Ursodeoxycholic Acid May Inhibit Deoxycholic Acid-Induced Apoptosis by Modulating Mitochondrial Transmembrane Potential and Reactive Oxygen Species Production

Abstract

Background: The hydrophilic bile salt ursodeoxycholate (UDCA) inhibits injury by hydrophobic bile acids and is used to treat cholestatic liver diseases. Interestingly, hepatocyte cell death from bile acid-induced toxicity occurs more frequently from apoptosis than from necrosis. However, both processes appear to involve the mitochondrial membrane permeability transition (MPT). In this study, we determined the inhibitory effect of UDCA on deoxycholic acid (DCA)-induced MPT in isolated mitochondria by measuring changes in transmembrane potential (APm) and production of reactive oxygen species (ROS). In addition, we examined the expression of apoptosis-associated proteins in mitochondria isolated from livers of bile acid-fed animals. Materials and Methods: Adult male rats were maintained on standard diet supplemented with DCA and/or UDCA for 10 days. Mitochondria were isolated from livers by sucrose/percoll gradient centrifugation and MPT was measured using spectrophotometric and fluorimetric assays. APm and ROS generation were determined by FACScan analysis. Cytoplasmic and mitochondrial protein abundance were determined by Western blot analysis.Results: DCA increased mitochondrial swelling 25-fold over controls (p < 0.001); UDCA reduced the swelling by >40% (p < 0.001). Similarly, UDCA inhibited DCAmediated release of calcein-loaded mitochondria by 50% (p < 0.001). APm was significantly decreased in mitochondria incubated with DCA but not with UDCA. ATm disruption was followed closely by increased superoxide anion and peroxides production (p < 0.01). Coincubation of mitochondria with UDCA significantly inhibited the changes associated with DCA (p < 0.05). In vivo, DCA feeding was associated with a 4.5-fold increase in mitochondria-associated Bax protein levels (p < 0.001); combination feeding with UDCA almost totally inhibited this increase (p < 0.001). Conclusion: UDCA significantly reduces DCA-induced disruption of ATm, ROS production, and Bax protein abundance in mitochondria, suggesting both shortand long-term mechanisms in preventing MPT. The results suggest a possible role for UDCA as a therapeutic agent in the treatment of both hepatic and nonhepatic diseases associated with high levels of apoptosis.

Consequences of bile salt biotransformations by intestinal bacteria

“…[107][108][109] UDCA treatment dilutes the concentration of DCA, and is thought to modulate the membrane and inflammatory effects of secondary bile acids on the colonic mucosa by increasing the hydrophilicity of the colonic bile acid milieu, and suppressing cell-signaling pathways activated by DCA. 110,111 Problematically, bile acid 7a-dehydroxylating bacteria, including Clostridium scindens and C. hiranonas are capable of converting UDCA to LCA through bile acid 7b-dehydroxylation. 8 Studies have shown that LCA is toxic, and causes DNA damage in bacteria 112 and colonocytes.…”

Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation

“…11 In fact, UDCA appears to regulate apoptosis in both hepatic and nonhepatic cells by preventing mitochondrial depolarization and reactive oxygen species production. 10,13 Interestingly, the ability of UDCA to inhibit apoptosis appears to be independent of the inducing agent and its pathway, suggesting a common antiapoptotic mechanism(s). In particular, while the pathway to execution may be more direct with DCA, the apoptosis from TGF-b1 and okadaic acid are equally sensitive to the inhibitory effects of UDCA.…”

“…In vivo DCA feeding was associated with a 4.5-fold increase in mitochondrial-associated Bax protein levels, while combination feeding with UDCA significantly inhibited these changes. 13 Like Bcl-2, UDCA appears to modulate Bax-induced events at several levels, including prevention of Bax redistribution, inhibition of cytochrome c release, and modulation of cytochrome cmediated downstream events such as DEVD-specific caspase activity and PARP cleavage. It remains to be determined whether other factors, such as Bid-induced conformational change of Bax and cytochrome c release, 64 are in some way modulated by UDCA.…”

“…The effect was observed for a variety of agents acting through different apoptotic pathways involving mitochondrial membrane perturbation. 10,13 It is widely recognized that alterations in mitochondrial function, and particularly the MPT, play a key role during apoptosis in many cell types. In fact, the loss of mitochondrial transmembrane potential (Dc m ), release of caspase activators and participation of pro-and antiapoptotic Bcl-2 family proteins appear to be critical events during apoptosis.…”

Modification of hepatic genomic DNA using RNA/DNA oligonucleotides

“…26 Extracts were prepared from nuclei and highly purified mitochondria isolated from rat liver, and the authenticity and purity of the extracts established by Western blot, enzymatic activity and electron microscopy. [28][29][30] Using this PCR-independent bacterial read-out system and extracts derived from rat liver, we have observed a chimeraplast-specific and mitochondrial protein extract dose-dependent growth of dual resistant colonies, ranging from 0.003 ± 0.002% to 0.05 ± 0.002%. This rate of repair in vitro did not differ significantly from that observed in parallel reactions using rat liver nuclear extract.…”