Decrease In ATP Content Research Articles

Mapping time-course mitochondrial adaptations in the kidney in experimental diabetes.

Oxidative phosphorylation (OXPHOS) drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homoeostasis. In diabetic kidney disease (DKD), mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics have not been previously documented. In the present study, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks), a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells (PTECs) of diabetic kidneys were clearly apparent, but no changes in urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide (H2O2) generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary kidney injury molecule-1 (KIM-1) excretion, where an increase in the Complex I-linked oxygen consumption rate (OCR), in the context of a decrease in kidney ATP, indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of DKD.

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis.

O-GlcNAcylation is implicated in modulating mitochondrial function, which is closely involved in regulating muscle metabolism. The presence of O-GlcNAcase (OGA), the enzyme involved in the removal of O-GlcNAc, in mitochondria was recently confirmed in rats. In the present study, we investigated the regulation of myogenesis and muscle insulin sensitivity to OGA in mice, with a focus on mitochondria. C57BL/6J mice fed a high-fat diet for 4months were used to observe mitochondrial density, activity and O-GlcNAcylation in muscle. Small interfering RNA and overexpression vectors were used to modulate protein content in vitro. High-fat feeding decreased the OGA level and largely increased mitochondrial O-GlcNAcylation in mouse skeletal muscle that was accompanied by decreased levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), decreased mitochondrial density and disrupted mitochondrial complex activities. Knockdown of OGA in C2C12 myoblasts promoted PGC-1α degradation, resulting in the suppression of mitochondrial biogenesis and myogenesis, whereas neither knockdown of O-GlcNAc transferase nor overexpression of OGA had significant effects on myogenesis. Mitochondrial dysfunction as evidenced by decreased ATP content and increased reactive oxygen species production, and increased lipid and protein oxidation was observed in both myoblasts and myotubes after OGA knockdown. Meanwhile, elevated O-GlcNAcylation through either OGA knockdown or treatment with the OGA inhibitor PUGNAc and the O-GlcNAc transferase substrate D-GlcNAc suppressed myotube insulin signalling transduction and glucose uptake. OGA overexpression had no significant effect on insulin sensitivity but sufficiently improved the insulin resistance induced by D-GlcNAc treatment. These data suggest that OGA can modulate mitochondrial density via PGC-1α and mitochondrial function via protein O-GlcNAcylation. In this manner, OGA appears to play a key role in myogenesis and the development of muscle insulin resistance.

Melatonin prevents abnormal mitochondrial dynamics resulting from the neurotoxicity of cadmium by blocking calcium-dependent translocation of Drp1 to the mitochondria.

Cadmium (Cd) is a persistent environmental toxin and occupational pollutant that is considered to be a potential risk factor in the development of neurodegenerative diseases. Abnormal mitochondrial dynamics are increasingly implicated in mitochondrial damage in various neurological pathologies. The aim of this study was to investigate whether the disturbance of mitochondrial dynamics contributed to Cd-induced neurotoxicity and whether melatonin has any neuroprotective properties. After cortical neurons were exposed to 10 μM cadmium chloride (CdCl2 ) for various periods (0, 3, 6, 12, and 24 hr), the morphology of their mitochondria significantly changed from the normal tubular networks into punctuated structures within 3 hr. Following this pronounced mitochondrial fragmentation, Cd treatment led to signs of mitochondrial dysfunction, including excess reactive oxygen species (ROS) production, decreased ATP content, and mitochondrial membrane potential (▵Ψm) loss. However, 1 mM melatonin pretreatment efficiently attenuated the Cd-induced mitochondrial fragmentation, which improved the turnover of mitochondrial function. In the brain tissues of rats that were intraperitoneally given 1 mg/kg CdCl2 for 7 days, melatonin also ameliorated excessive mitochondrial fragmentation and mitochondrial damage in vivo. Melatonin's protective effects were attributed to its roles in preventing cytosolic calcium ([Ca(2+) ]i ) overload, which blocked the recruitment of Drp1 from the cytoplasm to the mitochondria. Taken together, our results are the first to demonstrate that abnormal mitochondrial dynamics is involved in cadmium-induced neurotoxicity. Melatonin has significant pharmacological potential in protecting against the neurotoxicity of Cd by blocking the disbalance of mitochondrial fusion and fission.

Targeting the Wnt/β-catenin pathway in primary ovarian cancer with the porcupine inhibitor WNT974

Preclinical studies in ovarian cancer have demonstrated upregulation of the Wnt/β-catenin pathway promoting tumor proliferation and chemoresistance. Our objective was to evaluate the effect of the Wnt/β-catenin pathway inhibitor, WNT974, in primary ovarian cancer ascites cells. Ascites cells from patients with papillary serous ovarian cancer were isolated and treated with 1 μM WNT974±100 μM carboplatin. Viability was evaluated with the ATPlite assay. The IC50 was calculated using a dose–response analysis. Immunohistochemistry (IHC) was performed on ascites cells and tumor. Expression of R-spondin 2 (RSPO2), RSPO3, PORCN, WLS, AXIN2, and three previously characterized RSPO fusion transcripts were assessed using Taqman assays. Sixty ascites samples were analyzed for response to WNT974. The ascites samples that showed a decrease in ATP concentration after treatment demonstrated no difference from the untreated cells in percent viability with trypan blue staining. Flow cytometry demonstrated fewer cells in the G2 phase and more in the G1 and S phases after treatment with WNT974. Combination therapy with WNT974 and carboplatin resulted in a higher percentage of samples that showed ≥30% reduction in ATP concentration than either single drug treatment. IHC analysis of Wnt pathway proteins suggests cell cycle arrest rather than cytotoxicity after WNT974 treatment. QPCR indicated that RSPO fusions are not prevalent in ovarian cancer tissues or ascites. However, higher PORCN expression correlated to sensitivity to WNT974 (P=0.0073). In conclusion, WNT974 produces cytostatic effects in patient ascites cells with primary ovarian cancer through inhibition of the Wnt/β-catenin pathway. The combination of WNT974 and carboplatin induces cytotoxicity plus cell cycle arrest in a higher percentage of ascites samples than with single drug treatment. RSPO fusions do not contribute to WNT974 sensitivity; however, higher PORCN expression indicates increased WNT974 sensitivity.

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.

Partial Portal Vein Arterialization Attenuates Acute Bile Duct Injury Induced by Hepatic Dearterialization in a Rat Model

Hepatic infarcts or abscesses occur after hepatic artery interruption. We explored the mechanisms of hepatic deprivation-induced acute liver injury and determine whether partial portal vein arterialization attenuated this injury in rats. Male Sprague-Dawley rats underwent either complete hepatic arterial deprivation or partial portal vein arterialization, or both. Hepatic ischemia was evaluated using biochemical analysis, light microscopy, and transmission electron microscopy. Hepatic ATP levels, the expression of hypoxia- and inflammation-associated genes and proteins, and the expression of bile transporter genes were assessed. Complete dearterialization of the liver induced acute liver injury, as evidenced by the histological changes, significantly increased serum biochemical markers, decreased ATP content, increased expression of hypoxia- and inflammation-associated genes and proteins, and decreased expression of bile transporter genes. These detrimental changes were extenuated but not fully reversed by partial portal vein arterialization, which also attenuated ductular reaction and fibrosis in completely dearterialized rat livers. Collectively, complete hepatic deprivation causes severe liver injury, including bile infarcts and biloma formation. Partial portal vein arterialization seems to protect against acute ischemia-hypoxia-induced liver injury.

Quercetin Protects Rat L6 Myocytes from Antimycin A-Induced Mitochondrial Dysfunction

Objective: Mitochondrial dysfunction is often associated with various disorders such as diabetes, Alzheimer’s etc. Reactive oxygen species (ROS), aging, and reduction of mitochondrial biogenesis contribute to mitochondrial dysfunction. Antimycin-A (AMA) damages the mitochondria through inhibition of mitochondrial electron transport. The present study sought to investigate effects of quercetin on rat L6 cells and whether quercetin protects mitochondria against oxidative damage caused by AMA. Methods: Rat L6 myocytes were used for the study. Effects of quercetin on Antimycin-A induced mitochondrial dysfunction was studied using cytotoxicity, ATP levels, mitochondrial superoxide production and NDUFB8 mRNA expression. Results: In this study, exposure of L6 myocytes to AMA induced an increase cell death, decreased ATP content, followed by a decrease in mitochondrial superoxide, and decreased expression of NDUFB8. We found that quercetin protected myocytes from antimycin-A (AMA) induced L6 cell death as evidenced from increased lactate dehydrogenase (LDH) leakage into extracellular medium, protected ATP production, prevented increase in oxidative stress and restored levels of NDUFB8 mRNA expression implying improved mitochondrial function. Conclusion: These results suggest that the quercetin showed protective effect against AMA-induced mitochondrial dysfunction by increasing ATP production, decreasing oxidative stress, and restoring mitochondrial function.

Low-Dose Methylmercury-Induced Apoptosis and Mitochondrial DNA Mutation in Human Embryonic Neural Progenitor Cells.

Methylmercury (MeHg) is a long-lasting organic pollutant primarily found in the aquatic environment. The developing brain is particularly sensitive to MeHg due to reduced proliferation of neural stem cell. Although several mechanisms of MeHg-induced apoptosis have been defined in culture models, it remains unclear whether mitochondrial DNA (mtDNA) mutation is involved in the toxic effect of MeHg, especially in the neural progenitor cells. In the present study, the ReNcell CX cell, a human neural progenitor cells (hNPCs) line, was exposed to nanomolar concentrations of MeHg (≤50 nM). We found that MeHg altered mitochondrial metabolic function and induced apoptosis. In addition, we observed that MeHg induced ROS production in a dose-dependent manner in hNPCs cells, which was associated with significantly increased expressions of ND1, Cytb, and ATP6. To elucidate the mechanism underlying MeHg toxicity on mitochondrial function, we examined the ATP content and mitochondrial membrane potential in MeHg-treated hNPCs. Our study showed that MeHg exposure led to decreased ATP content and reduced mitochondrial membrane potential, which failed to match the expansion in mtDNA copy number, suggesting impaired mtDNA. Collectively, these results demonstrated that MeHg induced toxicity in hNPCs through altering mitochondrial function and inducing oxidative damage to mtDNA.

Antibodies to the RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein A1 Colocalize to Stress Granules Resulting in Altered RNA and Protein Levels in a Model of Neurodegeneration in Multiple Sclerosis.

ObjectiveMultiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system (CNS). Data suggest that antibodies to CNS targets contribute to the pathogenesis of MS. MS patients produce autoantibodies to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). hnRNP A1 is an RNA binding protein (RBP) overexpressed in neurons that functions in pre-mRNA splicing, mRNA trafficking, and translation. Previously, we showed that anti-hnRNP A1 antibodies entered neuronal cells (in vitro) via clathrin-mediated endocytosis, caused mislocalization of endogenous hnRNP A1 protein and increased markers of neurodegeneration including decreased ATP concentration and apoptosis. In this study, we hypothesized that anti-hnRNP A1 antibodies might cause stress granule formation and altered levels of RNAs and proteins that bind hnRNP A1.MethodsNeuronal cell lines were exposed to anti-hnRNP A1 and isotype-matched control antibodies in vitro and examined for neuronal granule formation, including stress granules, P bodies and transport granules. In addition, RNAs that bound hnRNP A1 were determined. Levels of RNA and their translated proteins were measured upon exposure to the anti-hnRNP A1 antibodies.ResultsAnti-hnRNP A1 antibodies induced and localized to stress granules, a marker of neurodegeneration, within a neuronal cell line. The anti-hnRNP A1 antibodies did not induce P bodies or neuronal granules. Clinically relevant RNAs were found to bind hnRNP A1. In addition, the anti-hnRNP A1 antibodies caused reduced levels of RNA and protein of the spinal paraplegia genes (SPGs) 4 and 7, which when mutated mimic progressive MS.ConclusionsTaken together, these data suggest potential mechanisms by which autoantibodies may contribute to neurodegeneration in MS.

ATP synthase subunit-β down-regulation aggravates diabetic nephropathy.

In this study, we investigated the role of ATP synthase subunit-β (ATP5b) in diabetic nephropathy. Histopathological changes, fibrosis, and protein expressions of α-smooth muscle actin (α-SMA), advanced glycation end-products (AGEs), and ATP5b were obviously observed in the kidneys of db/db diabetic mice as compared with the control db/m+ mice. The increased ATP5b expression was majorly observed in diabetic renal tubules and was notably observed to locate in cytoplasm of tubule cells, but no significant increase of ATP5b in diabetic glomeruli. AGEs significantly increased protein expression of ATP5b and fibrotic factors and decreased ATP content in cultured renal tubular cells via an AGEs-receptor for AGEs (RAGE) axis pathway. Oxidative stress was also induced in diabetic kidneys and AGEs-treated renal tubular cells. The increase of ATP5b and CTGF protein expression in AGEs-treated renal tubular cells was reversed by antioxidant N-acetylcysteine. ATP5b-siRNA transfection augmented the increased protein expression of α-SMA and CTGF and CTGF promoter activity in AGEs-treated renal tubular cells. The in vivo ATP5b-siRNA delivery significantly enhanced renal fibrosis and serum creatinine in db/db mice with ATP5b down-regulation. These findings suggest that increased ATP5b plays an important adaptive or protective role in decreasing the rate of AGEs-induced renal fibrosis during diabetic condition.

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility.

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.

MiR-34a is Involved in the Decrease of ATP Contents Induced by Resistin Through Target on ATP5S in HepG2 Cells.

Resistin is associated with metabolic syndrome and deciphering its developmental and molecular mechanisms may help the development of new treatments. MiRNAs serve as negative regulators in many physiological and pathological processes. Here, miRNA microarrays were used to detect differences in expression between resistin-treated and control mice, and results showed miR-34a to be upregulated by resistin. The purpose of this study was to determine whether miR-34a played a role in resistin-induced decrease of ATP contents. Transient transfection of miR-34a mimics was used to overexpress miR-34a and quantitative RT-PCR was used to detect its expression. Western blot analysis was used to determine the rate of expression at the protein level. ATP content was measured using an ATP assay kit. The target gene of miR-34a was analyzed using bioinformatics and confirmed with dual-luciferase report system. MiR-34a was upregulated by resistin in HepG2 cells, and overexpression of miR-34a was found to diminish ATP levels significantly. This study is the first to show that ATP5S is one of the target genes of miR-34a. Resistin diminishes ATP content through the targeting of ATP5S mRNA 3'UTR by miR-34a.

Mitochondrial fusion/fission process involved in the improvement of catalpol on high glucose-induced hepatic mitochondrial dysfunction.

Catalpol, an iridoid glycoside, has been shown to exert hypoglycemic effect by rescuing mitochondrial function, but the detailed mechanism remains unclear yet. In this study, the effect and mechanism of catalpol on the hepatic mitochondria under diabetic conditions were further examined. Oral administration of catalpol significantly reduced the blood glucose, triglyceride, and cholesterol levels in high-fat diet- and streptozotocin-induced diabetic mice. Additionally, catalpol attenuated the decrease in liver mitochondrial ATP content resulting from diabetes. Furthermore, the number of mitochondria possessing a long size was increased in catalpol-treated mice. Interestingly, the catalpol-induced recovery of mitochondrial function was associated with decreased fission protein 1 and dynamin-related protein 1 expression as well as increased mitofusin 1 expression in the liver. In HepG2 cells, catalpol alleviated the decrease of ATP content and mitochondrial membrane potential, and the increase of reactive oxygen species formation induced by high glucose. MitoTracker Green stain shows that the tubular feature of mitochondria was maintained when cells were treated with catalpol. Catalpol also decreased fission protein 1 and dynamin-related protein 1 expression and increased mitofusin 1 expression in HepG2 cells. The present results suggest that catalpol can ameliorate hepatic mitochondrial dysfunction under a diabetic state, and this may be related to its regulation of mitochondrial fusion and fission events.

Glutathione Depletion Induces Spermatogonial Cell Autophagy.

The development and survival of male germ cells depend on the antioxidant capacity of the seminiferous tubule. Glutathione (GSH) plays an important role in the antioxidant defenses of the spermatogenic epithelium. Autophagy can act as a pro-survival response during oxidative stress or nutrient deficiency. In this work, we evaluated whether autophagy is involved in spermatogonia-type germ cell survival during severe GSH deficiency. We showed that the disruption of GSH metabolism with l-buthionine-(S,R)-sulfoximine (BSO) decreased reduced (GSH), oxidized (GSSG) glutathione content, and GSH/GSSG ratio in germ cells, without altering reactive oxygen species production and cell viability, evaluated by 2',7'-dichlorodihydrofluorescein (DCF) fluorescence and exclusion of propidium iodide assays, respectively. Autophagy was assessed by processing the endogenous protein LC3I and observing its sub-cellular distribution. Immunoblot and immunofluorescence analysis showed a consistent increase in LC3II and accumulation of autophagic vesicles under GSH-depletion conditions. This condition did not show changes in the level of phosphorylation of AMP-activated protein kinase (AMPK) or the ATP content. A loss in S-glutathionylated protein pattern was also observed. However, inhibition of autophagy resulted in decreased ATP content and increased caspase-3/7 activity in GSH-depleted germ cells. These findings suggest that GSH deficiency triggers an AMPK-independent induction of autophagy in germ cells as an adaptive stress response.

Enhanced Production of Nitric Oxide Leads to ATP Collapse in the Retinas of Otsuka Long-Evans Tokushima Fatty Rats, a Model of Human Diabetes

Purpose: We determined nitric oxide (NO) production via inducible NO synthase (iNOS) by hyperglycemia using the retina of Otsuka Long-Evans Tokushima Fatty rats (OLETF rats), and investigated the relationship between ATP contents and NO production in the retinas of OLETF rats.Methods: Long-Evans Tokushima Otsuka rats (LETO rats, normal rats) and OLETF rats (model rat for diabetes mellitus) aged 60 weeks of age were used. Plasma glucose (Glu) levels were determined using an Accutrend GCT System, and NO levels were measured by the microdialysis method as nitrite (). Cytochrome c oxidase (CCO) activity was measured using a Mitochondrial Isolation Kit and Cytochrome c Oxidase Assay Kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200.Results: levels in the retinas of OLETF rats were significantly higher than in LETO rats, and the levels in the retinas of 60-week-old OLETF rats increased with increasing Glu. CCO activity in the retinas of OLETF rats showed no significant difference from that in LETO rats; however, ATP levels in the retinas of OLETF rats were significantly lower than those in LETO rats. The oral administration of aminoguanidine or disulfiram, an iNOS inhibitor, attenuated the decrease in ATP levels in the retinas of 60-week-old OELTF rats.Conclusion: The present study demonstrates that NO production via iNOS in the retinas of 60-week-old OLETF rats is caused by hyperglycemia, and that NO causes a decrease in ATP contents in the retinas of 60-week-old OELTF rats. It is possible that the low ATP contents caused by NO may affect the normal functioning of the retina in OLETF rats.

Evaluation of the impact of urban pollution on the quality of skin: a multicentre study in Mexico.

After pilot and preliminary studies aimed at identifying pertinent biochemical parameters, a multicenter clinical study was performed to evaluate the effect of pollution on human skin. The clinical study was performed in collaboration with the 'Centre Régional de lutte contre le cancer de Montpellier' and the 'National Institute of Public Health of Mexico' on 96 subjects in Mexico City (exposed to pollution) and 93 subjects in Cuernavaca (less exposed to pollution). Both biochemical and clinical skin parameters were studied. The study demonstrated significant quantitative and qualitative modifications of parameters related to sebum excretion in Mexico City compared to Cuernavaca one: An increased level of sebum excretion rate, a lower level of vitamin E and squalene in sebum, an increase of lactic acid and a higher erythematous index on the face of the subjects. In the stratum corneum, a significant higher level of carbonylated proteins and a lower level of IL 1α were noticed, as well as a decrease of ATP concentration with a decrease of chymotrysin like activity, without modifications of corneodesmosin content and trypsin like activity. From a clinical point of view, a higher frequency of atopic and urticarial skins, a higher frequency of red dermographism, an important seborrheic status at the forehead level and a lower level of dandruffs were noted in Mexico City population. The analysis taking into account the sex does not modify the observed results. The study demonstrated an important impact of polluted environmental conditions on skin quality, evidencing important modifications of superficial biochemical parameters. The cause/effects relationships of these modifications remain, however, to be further assessed by a complementary in vitro/in vivo approaches.

Brain Injury Alters Ectonucleotidase Activities and Adenine Nucleotide Levels in Rat Serum / Povreda Mozga Menja Ektonukleotidazne Aktivnosti I Nivo Adeninskih Nukleotida U Serumu Pacova

SummaryBackgroundCortical stab injury (CSI) induces changes in the activity, expression and cellular distribution of specific ectonucleotidases at the injury site. Also, several experimentally induced neuropathologies are associated with changes in soluble ectonucleotidase activities in the plasma and serum, whilst various insults to the brain alter purine compounds levels in cerebrospinal fluid, but also in serum, indicating that insults to the brain may induce alterations in nucleotides release and rate of their hydrolysis in the vascular system. Since adenine nucleotides and adenosine regulate diverse cellular functions in the vascular system, including vascular tone, platelet aggregation and inflammatory responses of lymphocytes and macrophages, alterations of ectonucleotidase activities in the vascular system may be relevant for the clinical outcome of the primary insult.MethodsWe explored ectonucleotidase activities using specific enzyme assays and determined adenine nucleotides concentrations by the UPLC method in the rat serum after cortical stab injury.ResultsAt 4-h post-injury, ATP and AMP hydrolysis increased by about 60% and 40%, respectively, while phosphodiesterase activity remained unchanged. Also, at 4-h post-injury a marked decrease in ATP concentration and more than 2-fold increase in AMP concentration were recorded.ConclusionsCSI induces rapid up-regulation of nucleotide catabolizing soluble ectonucleotidases in rat serum, which leads to the observed shift in serum nucleotide levels. The results obtained imply that ectonucleotidases and adenine nucleotides participate in the communication between the brain and the vascular system in physiological and pathological conditions and thereby may be involved in the development of various human neuropathologies.

Toxicity Mechanisms of Cigarette Smoke on Mouse Fetus Mitochondria.

Maternal smoking has been recognized as a common cause of low birth weight, preterm birth and the decrease of gestational age period. Unfortunately, there is an increasing interest within public especially woman in Iran in the tobacco products consumption. On the other hand, the deleterious effect of maternal smoking on human fetus in pregnancy period especially in the first trimester encouraged us to investigate toxicity mechanisms of cigarette smoke on mouse fetus mitochondria. For this purpose different concentrations (1, 10 and 100%) of standardized cigarette smoke extract (CSE) were administrated on mitochondria isolated from fetus of NMRI mice on the 15 day of gestation. Our results showed a significant increase in ROS (Reactive oxygen species) formation, lipid peroxidation, mitochondrial membrane potential collapse, mitochondrial swelling and finally a decrease in ATP concentration in the CSE-treated isolated fetus mitochondria. Our results suggest that CSE-induced embryo toxicity is the result of disruptive effect on mitochondrial respiratory chain that leads to ROS formation, lipid peroxidation, mitochondrial MMP (mitochondrial membrane potential) decline and decrease of ATP level which starts apoptosis signaling.

Differential Effects of Trovafloxacin on TNF-α and IL-6 Profiles in a Rat Hepatocyte–Kupffer Cell Coculture System

Immune-mediated chemical-induced hepatotoxicity is often overlooked as a potential mode of action due to the lack of appropriate in vitro models. For this reason, we have established a coculture system of rat primary hepatocytes and Kupffer cells (KCs) that can be used to examine chemical-induced inflammatory reactions resulting in acute hepatocellular toxicity. Cocultures were maintained for 24 or 48 hr with various concentrations of lipopolysaccharide (LPS) and trovafloxacin (TVX), a compound associated with immune-mediated hepatotoxicity in vivo. Cytokine (IL-6 and TNFα) levels, CYP3A activity, and cell viability (ATP, LDH release) were measured to monitor endotoxin- and TVX-induced changes in metabolic capacity and cell integrity. LPS-treated cocultures showed marked downregulation of CYP3A activity, which correlated with upregulation of IL-6 and TNF-α production. TVX/LPS-treated cocultures exhibited concentration-dependent hepatocellular injury, represented by decreased ATP content and increased LDH leakage. The IL-6-induced downregulation of CYP3A was impaired in cocultures cotreated with TVX/LPS, which coincided with a concentration-dependent decline in IL-6 production. However, TNF-α production was initially upregulated in TVX/LPS-treated cocultures and KC monocultures before showing a gradual decline. The data from these studies suggest that shifts in cytokine profiles, especially IL-6/TNF-α ratios, may play a role in immune-mediated chemical-induced hepatotoxicity. Coculture of hepatocytes and KC represents a valuable in vitro tool to assess adverse liver effects resulting from indirect adaptive immune responses during chemical exposure.

A comparison of toxicity mechanisms of cigarette smoke on isolated mitochondria obtained from rat liver and skin.

Previous studies demonstrated that CSE induces oxidative stress and its consequences on isolated mitochondria obtained from lung, heart and brain which may provide insight into the role of CSE in human health and disease. The present study was carried out to further characterize and compare toxic effect of CSE extract on isolated mitochondria obtained from either a directly contacting tissue (i.e. skin) or a vital visceral tissue (i.e. liver).We obtained Rat liver and skin mitochondria by differential ultracentrifugation and incubated the isolated mitochondria with different concentrations (1, 10 and 100%) ofstandardizedcigarette smoke extract (CSE). Our results were similar to our previous study which discovered CSE toxicity mechanisms on isolated mitochondria obtained from lung, heart and brain with minor changes.CSE induced a significant rise in ROS formation, lipid peroxidation and mitochondrial membrane potential collapse and mitochondrial swelling on isolated mitochondria obtained from both liver and skin. CSE induced Decrease in ATP concentration on isolated mitochondria obtained from both liver and skin did not include CSE lowest concentration (1%). Our findingsshowed that CSE-induced toxicity in liver and skin is due to disruptive effect on mitochondrial respiratory chain which canleads to cytochrome c release and apoptosis signaling.