Article Title: Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

doi: 10.1016/j.jpha.2023.08.004

Figure Lengend Snippet: Preceding treatment with pirfenidone (PFD) alleviated acetaminophen (APAP)-induced liver injury. (A) The mRNA expression of glutaredoxin-1 ( Glrx1 ) in mice liver after PFD treatment ( n = 5). (B) The protein expression of Glrx1 in mice liver, and the quantification of Glrx1 to β-actin after PFD treatment ( n = 3). (C) Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) after APAP treatment (saline: n = 5, APAP: n = 8). (D) Representative hematoxylin and eosin staining of the liver sections at 3, 12, and 24 h (left panel) and quantifications of the necrosis areas (right panel, n = 5). (E) Relative hepatic content of reduced glutathione (GSH) of mice treated with saline or APAP for 3 h (saline: n = 5, APAP: n = 6). (F) Western blotting analysis of the protein levels of extracellular signal-regulated kinases (ERK), phosphor-ERK (p-ERK), c-Jun N-terminal kinase (JNK) and phosphor-JNK (p-JNK) after APAP challenge for 3 h. (G) The quantification of the expression ration of p-ERK to ERK and p-JNK to JNK ( n = 3). (H) Relative mRNA expression of interleukin-1 beta ( Il-1β ), tumor necrosis factor alpha ( Tnfα ) and monocyte chemoattractant protein-1 ( Mcp-1 ) after APAP treatment ( n = 5). Data are presented as mean ± standard error of mean. ∗ P < 0.05, two-tailed Student's t -test.

Article Snippet: The standards of 3-cysteinylacetaminophen trifluoroacetic acid salt (APAP-CYS, C994750), 3-[ N -acetyl- L -cystein- S -yl] acetaminophen (APAP-NAC, P191100), and 6ʹ-hydroxychlorzoxazone (H825120) were obtained from Toronto Research Chemicals (Toronto, Canada).

Techniques: Expressing, Saline, Staining, Western Blot, Two Tailed Test