**3. Apoptosis induced by hydrophobic bile acids in rat hepatocytes**

Bile acids are synthesized from cholesterol in the liver, and act as surfactants that help digestion and absorption of lipids, and lipid-soluble vitamins. Major bile acids found in human bile are cholic and chenodeoxycholic acids, and they are secreted into bile as conjugates with taurine or glycine, via amide-bond. Most of bile acids secreted into the duodenum are reabsorbed by active transport in the terminal ileum, and returned to the liver. Bile acids are not so potently toxic as to injure hepatocytes in healthy subjects, but if the bile acid levels in the liver are too high or the ratio of hydrophobic bile acids to hydrophilic bile acids increases, as in the cases when there is some abnormality in bile acid synthesis, they induce apoptosis or necrosis [41]. The potency of their hepatotoxicity is, in the decreasing order, LCA > deoxycholic acid (DCA) > CDCA > CA > UDCA > dehydrocholic acid. The total bile acid in the liver tissue in normal subjects is not more than 10 µM when determined as serum bile acid level, but in patients with cholestasis, CDCA level in the liver tissue increases to about 20 times higher than in the normal case, and the serum bile acid level elevated to 10 to 30 times (100-300 µM) higher than normal, of which hydrophobic bile acid accounted for about 50-60 % [42, 19]. Cholestatic liver diseases are associated with bile duct obstruction by the formation of biliary stones, genetic defects, hepatotoxicity, hepatobiliary tumors [43]. Acute and chronic cholestasis induces hepatocelluar injury, biliary dilatation, hepatic fibrosis, cirrhosis, and ultimately hepatic failure [44]. Decrease in bile flow or total obstruction of bile duct upon cholestasis is induced by the stasis of metabolized products such as cholesterol and bile acids in the liver which are normally eliminated into bile. Especially some hydrophobic bile acids induce cytotoxicity in cultured hepatocytes [45]. In a major hypothesis on the mechanisms of hepatocyte injury, bile acids accumulated in the hepatocytes are regarded to be the major cause of cell death [23]. While bile acids such as taurolithocholic acid (TLCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA) are known to induce necrosis of hepatocytes via increase in oxidative stress mediated by hydroperoxide generation by mitochondria [23], attention is recently focused on apoptotic cell death associated with these bile acids [45, 46].

Some Findings on Apoptosis in Hepatocytes 209

On the other hand, oriental traditional pharmacognosy has utilized bear bile as a medicine for improving gastrointestinal symptoms. The effective ingredient of the bear bile is UDCA, which has been used widely as a choleretic or calculolytic drug for cholesterol gallstone. UDCA is now used for a wide range of liver disease treatment, and the evidence for its effectiveness has been reported one after another. The indications of UDCA include primary biliary sclerosis (PBC), calculolysis, prevention of gallstone formation and UDCA is demonstrated to be effective in most of these. However, there has been a report that in a clinical trial for reevaluation of effectiveness in PBC patients, the effectiveness of UDCA was not acknowledged [47]**.** In rat isolated primary cultured hepatocytes, when given in combination with hydrophobic bile acids, UDCA was reported to be cytotoxic [48] and activates the proapoptotic pathway [49]. We ourselves examined the action of UDCA on GCDCA-induced apoptosis in rat primary cultured hepatocytes [28]. When hepatocytes were incubated in the co-presence of UDCA and GCDCA, UDCA significantly inhibited GCDCA-induced apoptosis in a short incubation (4 hours), but a prolonged incubation (20 hour incubation) potentiated the apoptosis (Fig. 2). In the study of decrease in mitochondrial membrane potential difference, a further decrease in the potential was observed in coincubation of hepatocytes with UDCA and GCDCA even in a short incubation. It was suggested that when cholestatic condition is severe and hepatotoxicity of bile acids is more potent, UDCA might potentiate the toxicity and we pointed out the need for attention in the

**Figure 2. Effect of UDCA on ssDNA in GCDCA-induced apoptotic hepatocytes.** Each value

represents the mean+SEM of 5-15 samples. Other culture conditions were the same as in Fig. 1. \*p < 0.05: significant difference from untreated cells, # p < 0.05: significant difference from GCDCA-treated cells.

clinical use [28].

The mechanism and pathways of bile acid-induced apoptosis in hepatocytes are reported to include death receptor-mediated pathway [9] and increase in ROS generation [31]. We ourselves reported that, in primary cultured hepatocytes isolated from healthy rats, apoptosis is induced by a hydrophobic bile acid in a concentration- and time-dependent manner [28]. When hepatocyte was cultured with 200 µM of GCDCA for 6 hours, ssDNA and caspase-3 activity which are measures of apoptosis increased about 10 times higher than in the untreated cultured hepatocytes (Fig. 1). Also, in hepatocytes treated with 200 µM GCDCA for 4hours, the level of mRNA of Fas and activities of caspase-8 and caspase-3 were significantly increased compared with those in untreated hepatocytes. Further, mitochondrial membrane potential difference of 200 µM GCDCA-treated hepatocytes was decreased by 60% compared with the same in untreated hepatocytes. GDCDA, a hydrophobic bile acid, was demonstrated to induce an increase in Fas death receptors located in cell membrane to activate apoptotic pathway.

**Figure 1. The effect of GCDCA on detection of apoptotic cells.** Isolated hepatocytes (1x106 cells/well) were treated with GCDCA at 37 C. Formamide-denaturable DNA was detected in apoptotic hepatocytes. The ssDNA was stained with the primary antibody (anti-ssDNA-mAb) and peroxidaselabelled secondary antibody for color development. Each value represents the mean+SEM of 6-12 samples. Statistically significant changes are indicated as \*p<0.05 compared with the untreated hepatocytes.

On the other hand, oriental traditional pharmacognosy has utilized bear bile as a medicine for improving gastrointestinal symptoms. The effective ingredient of the bear bile is UDCA, which has been used widely as a choleretic or calculolytic drug for cholesterol gallstone. UDCA is now used for a wide range of liver disease treatment, and the evidence for its effectiveness has been reported one after another. The indications of UDCA include primary biliary sclerosis (PBC), calculolysis, prevention of gallstone formation and UDCA is demonstrated to be effective in most of these. However, there has been a report that in a clinical trial for reevaluation of effectiveness in PBC patients, the effectiveness of UDCA was not acknowledged [47]**.** In rat isolated primary cultured hepatocytes, when given in combination with hydrophobic bile acids, UDCA was reported to be cytotoxic [48] and activates the proapoptotic pathway [49]. We ourselves examined the action of UDCA on GCDCA-induced apoptosis in rat primary cultured hepatocytes [28]. When hepatocytes were incubated in the co-presence of UDCA and GCDCA, UDCA significantly inhibited GCDCA-induced apoptosis in a short incubation (4 hours), but a prolonged incubation (20 hour incubation) potentiated the apoptosis (Fig. 2). In the study of decrease in mitochondrial membrane potential difference, a further decrease in the potential was observed in coincubation of hepatocytes with UDCA and GCDCA even in a short incubation. It was suggested that when cholestatic condition is severe and hepatotoxicity of bile acids is more potent, UDCA might potentiate the toxicity and we pointed out the need for attention in the clinical use [28].

208 Apoptosis and Medicine

hepatocytes.

associated with these bile acids [45, 46].

located in cell membrane to activate apoptotic pathway.

cultured hepatocytes [45]. In a major hypothesis on the mechanisms of hepatocyte injury, bile acids accumulated in the hepatocytes are regarded to be the major cause of cell death [23]. While bile acids such as taurolithocholic acid (TLCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA) are known to induce necrosis of hepatocytes via increase in oxidative stress mediated by hydroperoxide generation by mitochondria [23], attention is recently focused on apoptotic cell death

The mechanism and pathways of bile acid-induced apoptosis in hepatocytes are reported to include death receptor-mediated pathway [9] and increase in ROS generation [31]. We ourselves reported that, in primary cultured hepatocytes isolated from healthy rats, apoptosis is induced by a hydrophobic bile acid in a concentration- and time-dependent manner [28]. When hepatocyte was cultured with 200 µM of GCDCA for 6 hours, ssDNA and caspase-3 activity which are measures of apoptosis increased about 10 times higher than in the untreated cultured hepatocytes (Fig. 1). Also, in hepatocytes treated with 200 µM GCDCA for 4hours, the level of mRNA of Fas and activities of caspase-8 and caspase-3 were significantly increased compared with those in untreated hepatocytes. Further, mitochondrial membrane potential difference of 200 µM GCDCA-treated hepatocytes was decreased by 60% compared with the same in untreated hepatocytes. GDCDA, a hydrophobic bile acid, was demonstrated to induce an increase in Fas death receptors

**Figure 1. The effect of GCDCA on detection of apoptotic cells.** Isolated hepatocytes (1x106 cells/well)

were treated with GCDCA at 37 C. Formamide-denaturable DNA was detected in apoptotic hepatocytes. The ssDNA was stained with the primary antibody (anti-ssDNA-mAb) and peroxidaselabelled secondary antibody for color development. Each value represents the mean+SEM of 6-12 samples. Statistically significant changes are indicated as \*p<0.05 compared with the untreated

**Figure 2. Effect of UDCA on ssDNA in GCDCA-induced apoptotic hepatocytes.** Each value represents the mean+SEM of 5-15 samples. Other culture conditions were the same as in Fig. 1. \*p < 0.05: significant difference from untreated cells, # p < 0.05: significant difference from GCDCA-treated cells.
