Shannon M. Bailey

Contribution of mitochondria to oxidative stress associated with alcoholic liver disease

The importance of oxidative stress in the development of alcoholic liver disease has long been appreciated. The mechanism by which ethanol triggers an increase in reactive oxygen species in the liver is complex, however, recent findings suggest that the mitochondrion may contribute significantly to the overall increase in oxidant levels in hepatocytes exposed to ethanol acutely or chronically. This review is focused on observations which indicate that the ability of ethanol to increase mitochondrial reactive oxygen species production is linked to its metabolism via oxidative processes and/or ethanol-related alterations to the mitochondrial electron transport chain. Furthermore, the capacity of ethanol-elicited increases in reactive oxygen species to oxidatively modify and inactivate mitochondrial proteins is highlighted as a mechanism by which ethanol might further disrupt the structure and function of mitochondria.The importance of oxidative stress in the development of alcoholic liver disease has long been appreciated. The mechanism by which ethanol triggers an increase in reactive oxygen species in the liver is complex, however, recent findings suggest that the mitochondrion may contribute significantly to the overall increase in oxidant levels in hepatocytes exposed to ethanol acutely or chronically. This review is focused on observations which indicate that the ability of ethanol to increase mitochondrial reactive oxygen species production is linked to its metabolism via oxidative processes and/or ethanol-related alterations to the mitochondrial electron transport chain. Furthermore, the capacity of ethanol-elicited increases in reactive oxygen species to oxidatively modify and inactivate mitochondrial proteins is highlighted as a mechanism by which ethanol might further disrupt the structure and function of mitochondria.

What Part of NO Don't You Understand? Some Answers to the Cardinal Questions in Nitric Oxide Biology

Nitric oxide (NO) regulates biological processes through signaling mechanisms that exploit its unique biochemical properties as a free radical. For the last several decades, the key aspects of the chemical properties of NO relevant to biological systems have been defined, but it has been a challenge to assign these to specific cellular processes. Nevertheless, it is now clear that the high affinity of NO for transition metal centers, particularly iron, and the rapid reaction of NO with oxygen-derived free radicals can explain many of its biological and pathological properties. Emerging studies also highlight a growing importance of the secondary metabolites of NO-dependent reactions in the post-translational modification of key metabolic and signaling proteins. In this minireview, we emphasize the current understanding of the biochemistry of NO and place it in a biological context.

High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo.

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Modification of the Mitochondrial Proteome in Response to the Stress of Ethanol-dependent Hepatotoxicity

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.

Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol

Increased production of reactive oxygen species (ROS) by the mitochondrion has been implicated in the pathogenesis of numerous liver diseases. However, the exact sites of ROS production within liver mitochondria and the electron transport chain are still uncertain. To determine the sites of ROS generation in liver mitochondria we evaluated the ability of a variety of mitochondrial respiratory inhibitors to alter the steady state levels of ROS generated within the intact hepatocyte and in isolated mitochondria. Treatment with myxothiazol alone at concentrations that significantly inhibit respiration dramatically increased the steady-state levels of ROS in hepatocytes. Similar results were also observed in isolated mitochondria oxidizing succinate. Coincubation with antimycin or rotenone had no effect on myxothiazol-induced ROS levels. Myxothiazol stimulation of ROS was mitochondrial in origin as demonstrated by the colocalization of MitoTracker Red and dichlorofluorescein staining using confocal microscopy. Furthermore, diphenyliodonium, an inhibitor that blocks electron flow through the flavin mononucleotide of mitochondrial complex I and other flavoenzymes, significantly attenuated the myxothiazol-induced increase in hepatocyte ROS levels. Together, these data suggest that in addition to the ubiquinone-cytochrome bc(1) complex of complex III, several of the flavin-containing enzymes or iron-sulfur centers within the mitochondrial electron transport chain should also be considered sites of superoxide generation in liver mitochondria.

Effects of alcohol and oxidative stress on liver pathology: the role of the mitochondrion.

This article represents the proceedings of a symposium at the 2001 Research Society on Alcoholism meeting in Montreal, Canada. The chairs were Alan Cahill and Carol C. Cunningham. The presentations were (1) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis: possible involvement of proapoptotic Bcl-2 family protein Bax, by Masayuki Adachi and Hiromasa Ishii; (2) Effects of ethanol on mitochondrial reactive oxygen species production and oxidative protein modification, by Shannon M. Bailey; (3) Acute ethanol binges elicit widespread oxidative mitochondrial DNA damage and depletion: protective effects of antioxidants and inhibitors of ethanol metabolism, by Bernard Fromenty; and (4) Effects of chronic ethanol consumption upon hepatic mtDNA oxidative modification and depletion, by Alan Cahill and Adrian Davies.

Circadian regulation of metabolism

In association with sleep-wake and fasting-feeding cycles, organisms experience dramatic oscillations in energetic demands and nutrient supply. It is therefore not surprising that various metabolic parameters, ranging from the activity status of molecular energy sensors to circulating nutrient levels, oscillate in time-of-day-dependent manners. It has become increasingly clear that rhythms in metabolic processes are not simply in response to daily environmental/behavioral influences, but are driven in part by cell autonomous circadian clocks. By synchronizing the cell with its environment, clocks modulate a host of metabolic processes in a temporally appropriate manner. The purpose of this article is to review current understanding of the interplay between circadian clocks and metabolism, in addition to the pathophysiologic consequences of disruption of this molecular mechanism, in terms of cardiometabolic disease development.

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the ‘canary in the coal mine’ by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value–the Bioenergetic Health Index (BHI)–can be calculated to represent the patients composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

The role of iNOS in alcohol‐dependent hepatotoxicity and mitochondrial dysfunction in mice

Nitric oxide (NO) is now known to control both mitochondrial respiration and organelle biogenesis. Under conditions of ethanol‐dependent hepatic dysfunction, steatosis is increased, and this is associated with increased expression of inducible nitric oxide synthase (iNOS). We have previously shown that after chronic exposure to ethanol, the sensitivity of mitochondrial respiration to inhibition by NO is enhanced, and we have proposed that this contributes to ethanol‐dependent hypoxia. This study examines the role of iNOS in controlling the NO‐dependent modification of mitochondrial function. Mitochondria were isolated from the livers of both wild‐type (WT) and iNOS knockout (iNOS−/−) mice that were fed an isocaloric ethanol‐containing diet for a period of 5 weeks. All animals that consumed ethanol showed some evidence of fatty liver; however, this was to a lesser extent in the iNOS−/− mice compared to controls. At this early stage in ethanol‐dependent hepatic dysfunction, infiltration of inflammatory cells and the formation of nitrated proteins was also decreased in response to ethanol feeding in the iNOS−/− animals. Mitochondria isolated from wild‐type ethanol‐fed mice showed a significant decrease in respiratory control ratio and an increased sensitivity to NO‐dependent inhibition of respiration relative to their pair‐fed controls. In contrast, liver mitochondria isolated from iNOS−/− mice fed ethanol showed no change in the sensitivity to NO‐dependent inhibition of respiration. In conclusion, the hepatic response to chronic alcohol‐dependent cytotoxicity involves a change in mitochondrial function dependent on the induction of iNOS. (HEPATOLOGY 2004;40:565–573.)

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.