A hepatotoxic dose of acetaminophen modulates expression of BCL-2, BCL-X(L), and BCL-X(S) during apoptotic and necrotic death of mouse liver cells in vivo.
The protein BCL-X(L) and protein product of proto-oncogene bcl-2 act as apoptosis antagonists, and BCL-X(S) serve as a dominant death promoter, including apoptosis following exposure to chemotherapeutic drugs. This investigation examined whether some aspects of the highly integrated process of acetaminophen (AAP)-induced hepatotoxicity involve down-regulation or upregulation of expression of BCL-2, BCL-X(L) and BCL-X(S) in mouse liver in vivo. Male ICR mice (CD-1; 35-45 g) were treated ip with a hepatotoxic dose of AAP (500 mg/kg) and sacrificed 0, 6, and 18 h later. Blood was collected upon sacrifice for determination of serum alanine aminotransferase (ALT) activity and the liver was sectioned for histopathological diagnosis of necrosis/apoptosis. Portions of liver tissues were also used for DNA extraction (for gel electrophoresis) and Western blot analysis. This study demonstrates that administration of a hepatotoxic dose of AAP to ICR mice results in severe liver injury (ALT leakage >200-fold at 6 h and >600-fold at 18 h) leading to massive cell death by apoptosis (diagnosed by nuclear ultrastructure, histopathology, and DNA ladder), in addition to necrosis coupled with spectacular changes in the BCL-X(L) expression (6 and 18 h after AAP administration). Western blot analysis of the liver proteins revealed that mouse liver expresses two proteins, BCL-X(L) and BCL-X(S), and does not express BCL-2. As the toxicity progressed, during 6 and 18 h post-AAP administration, the BCL-X(L) protein band shifted to a slower mobility band which might represent a phosphorylated form of BCL-X(L). Appearance of this higher molecular weight BCL-X(L) protein band correlated with massive apoptotic death of liver cells along with ladder-like DNA fragmentation. In the same time period, death inhibitory gene bcl-2 remained unexpressed, and the level of expression of BCL-X(S) remained unaltered. Whether the consistent level of expression of BCL-X(S) reflected inability of AAP to influence its expression remains unknown. Unaltered expression of BCL-X(S) in the near total absence of BCL-2 expression raises questions regarding the death promoting role of BCL-X(S) in vivo. The precise role of modified form of BCL-X(L) remains elusive. However, this study may have demonstrated for the first time drug-induced changes in the expression of anti-apoptotic gene BCL-X(L), and a positive link between AAP-induced apoptotic death and modification of BCL-X(L) protein in vivo.
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Molecular Toxicology Program, Department of Pharmacology, Toxicology, and Medicinal Chemistry, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY 11201, USA.
Jena
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D000082:Acetaminophen / Q000633:toxicity*; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D056486:Chemical and Drug Induced Liver Injury / Q000378:metabolism* / Q000473:pathology; D003593:Cytoplasm / Q000187:drug effects / Q000648:ultrastructure; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation; D005786:Gene Expression Regulation; D008099:Liver / Q000187:drug effects* / Q000473:pathology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009336:Necrosis; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D000082:Acetaminophen; D004247:DNA; D000410:Alanine Transaminase