Chronic ethanol consumption induces oxidative stress and elevates LPS/TNF-α levels in the liver, both of which play key roles in liver injury [27
]. Ethanol treatment elevates CYP2E1 levels and this increase in CYP2E1 may contribute to pathways by which ethanol induces oxidative stress [31
]. Increased CYP2E1 expression sensitized hepatocytes to TNF-α toxicity as mediated via a c-Jun cell death pathway [34
]. We have shown that increasing CYP2E1 levels via treatment with pyrazole sensitize rats and mice to LPS or TNF-α–induced hepatotoxicity, and that toxicity was associated with elevated oxidative stress, activation of p38 MAPK and JNK, and mitochondrial injury [4
]. The latter included increases in calcium-induced mitochondrial swelling, elevated MDA levels, lower GSH levels [6
]. It is not clear if the mitochondrial injury and/or MPT are important for the pyrazole plus LPS-induced liver injury or are consequences of the liver injury, i.e. are not central to the overall mechanism of liver injury in this model. The goal of this report was to evaluate whether CsA, a classic inhibitor of the MPT, would blunt the pyrazole plus LPS-induced mitochondrial damage, and if so, would prevent the liver injury.
Injecting pyrazole plus LPS-treated mice with CsA resulted in a decrease in mitochondrial swelling as compared to pyrazole plus LPS corn oil controls thus establishing that CsA lowers/prevents the MPT under these conditions. The CsA treatment prevented release of cytochrome c into the cytosolic fraction and also decreased mitochondrial MDA levels while elevating mitochondrial GSH levels. Thus, treatment with CsA protects against the pyrazole plus LPS-induced mitochondrial injury. Associated with this protection against mitochondrial injury is a protection against the pyrazole plus LPS-induced liver injury and formation of HNE and 3-NT protein adducts. This would suggest that damage to the mitochondria plays a central role in the pyrazole plus LPS liver injury and oxidative/nitrosative stress. The CsA treatment did not prevent induction of previously identified upstream factors from the mitochondria which mediate pyrazole plus LPS liver injury such as CYP2E1, TNF-α, iNOS and p38 MAPK and JNK [4
], further suggesting that the site of action for CsA protection is the mitochondria. These results indicate that mitochondria are the critical target of pyrazole plus LPS in mediating liver injury.
Since induction of CYP2E1, TNF-α, and iNOS increases ROS/reactive nitrogen species, it is not clear why CsA decreases HNE and 3-NT adducts in the liver after pyrazole plus LPS treatment when the CsA treatment is not blunting the increases in these agents. This would suggest that the mitochondria, besides being a target for ROS, are also important in generating additional ROS under these conditions i.e. the MPT is elevating ROS production, and CsA, by preventing the MPT, lowers ROS production. It has been proposed that MPT is a significant cause of ROS generation [35
] e.g. in calcium uptake studies by mitochondria, twenty one CsA analogs lowered mitochondrial ROS production with an efficacy which paralleled their potency of inhibiting the MPT [36
]. “ROS-induced ROS release (RIRR)” is generated by circuits requiring mitochondrial membrane channels including the MPT pore and the inner membrane anion channel (IMAC) [37
]. The accumulated exposure to ROS leads to an oxidant stress burden in mitochondria that can reach a threshold (MPT ROS threshold) level capable of inducing the MPT pore and can trigger the opening of one of the requisite mitochondrial channels (MPT pore or IMAC), which in turn leads to the simultaneous collapse of mitochondrial membrane potential and a transient increase in ROS generation by the electron transfer chain [37
]. Release of this ROS burst to the cytosol could potentially function as a “second messenger” to active RIRR in neighboring mitochondria. Thus mitochondria-to-mitochondria RIRR may constitute a positive feedback mechanism for enhanced ROS production leading to potentially significant mitochondrial and cellular injury [37
The release of cytochrome c from the mitochondria may also enhance mitochondrial ROS production as a result of a lower efficiency of electron transfer. The mechanisms of cytochrome c release remain controversial [41
]. One proposal is that pro-apoptotic BCL-2 family members, such as tBID, BAX and BAK, promote formation of specific cytochrome c release channels in the mitochondrial outer membrane [41
]. Another mechanism proposes that pores form in the inner membrane that nonspecifically conducts solutes up to 1500 Da. Opening of these pores, the MPT pore, leads to mitochondrial swelling. Consequently, the outer membrane ruptures to release intermembrane proteins such as cytochrome c [43
]. Anti-apoptotic BCL-2 family proteins (e.g. BCL-2, BCL-xL, BCL-W, A1, Mcl-1), contain BH domains 1-4 and are generally integrated within the outer mitochondrial membrane. These proteins function within the apoptotic pathway to directly bind and inhibit the pro-apoptotic BCL-2 family proteins. The pro-apoptotic BCL-2 family proteins are divided into two classes: the effector molecules (e.g. BAK and BAX), which contain BH1-3 domains and permeabilize the outer mitochondrial membrane by creating the proteolipid pore responsible for cytochrome c release; and the BH3-only proteins (e.g. BAD, BID, BIK, BIM, BMF, bNIP3, HRK, Noxa, PUMA) [44
]. While pyrazole plus LPS treatment increased the expression of pro-apoptotic proteins (e.g. BAX, BID, BCL-xS) 2-2.5 fold in liver mitochondria, which may contribute to development of the MPT, CsA did not attenuate these increases. Thus CsA does not prevent the MPT by modulating levels of BCL-2 family members.
Peroxynitrite induces the MPT [45
]. The elevated production of superoxide due to TNF-α plus CYP2E1 and the generation of nitric oxide due to induction of iNOS, promotes formation of peroxynitrite as evident from the increase in 3-NT protein adducts. We hypothesize that the initial generation of ROS/reactive nitrogen species from TNF-α, CYP2E1 and iNOS causes MPT, which then further elevates ROS production. CsA, by blocking MPT prevents this secondary enhanced production of ROS as reflected by CsA inhibition of 3-NT and HNE protein adduct formation.
presents a scheme to summarize what we believe are main features in the pyrazole plus LPS liver injury model. LPS induces Kupffer cells to release TNF-α, while pyrazole induces hepatocyte CYP2E1 expression. TNF-α plus CYP2E1 promote an elevated oxidative stress while activates iNOS, p38 MAPK and JNK2. iNOS, p38 MAPK and JNK are also present in the mitochondria and it is possible that the pyrazole plus LPS-induced oxidative stress activates these enzymes in the mitochondrial compartment in addition to the cytosol. This remains to be determined. Increases in ROS production and MAPK activation results in an imbalance between anti- and pro-apoptotic BCL-2 family proteins, MPT, release of cytochrome c, and further production of ROS. The MPT, the loss of cytochrome c, and the mitochondrial oxidative stress results in hepatocyte necrosis. CsA prevents the MPT, blocks cytochrome c release from mitochondria, and lowers mitochondrial oxidative stress thereby protecting the hepatocyte from necrosis. These results support the concept that blocking MPT could be a strategy to treat alcohol liver injury.
Fig. 7 Scheme for the protection by cyclosporin A (CsA) against pyrazole plus lipopolysaccharide (LPS) liver injury. LPS induces Kupffer cells to release TNF-α, while pyrazole induces hepatocyte CYP2E1 expression. TNF-α plus CYP2E1 enhance production (more ...)