cytochrome-c-t and cobaltiprotoporphyrin

Brain death (BD) leads to a marked increase in apoptosis, which influences the viability of donor organs. Induction of heme oxygenase 1 (HO-1) has been shown to exert beneficial effects in different liver injury models. Therefore, we examined the effect of pretreating rats with cobalt protoporphyrin (CoPP), an HO-1 inducer, on apoptosis in liver during BD and elucidated the mechanisms involved. First, rats were killed at 0, 1, 2, 4 and 6 h after BD induction to examine the expression of hepatic HO-1. Second, rats were randomly divided into four groups (n=6): (S group) rats undergoing sham operation, (CS group) rats pretreated with CoPP for 24 h before the sham operation, (B group) rats undergoing BD for 6 h, (CB group) rats pretreated with CoPP for 24 h before BD induction. The expression levels of hepatic HO-1 mRNA and protein in rats increased at 0, 1, 2, 4 and 6h after BD induction, compared with sham operated rats. In the CB group compared with the B group, the increased hepatic expression of HO-1 correlated with a significant decrease in serum ALT/AST levels, fewer apoptotic cells in liver, increased hepatic expression of Mcl-1 and Bcl-2, and decreased hepatic expression of Bax, cytosolic cytochrome c and cleaved caspase-3. CoPP inhibits apoptosis in liver of BD rats in part via modulating the mitochondrial apoptosis pathway. HO-1 may serve as a potential target for improving the quality of organs from BD donors.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; bcl-2-Associated X Protein; Brain Death; Caspase 3; Cytochromes c; Heme Oxygenase-1; Liver; Male; Membrane Proteins; Models, Animal; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Protoporphyrins; Rats, Sprague-Dawley; RNA, Messenger; Tissue and Organ Harvesting

Cadmium (Cd) is a well known environmental and industrial toxicant causing damaging effects in numerous organs. In this study, we examined the role of heme oxygenase-1 (HO-1) in modulating the Cd-induced apoptosis in human hepatoma (HepG2) cells after 24 h exposure.. HepG2 cells were exposed to 5 and 10 μM Cd as CdCl2 for 24 h while other sets of cells were pre-treated with either 10 μM Cobalt protoporphyrin (CoPPIX) or 10 μM Tin protoporphyrin (SnPPIX) for 24 h, or 50 μM Z-DEVD-FMK for 1 h before exposure to 5 and 10 μM CdCl2 for 24 h. Expressions of caspase 3, cytosolic cytochrome c, mitochondrial Bax and anti-apoptotic BCL-xl proteins were assessed by western blot. Intracellular reactive oxygen species (ROS) production was determined using the dihydrofluorescein diacetate (H2DFA) method. Cell viability was assessed by MTT assay, while a flow cytometry method was used to assess the level of apoptosis in the cell populations.. Our results show that there were a significant increase in the expression of cytosolic cytochrome c, mitochondrial Bax protein, and caspase 3 at 5 and 10 μM compared to the control, but these increases were attenuated by the presence of CoPPIX. The presence of SnPPIX significantly enhanced Cd-induced caspase 3 activities. CoPPIX significantly decreased the level of ROS production by 24.6 and 22.2 % in 5 and 10 μM CdCl2, respectively, but SnPPIX caused a significant increase in ROS production in the presence of CdCl2. HepG2 cell viability was also significantly impaired by 13.89 and 32.53 % in the presence of 5 and 10 μM CdCl2, respectively, but the presence of CoPPIX and Z-DEVD-FMK significantly enhanced cell survival, while SnPPIX enhanced Cd-impaired cell viability. The presence of CoPPIX and Z-DEVD-FMK also significantly decreased the population of apoptotic and necrotic cells compared with Cd.. In summary, the present study showed that HO-1 attenuates the Cd-induced caspase 3 dependent pathway of apoptosis in HepG2 cells, probably by modulating Cd-induced oxidative stress.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cadmium; Carcinoma, Hepatocellular; Caspase 3; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Heme Oxygenase-1; Hep G2 Cells; Humans; Liver Neoplasms; Metalloporphyrins; Mitochondria; Oligopeptides; Protoporphyrins; Reactive Oxygen Species