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 apo ptosis antagonists, and BCL-X-S serve as a dominant death promoter, includi ng apoptosis following exposure to chemotherapeutic drugs. This investigati on examined whether some aspects of the highly integrated process of acetam inophen (AAP)-induced hepatotoxicity involve down-regulation or upregulatio n 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 (50 0 mg/kg) and sacrificed 0, 6, and 18 h later. Blood was collected upon sacr ifice for determination of serum alanine aminotransferase (ALT) activity an d the liver was sectioned for histopathological diagnosis of necrosis/apopt osis. Portions of liver tissues were also used for DNA extraction (for gel electrophoresis) and Western blot analysis. This study demonstrates that ad ministration of a hepatotoxic dose of AAP to ICR mice results in severe liv er injury (ALT leakage >200-fold at 6 h and >600-fold at 18 h) leading to m assive cell death by apoptosis (diagnosed by nuclear ultrastructure, histop athology, and DNA ladder), in addition to necrosis coupled with spectacular changes in the BCL-X-L expression (6 and 18 h after AAP administration). W estern blot analysis of the liver proteins revealed that mouse liver expres ses two proteins, BCL-X-L and BCL-X-S, and does not express BCL-2. As the t oxicity 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 phos phorylated form of BCL-XL. Appearance of this higher molecular weight BCL-X L protein band correlated with massive apoptotic death of liver cells along with ladder-like DNB fragmentation. In the same lime period, death inhibit ory 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. Una ltered 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 st udy may have demonstrated for the first time drug-induced changes in the ex pression of anti-apoptotic gene BCL-X-L, and a positive link between AAP-in duced apoptotic death and modification of BCL-X-L protein in vivo.