**2. Cholestasis and hydrophilic or hydrophobic bile acids**

It has been demonstrated that hydrophobic bile acids damage cellular functions by affecting intracellular organelle and signaling system at the concentrations 100-500 µM, which are lower than those at which they show cytotoxic or detergent actions. Combettes and his coworkers reported that lithocholic acid (LCA) and taurolithocholic acid (TLCA) induce release of calcium ion (Ca2+) from the endoplasmic reticulum (ER) and the increased level of Ca2+ within the cell mediates cytotoxicity due to these hydrophobic bile acids [16]. Combettes and his colleagues speculated that this increase in intracellular Ca2+ levels occurs because LCA activates the inositol (1,4,5)-triphosphate (IP3) receptor, independent from IP3 itself, resulting in the release of Ca2+ from the intracellular organelle, ER [17]. On the other hand, there is a report that the increase in intracellular Ca2+ level induced by hydrophobic bile acid depends on extracellular Ca2+ level [18]. Spivey and his colleagues reported that at 250 µM, glycochenodeoxycholic acid (GCDCA) induces the impairment of mitochondrial function and cellular ATP depletion, followed by a sustained rise in cytosolic Ca2+ resulting from an influx of extracellular Ca2+ leading to the death of hepatocytes, and that this cytotoxicity decreases in the order of GCDCA>CDCA>tauro-CDCA [19].

Some Findings on Apoptosis in Hepatocytes 207

not clarified in detail. In vivo studies showed that UDCA is protecting hepatocytes from hydrophobic bile acid-induced apoptosis [31]. Furthermore, tauro-conjugated form of UDCA, tauro-UDCA (TUDCA), was shown to protect hepatocytes in ischemia-reperfusion injury in rats [32], and ethanol-fed rats [33]. When toxic bile acid is given to rats, apoptosis is induced in the liver, but when UDCA is given in combination, apoptosis is suppressed by inhibition of translocation of pro-apoptotic protein Bax from cytosol to mitochondria and ROS generation. This suppression is observed in cells other than hepatocytes, and UDCA was shown to act on classic mitochondrial-mediated pathway in different types of cells [34]. Taurourusodeoxycholic acid (TUDCA), a substance in which UDCA is conjugated with taurine, was shown to play an important role in various disorders including some liver diseases, type-II diabetes and metabolic syndrome [35, 36] possibly by its actions shown in isolated mitochondria to stabilize mitochondrial membrane directly through affecting channel formation by Bax [37], and bringing changes in ER stress-mediated pathways by

However, in a clinical trial for reevaluation of effectiveness in PBC patients, the effectiveness of UDCA was not acknowledged [38]. In rat isolated primary cultured hepatocytes, UDCA, given in combination with hydrophobic bile acids, was shown to be cytotoxic [39] and

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

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

decreasing caspase-12 activity and decrease in Ca2+ releases.

activates the pro-apoptotic pathway in some condition [40].

GCDCA is also reported to enhance the mitochondrial membrane permeability and induce cytotoxicity, while ursodeoxycholic acid (UDCA) shows suppression of GCDCA-induced enhancement of mitochondrial membrane permeability and cytotoxicity [20, 21]. Bile acidinduced enhancement of mitochondrial membrane permeability was also shown in the in vivo study of cholestasis [22]. Considering the data that increase in reactive oxygen species (ROS) generation in hepatocyte and generation of H2O2 stimulated by tauro-CDCA (TCDCA) in mitochondria preceded TCDCA-induced hepatocyte necrosis, it was speculated that generation of ROS by hydrophobic bile acid constitutes one of the causative factors of hepatic injury in cholestasis [23].

In cholestatic liver disease, loss of hepatocytes or appearance of apoptotic body in hepatocytes was morphologically observed, and involvement of apoptosis was suggested in the hepatocyte injury in cholestasis [24]. Patel and his colleagues reported for the first time that low concentration of glycodeoxycholic acid induced apoptosis in hepatocytes, and pointed out that bile acids may induce necrosis at higher concentrations and apoptosis at lower concentrations in hepatocytes [25, 26]. Furthermore, Sokol and his colleagues reported that hydrophobic bile acid induced lipid peroxidation and mitochondrial dysfunction via enhancement of mitochondrial membrane permeability [27], and we ourselves also reported mitochondria-mediated time- and concentration-dependent apoptotic cell death and endoplasmic reticulum (ER) stress-mediated apoptosis of hepatocytes induced by GCDCA [28, 29].

Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has been widely used as a therapeutic agent for primary biliary cirrhosis (PBC) or cholestasis [30]. Suggested mechanism of action of UDCA includes promotion of bile secretion, detoxification metabolism in the liver, and antioxidant stress response, but its molecular mechanism is still not clarified in detail. In vivo studies showed that UDCA is protecting hepatocytes from hydrophobic bile acid-induced apoptosis [31]. Furthermore, tauro-conjugated form of UDCA, tauro-UDCA (TUDCA), was shown to protect hepatocytes in ischemia-reperfusion injury in rats [32], and ethanol-fed rats [33]. When toxic bile acid is given to rats, apoptosis is induced in the liver, but when UDCA is given in combination, apoptosis is suppressed by inhibition of translocation of pro-apoptotic protein Bax from cytosol to mitochondria and ROS generation. This suppression is observed in cells other than hepatocytes, and UDCA was shown to act on classic mitochondrial-mediated pathway in different types of cells [34]. Taurourusodeoxycholic acid (TUDCA), a substance in which UDCA is conjugated with taurine, was shown to play an important role in various disorders including some liver diseases, type-II diabetes and metabolic syndrome [35, 36] possibly by its actions shown in isolated mitochondria to stabilize mitochondrial membrane directly through affecting channel formation by Bax [37], and bringing changes in ER stress-mediated pathways by decreasing caspase-12 activity and decrease in Ca2+ releases.

206 Apoptosis and Medicine

**2. Cholestasis and hydrophilic or hydrophobic bile acids** 

cytotoxicity decreases in the order of GCDCA>CDCA>tauro-CDCA [19].

hepatic injury in cholestasis [23].

[28, 29].

It has been demonstrated that hydrophobic bile acids damage cellular functions by affecting intracellular organelle and signaling system at the concentrations 100-500 µM, which are lower than those at which they show cytotoxic or detergent actions. Combettes and his coworkers reported that lithocholic acid (LCA) and taurolithocholic acid (TLCA) induce release of calcium ion (Ca2+) from the endoplasmic reticulum (ER) and the increased level of Ca2+ within the cell mediates cytotoxicity due to these hydrophobic bile acids [16]. Combettes and his colleagues speculated that this increase in intracellular Ca2+ levels occurs because LCA activates the inositol (1,4,5)-triphosphate (IP3) receptor, independent from IP3 itself, resulting in the release of Ca2+ from the intracellular organelle, ER [17]. On the other hand, there is a report that the increase in intracellular Ca2+ level induced by hydrophobic bile acid depends on extracellular Ca2+ level [18]. Spivey and his colleagues reported that at 250 µM, glycochenodeoxycholic acid (GCDCA) induces the impairment of mitochondrial function and cellular ATP depletion, followed by a sustained rise in cytosolic Ca2+ resulting from an influx of extracellular Ca2+ leading to the death of hepatocytes, and that this

GCDCA is also reported to enhance the mitochondrial membrane permeability and induce cytotoxicity, while ursodeoxycholic acid (UDCA) shows suppression of GCDCA-induced enhancement of mitochondrial membrane permeability and cytotoxicity [20, 21]. Bile acidinduced enhancement of mitochondrial membrane permeability was also shown in the in vivo study of cholestasis [22]. Considering the data that increase in reactive oxygen species (ROS) generation in hepatocyte and generation of H2O2 stimulated by tauro-CDCA (TCDCA) in mitochondria preceded TCDCA-induced hepatocyte necrosis, it was speculated that generation of ROS by hydrophobic bile acid constitutes one of the causative factors of

In cholestatic liver disease, loss of hepatocytes or appearance of apoptotic body in hepatocytes was morphologically observed, and involvement of apoptosis was suggested in the hepatocyte injury in cholestasis [24]. Patel and his colleagues reported for the first time that low concentration of glycodeoxycholic acid induced apoptosis in hepatocytes, and pointed out that bile acids may induce necrosis at higher concentrations and apoptosis at lower concentrations in hepatocytes [25, 26]. Furthermore, Sokol and his colleagues reported that hydrophobic bile acid induced lipid peroxidation and mitochondrial dysfunction via enhancement of mitochondrial membrane permeability [27], and we ourselves also reported mitochondria-mediated time- and concentration-dependent apoptotic cell death and endoplasmic reticulum (ER) stress-mediated apoptosis of hepatocytes induced by GCDCA

Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has been widely used as a therapeutic agent for primary biliary cirrhosis (PBC) or cholestasis [30]. Suggested mechanism of action of UDCA includes promotion of bile secretion, detoxification metabolism in the liver, and antioxidant stress response, but its molecular mechanism is still However, in a clinical trial for reevaluation of effectiveness in PBC patients, the effectiveness of UDCA was not acknowledged [38]. In rat isolated primary cultured hepatocytes, UDCA, given in combination with hydrophobic bile acids, was shown to be cytotoxic [39] and activates the pro-apoptotic pathway in some condition [40].
