**5. Detailed mechanism of hydrophobic bile acid-induced apoptosis in HepG2 cells**

A hydrophobic bile acid GCDCA activates caspase-8 in hepatocytes via Fas death receptor in the cell membrane. Activated caspase-8 enhances mitochondrial membrane permeability to facilitate the release of cytochrome c, a pro-apoptotic protein, activates caspase-9 and caspase-3 to induce apoptosis. Caspase-8 activated by GCDCA cleaves BAP31 protein in ER membrane and may possibly be associated with Ca2+ release from mitochondria from ER. BAP31 is cleaved to be BAP20 which is an activated form. As shown in Fig. 3, intact (uncleaved) BAP31 protein is observed in ER in untreated hepatic cells, but in GCDCA-treated hepatic cells, the content of intact BAP31 protein is decreased. The decrease in intact BAP31 observed in GCDCA-treated hepatocytes was suppressed by pretreatment with z-IETD-FMK. When recombinant active caspase-8 was added to hepatocytes, decrease in intact BAP31 was observed. GCDCA-induced increased intracellular Ca2+ interacted with mitochondria and caused its dysfunction, followed by increase in mitochondrial membrane permeability and release of pro-apoptotic factors from mitochondria. Also, calpain was activated by the increase in Ca2+ release followed by activation of caspases. Furthermore, ER chaperone BIP is usually bound to an ER stress sensor located within the lumen of ER, but GCDCA treatment causes the increase in unfolded protein, and promotes the release of BIP from the stress sensor and increases the expression of CHOP, an ER stress-related transcription factor that works in the downstream of PERK, an ER stress sensor (Fig. 4). CHOP induces the transcription of Bim, a pro-apoptotic BH3 only protein [55]. Membranes of mitochondria and ER are connected by complexes composed of tethering proteins [50].

These complexes facilitate the transport of calcium and phospholipids and possibly are associated with apoptosis. GCDCA is known to induce apoptosis via mitochondria- and ER stress-mediated pathways via death receptor, but mitochondria and ER themselves seem to be interrelated to each other.

Some Findings on Apoptosis in Hepatocytes 213

**Figure 4. The effect of GCDCA on the CHOP mRNA expression in isolated heaptocytes.** (A) CHOP mRNA expression was determined by semiquantitative PCR. Line graphs representation of the observed fold induction normalized to β-actin for each time point. (B) RT-PCR analysis of CHOP and βactin expression at each incubation time after treatment with GCDCA. (C) Reaction cycles-PCR product yield curves of each reaction mixture were plotted. The intensity of fluorescence was fitted to the data in the linear portion of curves. The resulting CHOP mRNA / β-actin mRNA ratio is represented as the mean+SEM of 8-15 samples. \*p<0.05 and \*\*p<0.01: significant difference from untreated hepatocytes

hepatitis or alcoholic liver cirrhosis [3, 59]. Clinical studies suggested similar findings. Various factors including cytotoxicity by alcohol and its metabolites, changes in metabolizing enzymes, invasion of inflammatory cells, reactive oxygen species, cytokines, hepatic microcirculation, nutritional factors, etc. are involved in the onset of apoptosis. Among others, correlation between ROS or oxidative stress and hepatocyte apoptosis is attracting attention. Orally fed ethanol is absorbed in the upper digestive organs, mainly small intestine, and 90% of it is metabolized in the liver. Upon metabolization, ROS is generated in the liver by alcohol dehydrogenase, microsome-ethanol oxydizing system (MEOS) via cytochrome P450, and NADPH oxidase (NOX) in the cell membrane, which is a center for ROS generation (Fig. 5). In general, oxidative stress inflicts cytotoxicity when excessive ROS is generated in the cells. Antioxidant was shown to reduce hepatocyte apoptosis in acute ethanol-addicted rats [60]. On the other hand, several death receptors and their ligands (especially Fas/FasL) are over-expressed in the liver cells of alcoholic hepatitis patients compared with those in healthy subjects. The levels of Fas and FasL are increased in serious alcoholic hepatitis patients, but there are still many uncertainties about the details of their mediators and biological importance [61]. The increase in FasL might be mediated by ROS or increase in NF-кB which increases the transcription of Fas and FasL genes based on

(control).
