Decrease In Cytochrome C Oxidase Activity Research Articles

Methylene blue and photobiomodulation recover cognitive impairment in hepatic encephalopathy through different effects on cytochrome c-oxidase

Mitochondrial dysfunction plays a central role in hepatic encephalopathy (HE), due to changes in enzyme cytochrome c-oxidase (CCO), causing a decline in brain metabolism. We used an HE animal model and applied intracranial administration of methylene blue (MB) and transcranial photobiomodulation (PBM), both targeting CCO, to determine their differential effects on recovering cognition. Five groups of rats were used: sham-operated group + saline (SHAM + SAL, n = 6), hepatic encephalopathy + SAL (HE + SAL, n = 7), SHAM + methylene blue (SHAM + MB, n = 7), HE + MB (n = 7), HE + PBM (n = 7). PBM animals were exposed transcranially to 670 +/− 10 nm LED light at a dose of 9 J/cm2 once a day for 7 days, and the MB and SAL groups were injected with 2.2 μg/0.5 μL in the accumbens. Cognitive dysfunction was evaluated on a striatal stimulus-response task using the Morris water maze. Our results showed cognitive improvement in the HE group when treated with MB. This improvement was accompanied by a decrease in CCO activity in the prefrontal cortex, dorsal striatum, and dorsal hippocampus. When comparing MB and PBM, we found that, although both treatments effectively improved the HE-memory deficit, there was a differential effect on CCO. A general decrease in CCO activity was found in the prefrontal and entorhinal cortices, dorsal striatum, and hippocampus when PBM, compared to MB, was applied. Our results suggest that mitochondrial dysfunction and brain metabolic decline in HE might involve CCO alteration and can be improved by administering MB and PBM.

Changes in COX histochemistry in the brain of mice and rats exposed to chronic subcutaneous rotenone

Exposure of experimental animals to the mitochondrial toxin rotenone is considered to be a model of environmental progression of Parkinson's disease (PD). We investigated the differential vulnerability of various brain regions to generalized inhibition of complex I, induced by subcutaneous rotenone injections for the duration of 1, 3 and 7 days in both rats (2 mg/kg dosage) and mice (4 mg/kg dosage). To examine patterns of metabolic activity changes in the brain, histochemical evaluation of cytochrome C oxidase (COX) activity was performed in post mortem brain sections. Animals displayed a similar time course of neuronal loss in substantia nigra pars compacta (SNpc), reaching 44 % in mice and 42 % in rats by the 7th day. The pattern of COX activity changes, however, was different for the two species. In both experiments, metabolic changes were evident not only in the substantia nigra, but also in non-specific structures (cortex and hippocampus). In mice, a decrease in COX activity was shown mostly for the non-specific areas (V1 cortex and ventral hippocampus) after the single exposure to rotenone. Data from the experiment conducted on rats demonstrated both an acute metabolic decrease in mesencephalic structures (SNpc and nucleus ruber) after a single injection of rotenone and secondary changes in cortical structures (S1 cortex and dorsal hippocampus) after chronic 7 day exposure. These changes reflect the general effect of rotenone on neuronal metabolic rate.

The mitochondrial metallochaperone SCO1 maintains CTR1 at the plasma membrane to preserve copper homeostasis in the murine heart.

SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.

A positive feedback loop between nitric oxide and amyloid β (1-42) accelerates mitochondrial damage in human lens epithelial cells.

We have reported that excessive nitric oxide (NO), like other reactive oxygen species (ROS), causes a decrease in cytochrome c oxidase (CCO) activity and ATP levels (mitochondrial damage) resulting in lens opacity. In addition, previous reports have shown that oxidative stress caused by ROS enhances amyloid β (Aβ) production in mammalian lenses, and that Aβ1-42 stimulates inducible nitric oxide synthase (iNOS) promoter activity. Based on these reports, we investigated the relationship between NO and Aβ1-42 production in human lens epithelial (HLE) cells. iNOS was induced by the co-incubation of HLE cells with 1000 IU interferon-γ (IFN-γ) and 100ng/ml lipopolysaccharide (LPS) for 48h. This led to enhanced NO release, an increase in the gene expression levels of proteins related to Aβ production, and the cellular accumulation of Aβ1-42. Moreover, both aminoguanidine (AG, a selective inhibitor of iNOS) and diethyldithiocarbamate (DDC, a nuclear factor-kappa B (NFκB) inhibitor) attenuated these changes in IFN-γ and LPS stimulated HLE cells. Based on our finding that Aβ1-42 accumulation is induced by co-incubation of HLE cells with both IFN-γ and LPS, we prepared a HLE cell model with Aβ1-42 accumulation (Aβ-accumulated-HLE cell model) by pre-stimulating cells with IFN-γ and LPS for 48h. Aβ1-42 accumulation caused NO production via iNOS, resulting in an enhancement in the mRNA levels for enzymes necessary for the proteolysis of amyloid precursor protein (APP) to Aβ in HLE cells. In addition, excessive NO produced in response to Aβ1-42 accumulation led to a decrease in CCO activity and ATP levels. Taken together, we hypothesize that excessive NO production in the lens epithelium enhances Aβ1-42 production, and that this enhancement accelerates NO release. The enhancement in NO production in the lens epithelium based on positive feedback (NO-Aβ positive feedback loop, a vicious cycle) may promote the onset of cataracts (lens opacification) via the decrease in CCO activity and ATP levels. These findings provide significant information that can be used to design further studies aimed at developing anti-cataract drugs.

Novel Mutations inSCO1as a Cause of Fatal Infantile Encephalopathy and Lactic Acidosis

Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele-specific defects in SCO1 function might underlie the genotype-phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1.

Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity

The inducible form of Heme Oxygenase-1 (HO-1), a major endoplasmic reticulum (ER) associated heme protein, is known to play important roles in protection against oxidative and chemical stress by degrading free heme released from degradation of heme proteins. In this study we show that induced expression of HO-1 by subjecting macrophage RAW-264.7 cells to chemical or physiological hypoxia resulted in significant translocation of HO-1 protein to mitochondria. Transient transfection of COS-7 cells with cloned cDNA also resulted in mitochondrial translocation of HO-1. Deletion of N-terminal ER targeting domain increased mitochondrial translocation under the transient transfection conditions. Mitochondrial localization of both intact HO-1 and N-terminal truncated HO-1 caused loss of heme aa-3 and cytochrome c oxidase (CcO) activity in COS-7 cells. The truncated protein, which localizes to mitochondria at higher levels, induced substantially steeper loss of CcO activity and reduced heme aa3 content. Furthermore, cells expressing mitochondria targeted HO-1 also induced higher ROS production. Consistent with dysfunctional state of mitochondria induced by HO-1, the mitochondrial recruitment of autophagy markers LC-3 and Drp-1 was also increased in these cells. Chronic ethanol feeding in rats also caused an increase in mitochondrial HO-1 and decrease in CcO activity. These results show that as opposed to the protective effect of the ER associated HO-1, mitochondria targeted HO-1 under normoxic conditions induces mitochondrial dysfunction.

Homocysteine Restricts Copper Availability Leading to Suppression of Cytochrome C Oxidase Activity in Phenylephrine-Treated Cardiomyocytes

Cardiomyocyte hypertrophy induced by phenylephrine (PE) is accompanied by suppression of cytochrome c oxidase (CCO) activity, and copper (Cu) supplementation restores CCO activity and reverses the hypertrophy. The present study was aimed to understand the mechanism of PE-induced decrease in CCO activity. Primary cultures of neonatal rat cardiomyocytes were treated with PE at a final concentration of l00 µM in cultures for 72 h to induce cell hypertrophy. The CCO activity was determined by enzymatic assay and changes in CCO subunit COX-IV as well as copper chaperones for CCO (COX17, SCO2, and COX11) were determined by Western blotting. PE treatment increased both intracellular and extracellular homocysteine concentrations and decreased intracellular Cu concentrations. Studies in vitro found that homocysteine and Cu form complexes. Inhibition of the intracellular homocysteine synthesis in the PE-treated cardiomyocytes prevented the increase in the extracellular homocysteine concentration, retained the intracellular Cu concentration, and preserved the CCO activity. PE treatment decreased protein concentrations of the COX-IV, and the Cu chaperones COX17, COX11, and SCO2. These PE effects were prevented by either inhibition of the intracellular homocysteine synthesis or Cu supplementation. Therefore, PE-induced elevation of homocysteine restricts Cu availability through its interaction with Cu and suppression of Cu chaperones, leading to the decrease in CCO enzyme activity.

LRPPRC mutation suppresses cytochrome oxidase activity by altering mitochondrial RNA transcript stability in a mouse model

LRPPRC (leucine-rich pentatricopeptide repeat-containing) has been shown to be essential for the maturation of COX (cytochrome c oxidase), possibly by stabilizing RNA transcripts of COXI, COXII and COXIII genes encoded in mtDNA (mitochondrial DNA). We established a mouse 'gene-trap' model using ES cells (embryonic stem cells) in which the C-terminus of LRPPRC has been replaced with a β-geo construct. Mice homozygous for this modification were found to be subject to embryonic lethality, with death before 12.5 dpc (days post-coitum). Biochemical analysis of MEFs (mouse embryonic fibroblasts) isolated from homozygous mutants showed a major decrease in COX activity, with slight reductions in other respiratory chain complexes with mtDNA encoded components. Constructs of LRPPRC containing different numbers of PPRs (pentatricopeptide repeats) were expressed as recombinant proteins and tested for their ability to bind to the COXI mRNA transcript. Full binding required the first 19 PPR motifs. A specific segment of COXI mRNA was identified as the binding target for LRPPRC, encoded by mouse mtDNA nucleotides 5961-6020. These data strongly suggest that LRPPRC is involved in the maturation of COX, and is involved in stabilizing of mitochondrial mRNAs encoding COX transcripts.

Involvement of cytochrome c oxidase subunits Va and Vb in the regulation of cancer cell metabolism by Bcl-2

Bcl-2 has been shown to promote survival of cancer cells by maintaining a slight pro-oxidant state through elevated mitochondrial respiration during basal conditions. On oxidative stress, Bcl-2 moderates mitochondrial respiration through cytochrome c oxidase (COX) activity to prevent an excessive buildup of reactive oxygen species (ROS) by-production from electron transport activities. However, the underlying molecular mechanism(s) of Bcl-2-mediated ROS regulation and its impact on carcinogenesis remain unclear. In this study, we show that Bcl-2 expression positively influences the targeting of nuclear-encoded COX Va and Vb to the mitochondria of cancer cells. In addition, evidence is presented in support of a protein-protein interaction between COX Va and Bcl-2, involving the BH2 domain of Bcl-2. Interestingly, episodes of serum withdrawal, glucose deprivation or hypoxia aimed at inducing early oxidative stress triggered Bcl-2-overexpressing cells to preserve mitochondrial levels of COX Va while depressing COX Vb, whereas the reverse was observed in mock-transfected cells. The unique manner in which Bcl-2 adjusted COX subunits during these physiological stress triggers had a profound impact on the resultant decrease in COX activity and maintenance of mitochondrial ROS levels, thus delineating a novel mechanism for the homeostatic role of Bcl-2 in the redox biology and metabolism of cancer cells.

Complex I Function Is Defective in Complex IV-deficient Caenorhabditis elegans

Cytochrome c oxidase (COX) is hypothesized to be an important regulator of oxidative phosphorylation. However, no animal phenotypes have been described due to genetic defects in nuclear-encoded subunits of COX. We knocked down predicted homologues of COX IV and COX Va in the nematode Caenorhabditis elegans. Animals treated with W09C5.8 (COX IV) or Y37D8A.14 (COX Va) RNA interference had shortened lifespans and severe defects in mitochondrial respiratory chain function. Amount and activity of complex IV, as well as supercomplexes that included complex IV, were decreased in COX-deficient worms. The formation of supercomplex I:III was not dependent on COX. We found that COX deficiencies decreased intrinsic complex I enzymatic activity, as well as complex I-III enzymatic activity. However, overall amounts of complex I were not decreased in these animals. Surprisingly, intrinsic complex I enzymatic activity is dependent on the presence of complex IV, despite no overall decrease in the amount of complex I. Presumably the association of complex I with complex IV within the supercomplex I:III:IV enhances electron flow through complex I. Our results indicate that reduction of a single subunit within the electron transport chain can affect multiple enzymatic steps of electron transfer, including movement within a different protein complex. Patients presenting with multiple defects of electron transport may, in fact, harbor a single genetic defect.

β1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.

Oxidative metabolism of limbic structures after acute administration of diazepam, alprazolam and zolpidem

The effects of acute administration of two benzodiazepines and a non-benzodiazepine hypnotic on behavior and brain metabolism were evaluated in rats. After testing the behavioral action of the benzodiazepines on the open field and the elevated plus-maze, the effects of the three drugs on neuronal metabolism of particular limbic regions were measured using cytochrome c oxidase (CO) histochemistry. Diazepam (5 mg/kg i.p.) and alprazolam (0.5 mg/kg i.p.) induced clear anxiolytic effects and a decrease in locomotion, whereas zolpidem (2 mg/kg i.p.) caused an intense hypnotic effect. The anxiolytic effects of alprazolam were distinguishable from diazepam due to the pharmacological and clinical profile of this triazolobenzodiazepine. CO activity decreased significantly in almost all the limbic regions evaluated after zolpidem administration. However, significant prominent decreases in CO activity were found after diazepam treatment in the medial mammillary nucleus, anteroventral thalamus, cingulate cortex, dentate gyrus and basolateral amygdala. Alprazolam caused similar decreases in CO activity, with the exception of the prelimbic and cingulate cortices, where significant increases were detected. In agreement with previous studies using other functional mapping techniques, our results indicate that particular benzodiazepines and non-benzodiazepine hypnotics induce selective changes in brain oxidative metabolism.

352 Quantitative and qualitative mitochondrial adaptations of substrates utilization in cardiac and skeletal muscles

have recently shown that knocking out of endothelium NOS (eNOS) induces an important reduction of mitochondrial oxidative capacity specifically in slow-twitch muscle (soleus, Sol) (Momken et al. 2002, Biochem J, 368:341-7). To examine the functional consequences of eNOS inhibition, physical capacity of mice, null for eNOS isoform, was estimated using a voluntary wheel-running program. In parallel, we studied energy metabolism enzyme profiles and their response to voluntary exercise in cardiac, Sol and fast-twitch gastrocnemius (Gast) skeletal muscles. Running distance, work, running speed and distance per run (mean distance the mice run each time they use the wheel) over the 8 week program were calculated after continuous recording of the output voltage of the DC generator on a PC computer. Weekly averaged work and running distance were two times lower for eNOS-/mice (608±65 J/day and 4.09±0.42 km/day) than for control (1207±74 J/day, p<0.01 and 7.74±0.42 km/day, p<0.01). The average maximal running speed was 19% (p<0.01) and the distance per run was 50% (p<0.01) lower in eNOS-/-mice. Furthermore, our study showed that physical activity influenced the adaptation to exercise specifically in Sol muscle. Physical activity increased significantly Sol weight by 22% (p<0.05) in wild-type mice but not in eNOS-/mice. Control Sol did not change its metabolic profile, in contrast to eNOS-/muscle where exercise decreased cytochrome c oxidase (COX) (-36%, p<0.05), citrate synthase (-37%, p<0.06) and creatine kinase (-24%, p<0.01) activities. Exercise changed energy enzyme profile neither in heart nor in Gast muscle (the only exception was a 44%, p<0.05 decrease in COX activity in active Gast muscle). Thus, in the absence of eNOS-derived NO, exercise induces a down-regulation of energy metabolism specifically in slow-twitch muscle. These results suggest that eNOS might be important for maintaining a suitable physical capacity and that its down-regulation might participate in intolerance to exercise.

Mitochondrial impairment in p53-deficient human cancer cells.

The mechanism linking p53 inactivation to human cell malignancy remains unclear. Studies have indicated that mitochondrial dysfunction is involved in carcinogenesis. In this study we investigated the role of p53 in mitochondrial DNA (mtDNA) mutation and maintenance of proper mitochondrial function. We measured mtDNA mutation and found no difference in frequency of mutation between the p53(+/+) and p53(-/-) cell lines. However, mitochondrial cytochrome c oxidase (COX) activity was significantly diminished in p53(-/-) cells. This decrease in COX activity was attributed to decreased protein levels of the COXII subunit encoded by the mitochondrial genome and was not due to mutation in the mitochondrial COXII gene. Further investigation revealed no concomitant decrease in COXII mRNA levels in p53(-/-) cells and the stability of mRNA in p53(-/-) cells was unaffected. This study suggests that decreased COX activity is likely due to post-transcriptional regulation of the COXII subunit by p53. COX is a critical enzyme in the mitochondrial electron transport chain and reduced COX activity may affect mitochondrial structure. However, examination of mitochondrial ultrastructure revealed no obvious differences between p53(+/+) and p53(-/-) cell lines. Together, our study suggests that p53 is involved in regulation of COXII at the protein level but not at the mRNA level. p53 does not affect mtDNA mutation or mitochondrial ultrastructure.

Cytochrome c oxidase is decreased in Alzheimer’s disease platelets

Cytochrome c oxidase (COX) activity reportedly is reduced in Alzheimer’s disease (AD) brain and platelets. The reasons for the defect in either tissue are unknown, but its presence in a non-degenerating tissue suggests it is not simply a consequence of neurodegeneration. We now offer confirmation of the AD platelet COX defect. Compared to age-matched controls, in mitochondria isolated from AD platelets there was a 15% decrease in COX activity despite the fact that COX subunits were present at normal levels. Platelet ATP levels were diminished in AD (from 11.33±0.52 to 9.11±0.72 nmol/mg), while reactive oxygen species (ROS) were increased (from 97.03±25.9 to 338.3±100 K/mg). Platelet membrane fluidity, Vitamin E, and cholesterol content were similar between groups. We conclude that COX catalytic activity is indeed diminished in AD platelet mitochondria, does not result from altered membrane fluidity, and is associated with ROS overproduction and ATP underproduction.

Myocardial cytochrome c oxidase activity in Swedish moose ( Alces alces L.) affected by molybdenosis

Since the mid-1980s, a ‘mysterious’ wasting disease has been afflicting the moose ( Alces alces L.) population of south-western Sweden. In 1994, molybdenosis combined with copper deficiency was suggested as the cause of this complex syndrome of clinical signs, diversity of necropsy findings and changes in trace element concentrations. These findings were corroborated by scientists in many countries by similar observations in other ruminants, particularly cattle and sheep, and also by changes in trace element concentrations and clinical chemical findings in our model experiments with goats. The biochemistry of copper is dependent on a number of copper-dependent enzymes in the animal organism. An important example is cytochrome c oxidase (COX), responsible for oxidative phosphorylation and energy production within the cell. In the present study, COX activity and trace element concentrations were determined in myocardium from affected and healthy moose. Citrate synthase (CS) activity was also measured for use as a mitochondrial marker. COX activity had decreased by 45% and the COX/CS ratio by 37%, while Mo and Na were found to have increased by 140% and 25%, respectively. The increase in Na was indicative of the frequently reported oedematous changes in ‘flabby’ moose heart. The concentrations of the elements Cu, Mg, Mn, P and Zn had decreased by 20%, 20%, 35%, 7% and 19%, respectively. The simultaneous decrease in COX activity and Cu concentration and the increase in Mo further support the hypothesis that molybdenosis is the cause of the moose disease.

Formation of Malondialdehyde Adducts in Livers of Rats Exposed to Ethanol: Role in Ethanol-Mediated Inhibition of Cytochrome c Oxidase

Background: Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. Methods and Results: It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 μM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. Conclusions: The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.

Formation of Malondialdehyde Adducts in Livers of Rats Exposed to Ethanol: Role in Ethanol‐Mediated Inhibition of Cytochrome c Oxidase

Previous studies in our laboratory demonstrated that short-term ethanol consumption by maternal rats increased the hepatic levels of 4-hydroxynonenal (HNE) in both the adult and the fetus. Additionally, HNE inhibited cytochrome c oxidase (COX) by forming adducts with the enzyme subunits. The present study examined modification of COX by another major aldehydic lipid peroxidation product, malondialdehyde (MDA), and its role in COX inhibition by ethanol. It is demonstrated in vitro that MDA inhibits the activity of purified COX while forming adducts with the enzyme. Compared with HNE, MDA is a more potent inhibitor of COX. Overnight incubation at room temperature caused an 80% decrease in COX activity by MDA versus a 67% decrease by HNE. MDA produced marked inhibition of COX activity at physiologically relevant concentrations, e.g., 43% inhibition at 10 microM. Although our previous studies documented that HNE formed adducts primarily with subunit IV of COX via histidine residues, the current report showed that MDA forms adducts with both subunit IV and subunit V via lysine residues. Furthermore, both aldehydes induce carbonyl formation in subunit IV. The in vivo role of MDA in the impairment of COX by ethanol is assessed in both adult and fetal liver after maternal ethanol consumption. The results showed that: (1) there are significant increases in MDA levels in liver homogenate as well as mitochondria in both adult and fetal livers after ethanol exposure; (2) these MDA levels are in the nanomole/mg protein range, in contrast to picomole/mg protein range of HNE in identical setting; and (3) ethanol-induced production of MDA is accompanied by enhanced formation of MDA adducts with COX. These findings suggest that MDA may play at least as equally an important role as HNE in ethanol-induced inhibition of COX.

Formation of 4-hydroxynonenal adducts with cytochrome c oxidase in rats following short-term ethanol intake.

This study addresses the role of the lipid peroxidation product, 4-hydroxynonenal (HNE), in ethanol-related damage of cytochrome c oxidase (COX) in vivo. It utilizes an animal model with acute ethanol exposure in which HNE levels in liver mitochondria are strikingly increased. Pregnant female Sprague-Dawley rats were administered 5 doses of ethanol (4 gm/kg, po at 12-hour intervals) beginning on day 17 of gestation and were sacrificed on day 19. Controls were pair-fed and received dextrose isocaloric to ethanol. Mitochondria were isolated from maternal and fetal livers and COX activities were measured spectrophotometrically. Compared with the pair-fed controls, COX activity was decreased with exposure to ethanol by 25% in maternal rats and 43% in fetal rats (P<.05). Western Blot with an HNE-Histidine antibody showed enhanced formation of HNE adducts with COX from ethanol-exposed rats, which was more pronounced in fetal than in adult livers. The HNE adducts were mainly with subunit IV of COX. The cause and effect relationship between HNE adduct formation and COX inhibition was examined in vitro by incubating purified COX with HNE. COX inhibition was accompanied by concentration-dependent HNE adduct formation that was consistent with those found in in vivo ethanol-exposed samples. These results suggest that the ethanol-related decreases in COX activity found in liver mitochondria could be attributable to HNE adduct formation with the enzyme complex. This could be an important mechanism by which modification of proteins occur in in vivo oxidative stress.

Zidovudine (AZT) induced alterations in mitochondrial biogenesis in rat striated muscles.

Zidovudine (AZT) and didanosine (ddI), two drugs used in the treatment of AIDS, are also known to cause mitochondrial abnormalities. We investigated the physiological relevance of the mitochondrial defects by measuring in situ skeletal muscle performance and cytochrome c oxidase (CYTOX) enzyme activity in heart muscle, red highoxidative (RG) and white low-oxidative (WG) portions of the gastrocnemius muscle of control (n = 17), AZT-(n = 14), or ddI-treated (n = 11) rats for 28 days. We also evaluated the hypothesis that AZT treatment could alter the expression of the mitochondrial transcription factor A (mtTFA), a key molecule involved in mitochondrial DNA (mtDNA) replication and transcription. AZT had a pronounced effect on blood pressure and skeletal muscle performance, which were significantly decreased during contractile activity at 2 and 5 Hz, compared with control. A significant decrease in CYTOX activity in heart and RG, but not WG muscles, was also evident. In the heart, this was accompanied by an apparent compensatory increase in mtTFA mRNA level that could not be attributed to enhanced transcriptional activation mediated by nuclear respiratory factor 1 (NRF-1). In contrast with AZT, no effect of ddI was found on the extent of fatigue or muscle enzyme activity. These results indicate that AZT induces mitochondrial defects primarily in muscles with the highest oxidative capacities (heart and RG). The long-term effects of AZT on mitochondrial biogenesis have the potential to reduce muscle performance, but the effects on performance in this short-term study were likely due to an inability of the AZT-treated animals to maintain blood pressure during contractile activity.