Mitochondrial GSH Depletion Research Articles

Hepatotoxic effects of acetaminophen. Protective properties of tryptophan derivatives

Rat intoxication with acetaminophen (APAP) (500–1500 mg/kg body weight, intragastrically) caused a considerable dose-dependent decrease in reduced glutathione (GSH) level in both liver cell cytoplasm and mitochondria (at the dose 1500 mg/kg body weight by 60% and 33%, respectively). The decrease in cytoplasmic GSH level was more pronounced than in mitochondria. Despite of significant mitochondrial GSH depletion we did not observe any inactivation of the mitochondrial enzymes: succinate dehydrogenase, α-ketoglutarate dehydrogenase, glutathione peroxidase, and also any decrease in the respiratory activity of liver mitochondria isolated from APAP-intoxicated rats. We have investigated hepatoprotector properties of tryptophan derivatives, melatonin and N-acetyl-nitrosotryptophan (a nitric oxide donor). The pineal gland hormone, melatonin, a known antioxidant (10 mg/kg body weight), did not prevent intramitochondrial GSH, but decreased the APAP hepatotoxicity evaluated as the decrease in the activity of marker enzymes of hepatic damage, ALT and AST and total bilirubin content in blood plasma of intoxicated rats, whereas NNT did not exhibit any hepatoprotective effects.

Pathologic role of stressed-induced glucocorticoids in drug-induced liver injury in mice

We previously reported that acetaminophen (APAP)-induced liver injury (AILI) in mice is associated with a rise in serum levels of the glucocorticoid (GC), corticosterone. In the current study, we provide evidence that endogenous GC play a pathologic role in AILI. Specifically, pretreatment of mice with the GC receptor (GCR) inhibitor, RU486 (mifepristrone), protected normal but not adrenalectomized mice from AILI, while pretreatment with dexamethasone, a synthetic GC, exacerbated AILI. RU486 did not affect the depletion of whole liver reduced GSH or the formation of APAP–protein adducts. It also had no effects on the formation of reactive oxygen species or the depletion of mitochondrial GSH or ATP. While RU486 pretreatment also protected against halothane-induced liver injury, it exacerbated concanavalin A (ConA)- and carbon tetrachloride (CCl 4)-induced liver injury, demonstrating the complexity of GC effects in different types of liver injury. Conclusion: These results suggest that under certain conditions, elevated levels of GC might represent a previously unappreciated risk factor for liver injury caused by APAP and other drugs through the diverse biological processes regulated by GCR.

Mitochondria, cholesterol and amyloid β peptide: a dangerous trio in Alzheimer disease

The molecular mechanisms of Alzheimer's disease (AD) are not fully understood. Extensive evidence from experimental models has involved the overgeneration and accumulation of toxic amyloid beta peptides (Abeta) in the onset and progression of the disease. The amyloidogenic processing of amyloid precursor protein into pathogenic Abeta fragments is thought to occur in specific domains of the plasma membrane and favored by cholesterol enrichment. Intracellular Abeta accumulation is known to induce oxidative stress, predominantly via mitochondria targeting of toxic Abeta. Recent evidence using mouse models of cholesterol loading has demonstrated that the specific mitochondrial cholesterol pool sensitizes neurons to Abeta-induced oxidant cell death and caspase-independent apoptosis due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics. mGSH replenishment by permeable precursors such as glutathione ethyl ester protected against Abeta-mediated neurotoxicity and inflammation. Thus, these novel data expand the pathogenic role of cholesterol in AD indicating that in addition to fostering Abeta generation, mitochondrial cholesterol determines Abeta neurotoxicity via mGSH regulation.

Malarial infection develops mitochondrial pathology and mitochondrial oxidative stress to promote hepatocyte apoptosis

Activation of the mitochondrial apoptosis pathway by oxidative stress has been implicated in hepatocyte apoptosis during malaria. Because mitochondria are the source and target of reactive oxygen species (ROS), we have investigated whether hepatocyte apoptosis is linked to mitochondrial pathology and mitochondrial ROS generation during malaria. Malarial infection induces mitochondrial pathology by inhibiting mitochondrial respiration, dehydrogenases, and transmembrane potential and damaging the ultrastructure as evident from transmission electron microscopic studies. Mitochondrial GSH depletion and formation of protein carbonyl indicate that mitochondrial pathology is associated with mitochondrial oxidative stress. Fluorescence imaging of hepatocytes documents intramitochondrial superoxide anion (O 2 − ) generation during malaria. O 2 − inactivates mitochondrial aconitase to release iron from iron–sulfur clusters, which forms the hydroxyl radical ( OH) interacting with H 2O 2 produced concurrently. Malarial infection inactivates mitochondrial aconitase, and carbonylation of aconitase is evident from Western immunoblotting. The release of iron has been documented by fluorescence imaging of hepatocytes using Phen Green SK, and mitochondrial OH generation has been confirmed. During malaria, the depletion of cardiolipin and formation of the mitochondrial permeability transition pore favor cytochrome c release to activate caspase-9. Interestingly, mitochondrial OH generation correlates with the activation of both caspase-9 and caspase-3 with the progress of malarial infection, indicating the critical role of OH.

Hydroxychalcones induce apoptosis in B16-F10 melanoma cells via GSH and ATP depletion

Searching for leading compounds of new drugs for cancer therapy, we studied the toxicity of 13 hydroxychalcones never tested before toward melanoma cell line (B16-F10). The compounds were obtained by aldolic condensation between aldehydes and hydroxylated acetophenones, in alkaline conditions. Three of them showed cytotoxicity to the cell line. Two of them induced mitochondrial GSH and ATP depletion and promoted cell death through apoptosis in melanoma cells. One of the compounds induced cell death through necrosis but did not significantly decrease the intracellular mitochondrial GSH and ATP levels in melanoma cells. The results suggest that the predominant factor for the activity is the molecule shape, and secondarily the number of hydroxyl groups.

Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.

Essential Roles of the PI3 Kinase/Akt Pathway in Regulating Nrf2-Dependent Antioxidant Functions in the RPE

To investigate functional interactions between the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant system in cultured human retinal pigment epithelium (RPE) cells. Cultured ARPE-19 cells were treated with different concentrations of PI3K inhibitors, followed by exposure to sulforaphane, an Nrf2 inducer. Akt phosphorylation was detected by Western blot analysis. Intracellular glutathione (GSH) content was measured by HPLC. Expression of genes downstream of Nrf2, including glutamate-cysteine ligase (GCL) and glutathione S-transferase, was measured by quantitative RT-PCR. Nrf2 activity was measured by a dual luciferase assay after transfection of a reporter plasmid containing the antioxidant response element (ARE). The small interference RNA approach was used to knock down Nrf2 in the RPE. Nrf2 localization was determined by subcellular fractionation and Western blot analyses. PI3K inhibitors wortmannin and LY294002 caused dose-dependent cellular and mitochondrial GSH depletion and downregulation of the modulatory subunit of GCL in cultured RPE cells. Both the basal and the induced Nrf2 activities were inhibited by wortmannin and LY294002. Overexpression of a constitutively active form of Akt potentiated Nrf2 activation, and the effect of Akt was blocked by siRNA that knocked down Nrf2. LY294002 also inhibited sulforaphane-induced Nrf2 nuclear translocation. The PI3K/Akt pathway plays key roles in regulating Nrf2-ARE-dependent protection against oxidative stress in the RPE.

Ester derivatives of gallic acid with potential toxicity toward L1210 leukemia cells

Gallic acid and gallates with the same number of hydroxyl groups and varying the length of the side carbon chain, with respective lipophilicity being defined through the C log P values, were examined for their ability to induce apoptosis (through the DNA ladder fragmentation pattern), mitochondrial and cytoplasmic GSH depletion and NF-κB activation in murine lymphoblastic L1210 leukemia cells. A relationship between cytotoxic effect and a limited degree of lipophilicity was observed.

A new acetylcholinesterase inhibitor with anti-PAF activity modulates oxidative stress and pro-inflammatory mediators release in stimulated RAW 264.7 macrophage cells. Comparison with tacrine

Inflammatory injury and induction of oxidative stress have been implicated as causative factors in neurodegenerative diseases such as Alzheimer's disease (AD). Using LPS-stimulated RAW 264.7 macrophages as a model of inflammatory injury, LPS was found to stimulate ROS production (159%), GSH depletion (15%) and loss of mitochondrial activity (32%) as well as TNFα release (40%), and NO production (13.7 times), all parameters involved in AD. PMS777, a tetrahydrofuran derivative, designed as a dual PAF and acetylcholinesterase inhibitor, was found to decrease ROS (up to 32%) and NO production (up to 5 times), TNFα release (33%). PMS777 also prevents loss of mitochondrial activity, and GSH depletion. In contrast, tacrine was found to decrease ROS production (57% up to 102%) and TNFα level (up to 30%). It decreases NO release only at the highest concentrations without preventing loss of mitochondrial activity and GSH depletion. In this study, we show that PMS777 is strongly anti-inflammatory against LPS-induced responses in RAW 264.7. Differential effects between PMS777 and tacrine could be attributed to the anti-PAF activity of PMS777 which was able to fight inflammatory events and oxidative injury whereas tacrine only minimizes them. PMS777 could open a new approach in the treatment of AD.

Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl 2 (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24 h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond. The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.

Subcellular compartmentalization of glutathione: Correlations with parameters of oxidative stress related to genotoxicity

Glutathione (GSH) is a major component of the antioxidant defence system of mammalian cells and is found in subcellular pools within the cytoplasm, nucleus and mitochondria. To evaluate the relationships between these pools and parameters of oxidative stress related to genotoxicity, wild type (WT) and 8-oxo-2'-deoxyguanosine glycosylase 1 (OGG1)-null (mOGG1(-/-)) mouse embryonic fibroblasts (MEF) were treated with buthionine sulphoximine (BSO; 0-1000 microM, 24 h), an inhibitor of GSH biosynthesis. BSO treatment resulted in a concentration-dependent depletion of GSH from the cytoplasm, but depletion of mitochondrial and nuclear GSH occurred only at concentrations > or =100 microM. GSH levels were correlated with reactive oxygen species (ROS), lipid peroxidation (measured as the increase in the genotoxic end-product malondialdehyde (MDA)) and oxidative DNA modifications, measured as both frank DNA strand-breaks (FSB) and oxidized purine lesions (OxP) using the alkaline comet assay with formamidopyrimidine DNA glycosylase (FPG) modification; this system allowed for the identification of BSO-induced DNA modifications as primarily mutagenic 8-oxo-2'-deoxyguanosine lesions. A number of significant correlations were observed. First, negative linear correlations were observed between mitochondrial GSH and ROS (r = -0.985 and r = -0.961 for WT and mOGG1(-/-) MEF, respectively), and mitochondrial GSH and MDA (r = -0.967 and r = -0.963 for WT and mOGG1(-/-) MEF, respectively). Second, positive linear correlations were observed between ROS and MDA (r = 0.996 and r = 0.935 for WT and mOGG1(-/-) MEF, respectively), and ROS and OxP (r = 0.938 and r = 0.981 for WT and mOGG1(-/-) MEF, respectively). Finally, oxidative DNA modifications displayed a negative linear correlation with nuclear GSH (r = -0.963 and -0.951 between nuclear GSH and FSB and OxP, respectively, for WT MEF and r = -0.960 between nuclear GSH and OxP in mOGG1(-/-) MEF), thus, demonstrating the genotoxic potential of compounds that deplete GSH. The findings highlight the critical roles of the mitochondrial and nuclear GSH pools in protecting cellular components, particularly DNA, from oxidative modification.

Mitochondrial glutathione: Hepatocellular survival–death switch

Steatohepatitis represents an advanced stage of fatty liver disease that encompasses alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). The progression from steatosis to steatohepatitis is poorly understood. One of the clues to this progression is the sensitization of hepatocytes to oxidative stress and cytokine-induced cell death. Mitochondrial glutathione (mGSH), which plays a central role in the control of mitochondrial reactive oxygen species (ROS) generation, modulates the sensitivity to cell death pathways. Mitochondrial GSH depletion due to alcohol-mediated alteration in mitochondrial membrane dynamics underlies the susceptibility of hepatocytes from alcohol-fed models to tumor necrosis factor (TNF), and in nutritional and genetic models of hepatic steatosis, mGSH depletion occurs due to the enrichment of mitochondria in free cholesterol, resulting in decreased mitochondrial membrane fluidity. The signaling of TNF through its membrane receptor TNFR1 from complex I to complex II is similar in hepatocytes depleted or not depleted in mGSH, yet hepatocellular susceptibility to TNF occurs if mGSH is depleted. Thus, mGSH is a critical factor in the development of steatohepatitis through sensitization of hepatocytes to inflammatory cytokines, and understanding the homeostasis of cholesterol and its trafficking to mitochondria may be of relevance in the pathophysiology of ASH and NASH.

Inducible nitric oxide synthase up‐regulation and mitochondrial glutathione depletion mediate cyanide‐induced necrosis in mesencephalic cells

We have previously shown in rat primary cultured mesencephalic cells that cyanide induces a high level of oxidative stress and necrotic death. To evaluate the mechanism of the cytotoxicity, the effects of cyanide on intracellular glutathione (GSH) pools and inducible nitric oxide synthase (iNOS)-mediated reactive nitrogen species (RNS) generation were studied. Cyanide rapidly depleted intracellular GSH. Restoration of GSH blocked cell death, whereas depletion of GSH by synthesis inhibition increased the necrosis. Selective depletion of mitochondrial GSH (mtGSH) increased oxidative stress and enhanced cell death, whereas the cytoplasmic pool was not critical to cell survival. These actions were accompanied by increased iNOS expression as determined by Western blot analysis, RT-PCR and immunohistochemistry. Up-regulation of iNOS led to increased generation of NO as reflected by elevated nitrite levels (an end product of NO metabolism). It was determined by use of a selective inhibitor that up-regulation of iNOS expression was transcriptionally regulated by activation of nuclear factor-kappaB, a redox-sensitive transcription factor. It was concluded that, in cyanide-mediated neurotoxicity, mtGSH is a vital component of the cellular antioxidant defense, and its depletion can lead to oxidative stress-mediated iNOS up-regulation, thus enhancing RNS generation and necrosis.

P3-371: Mitochondrial GSH depletion sensitizes human neuroblastoma cells to beta-amyloid peptide-induced oxidative stress and apoptotic cell death

Comunicacion presentada en la Alzheimer's Association 10th International Conference on Alzheimer's Disease and Related Disorders, celebrada del 15 al 20 de julio de 2006 en Madrid (Espana)

Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion

Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts and lead to heart disease. At present, the source of these cardiac ROS or the subcellular site of cardiac GSH loss [i.e., cytosolic (cGSH) or mitochondrial (mGSH) GSH] has not been completely elucidated. With the use of rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced diabetic rat heart, the mitochondrial origin of ROS was established. Furthermore, mitochondrial damage, as evidenced by loss of membrane potential, increases in oxidative stress, and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and -3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, L-buthionine-sulfoximine (BSO; 10 mmol/kg ip), which blocks GSH synthesis, or diethyl maleate (DEM; 4 mmol/kg ip), which inactivates preformed GSH, was administered in diabetic rats for 4 days after STZ administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were coadministered in diabetic rats. In this setting, mGSH was undetectable and cardiac apoptosis was further aggravated compared with the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification of mGSH in diabetic rat hearts and prevented apoptosis. Our data suggest for the first time that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.

Protection of rats against 3-butene-1,2-diol-induced hepatotoxicity and hypoglycemia by N-acetyl- l-cysteine

3-Butene-1,2-diol (BDD), an allylic alcohol and major metabolite of 1,3-butadiene, has previously been shown to cause hepatotoxicity and hypoglycemia in male Sprague–Dawley rats, but the mechanisms of toxicity were unclear. In this study, rats were administered BDD (250 mg/kg) or saline, ip, and serum insulin levels, hepatic lactate levels, and hepatic cellular and mitochondrial GSH, GSSG, ATP, and ADP levels were measured 1 or 4 h after treatment. The results show that serum insulin levels were not causing the hypoglycemia and that the hypoglycemia was not caused by an enhancement of the metabolism of pyruvate to lactate because hepatic lactate levels were either similar (1 h) or lower (4 h) than controls. However, both hepatic cellular and mitochondrial GSH and GSSG levels were severely depleted 1 and 4 h after treatment and the mitochondrial ATP/ADP ratio was also lowered 4 h after treatment relative to controls. Because these results suggested a role for hepatic cellular and mitochondrial GSH in BDD toxicity, additional rats were administered N-acetyl- l-cysteine (NAC; 200 mg/kg) 15 min after BDD administration. NAC treatment partially prevented depletion of hepatic cellular and mitochondrial GSH and preserved the mitochondrial ATP/ADP ratio. NAC also prevented the severe depletion of serum glucose concentration and the elevation of serum alanine aminotransferase activity after BDD treatment without affecting the plasma concentration of BDD. Thus, depletion of hepatic cellular and mitochondrial GSH followed by the decrease in the mitochondrial ATP/ADP ratio was likely contributing to the mechanisms of hepatotoxicity and hypoglycemia in the rat.

S-Adenosylmethionine Protects against Acetaminophen-Induced Hepatotoxicity in Mice

An overdose of acetaminophen (APAP) is the most frequent cause of fulminant liver failure in the United States. Increasing evidence demonstrates that oxidative stress plays an important etiologic role in APAP-induced liver injury. S-Adenosylmethionine (SAMe) is a key intermediate in the hepatic trans-sulfuration pathway and serves as a precursor for glutathione (GSH) as well as the methyl donor in most transmethylation reactions. In the present study, we investigated effects of SAMe on liver injury induced by APAP administration in male C57BL/6 mice. Two related studies were performed. In the first experiment, SAMe (1g/kg BW) was injected intraperitoneally 4 h before APAP (600 mg/kg BW) administration. In the second experiment, SAMe was injected intraperitoneally 1 h after APAP administration. Our results showed that APAP administration induced changes typical of confluent centrilobular necrosis by histological examination and a marked elevation in serum alanine aminotransferase (ALT) activity. APAP administration induced significant decreases in both hepatic and blood SAMe concentrations. In addition, APAP decreased intracellular (both cytosolic and mitochondrial) GSH concentrations along with increased lipid peroxidation in conjunction with mitochondrial dysfunction as documented by Ca²⁺-induced mitochondrial permeability transition. SAMe treatment (both before and after APAP) significantly attenuated the liver injury. Exogenous SAMe prevented the decrease in liver and blood SAMe concentrations. Moreover, SAMe treatment attenuated both cytosolic and mitochondrial GSH depletion as well as mitochondrial dysfunction. We conclude that SAMe at least in part protects the liver from APAP-induced injury by preventing intracellular GSH depletion and mitochondrial dysfunction.

Usnic acid-induced necrosis of cultured mouse hepatocytes: inhibition of mitochondrial function and oxidative stress

Usnic acid, a lichen acid, is a compound found in crude medicines and dietary supplements, including Lipokinetix ®, a supplement marketed as a weight loss agent that caused hepatotoxicity and acute liver failure in patients. In this study, we examined the toxicity of usnic acid and assessed whether usnic acid may be contributing to hepatotoxicity caused by Lipokinetix. In primary cultured murine hepatocytes, usnic acid treatment (5 μM) resulted in 98% necrosis within 16 hr (no apoptosis was detected). Usnic acid treatment was associated with early inhibition and uncoupling of the electron transport chain in mitochondria of cultured hepatocytes. This inhibition of mitochondria by usnic acid corresponded with a fall in ATP levels in hepatocytes. In isolated liver mitochondria, usnic acid was observed to directly inhibit and uncouple oxidative phosphorylation. Oxidative stress appears to be central in usnic acid-induced hepatotoxicity based on the following findings: (1) pretreatment with antioxidants (butylated hydroxytoluene + Vitamin E) decreased usnic acid-induced necrosis by nearly 70%; (2) depletion of mitochondrial GSH with diethylmaleate increased susceptibility of hepatocytes to usnic acid; (3) usnic acid treatment was associated with increase free radical generation, measured using the fluorescent probe, dichlorodihydrofluorescin. The source of reactive oxygen species after usnic acid treatment include autoxidation of usnic acid and increased hydrogen peroxide generation by mitochondria caused by usnic acid inhibition of the respiratory chain, with the latter playing a more prominent role. Taken together, our results suggest that usnic acid is a strong hepatotoxic agent that triggers oxidative stress and disrupts the normal metabolic processes of cells. Usnic acid therefore may contribute to the hepatotoxic effects of Lipokinetix and its use in any supplement must come into question.

Mitochondrial permeability transition induced by reactive oxygen species is independent of cholesterol-regulated membrane fluidity

Cholesterol enrichment of rat liver mitochondria (CHM) impairs atractyloside-induced mitochondrial permeability transition (MPT) due to decreased membrane fluidity. In this study we addressed the effect of cholesterol enrichment on MPT induced by reactive oxygen species (ROS). Superoxide anion generated by xanthine plus xanthine oxidase triggered mitochondrial swelling and cytochrome c release in CHM, which was prevented by butylated hydroxytoluene, an anti-voltage-dependent anion channel antibody, or cyclosporin A. Furthermore, hydrogen peroxide generated by the combination of ganglioside GD3 and mitochondrial GSH depletion elicited mitochondrial swelling and release of cytochrome c, Smac/Diablo and apoptosis-inducing factor in control mitochondria and CHM. Thus, ROS induce MPT and apoptosome activation regardless of decreased mitochondrial membrane dynamics due to cholesterol enrichment.

The role of carotenoids in the prevention of human pathologies

Reactive oxygen species (ROS) and oxidative damage to biomolecules have been postulated to be involved in the causation and progression of several chronic diseases, including cancer and cardiovascular diseases, the two major causes of morbidity and mortality in Western world. Consequently dietary antioxidants, which inactivate ROS and provide protection from oxidative damage are being considered as important preventive strategic molecules. Carotenoids have been implicated as important dietary nutrients having antioxidant potential, being involved in the scavenging of two of the ROS, singlet molecular oxygen ( 1O 2) and peroxyl radicals generated in the process of lipid peroxidation. Carotenoids are lipophilic molecules which tend to accumulate in lipophilic compartments like membranes or lipoproteins. Chronic ethanol consumption significantly increases hydrogen peroxide and decreases mitochondrial glutathione (GSH) in cells overexpressing CYP2E1. The depletion of mitochondrial GSH and the rise of hydrogen peroxide are responsible for the ethanol-induced apoptosis. Increased intake of lycopene, a major carotenoid in tomatoes, consumed as the all- trans-isomer attenuates alcohol induced apoptosis in 2E1 cells and reduces risk of prostate, lung and digestive cancers. Cancer-preventive activities of carotenoids have been associated as well as with their antioxidant properties and the induction and stimulation of intercellular communication via gap junctions which play a role in the regulation of cell growth, differentiation and apoptosis. Gap junctional communication between cells which may be a basis for protection against cancer development is independent of the antioxidant property.