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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 451 records · Page 25
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Cyclosporin A inhibits mitochondrial biogenesis in Hep G2 cells

Highlights: • Cyclosporine A (CsA) reduced the expressions of PGC-1α, NRF1 and TFAM. • CsA reduced mtDNA/nDNA, Mitochondrial mass, ATP, and cytochrome C oxidase activity. • Overexpression of PGC-1α prevented CsA-induced reduction in mitochondrial biogenesis. • CsA-induced reduction of PGC-1α expression was mediated by CREB. Dysregulation of mitochondrial biogenesis is associated with pathogenesis in many diseases, including liver diseases. Cyclosporine A (CsA), one of the most commonly used drug to treat many autoimmune diseases and to prevent allograft rejection after organ transplantation, has been reported to cause mitochondrial dysfunction. However, the cellular mechanisms underlying CsA on mitochondrial dysfunction remain at present not completely elucidated. In this study, we found that CsA reduced the expression of PGC-1α at both the mRNA and protein levels in HepG2 cells. Correspondingly, the expressions of its target genes NRF 1 and TFAM were reduced in response to CsA treatment. In addition, mtDNA/nDNA, mitochondria mass, ATP production, and cytochrome C oxidase activity were significantly reduced by treatment with CsA. Over-expression of PGC-1α was found to rescue the negative effect of CsA administration on mitochondrial biogenesis. Mechanistically, CREB was involved in the inhibitory effects of CsA in mitochondrial biogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Ceramidase critically affects GPVI-dependent platelet activation and thrombus formation

Highlights: • Neither ceramide synthase nor serine palmitoyltransferase inhibition modifies GPVI-dependent platelet function. • Ceramidase inhibition impairs platelet aggregation, dense granule secretion and in vitro thrombus formation. • Impaired platelet function during ceramidase inhibition could be overcome by exogenous d-sphingosine. Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700{sup −sec}. Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Functional characterization of lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p)

Highlights: • A mutation system for analyzing lethal mutations in Dyh3p was established. • The P1 mutant dynein localizes in the oral apparatus and the proximal region of the cilia. • The P2 mutant dynein localizes only in the oral apparatus. • The P1 and P2 mutations decrease the swimming and feeding of Tetrahymena cells. • The P1 and P2 mutant dyneins show similar microtubule-binding properties. Mutational analyses of axonemal dyneins are useful for elucidating the molecular mechanism of ciliary motility. This study demonstrates a mutation system for characterizing lethal P-loop mutations in Tetrahymena outer arm dynein (Dyh3p). The viable DYH3-knockout (vKO-DYH3) cells isolated in this study enabled the examination of lethal mutations in P-loops 1 and 2. The P1 mutant dynein localized in the oral apparatus and the proximal region of the cilia, and the P2 mutant dynein localized only in the oral apparatus. Both results are different from the localization of wild-type Dyh3p. In addition, a co-precipitation assay showed that the P1 and P2 mutant dyneins did not dissociate from microtubules in ATP plus vanadate or in no-ATP conditions, in contrast to wild-type Dyh3p. This mutation system is useful for further molecular studies of axonemal dyneins and ciliary motility.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

ALK is required for NLRP3 inflammasome activation in macrophages

Highlights: • ALK promotes LPS-induced NLRP3 priming. • ALK promotes ATP-induced NLRP3 activation. • ALK promotes NLRP3-NEK7 complex formation. • ALK-mediated lipid peroxidation promotes NLRP3 activation. The NLRP3 inflammasome is a key mediator of host immune responses through the induction of pyroptosis and the release of cytokines. Although the pathologic role of inflammasome in infection and sterile inflammation is well known, the mechanism and regulation of NLRP3 inflammasome activation remains obscure. Here, we report that anaplastic lymphoma kinase (ALK) is a novel regulator of NLRP3 inflammasome activation in macrophages. Pharmacologic or genetic inhibition of ALK through targeted drugs (ceritinib and lorlatinib) or RNAi blocked extracellular ATP-induced NLRP3 inflammasome activation in macrophages. Mechanically, ALK-mediated NF-κB activation was required for the priming step of NLRP3 upregulation, whereas ALK-mediated lipid peroxidation contributed to the sensing step of NLRP3-NEK7 complex formation. These studies indicate that inhibition of ALK could be utilized to treat NLRP3-related inflammatory diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Erybraedin A is a potential Src inhibitor that blocks the adhesion and viability of non-small-cell lung cancer cells

Highlights: • Erybraedin A inhibited the adhesion of lung cancer cells to fibronectin. • Erybraedin A suppressed the activation of Src and its downstream targets. • Erybraedin A inhibited the interaction between integrin β1 or integrin β3 and Src. • Erybraedin A interacted with the ATP-binding pocket and the regulatory site of Src. • Erybraedin A inhibited the viability of lung cancer cells by inducing apoptosis. The adhesion of cancer cells to the extracellular matrix (ECM) is crucial for cell proliferation, survival, and metastasis. Thus, it is necessary to inhibit cell-ECM adhesion by blocking the activation of the associated signaling to control cancer. Here, we identify erybraedin A (EBA) as a potential Src inhibitor that blocks cell adhesion and viability in non-small-cell lung cancer (NSCLC). EBA significantly inhibited the adhesion of NSCLC cells to fibronectin. EBA also markedly inhibited the activation of Src and its downstream targets, including FAK and Akt. The interaction between integrin β1 or integrin β3 and Src was inhibited by EBA treatment. A docking study revealed the bindings of EBA to the ATP-binding pocket and the allosteric regulatory site of the Src kinase. Additionally, EBA markedly inhibited the viability and the colony formation of NSCLC cells and induced apoptotic cell death. These results describe novel biological properties of EBA, which can block the Src-mediated adhesion and survival of NSCLC cells, suggesting the potential of EBA as an anticancer Src inhibitor that warrants further development in advanced preclinical and clinical settings.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Lactic acid induces lactate transport and glycolysis/OXPHOS interconversion in glioblastoma

Highlights: • Lactic acidosis induces lactate transportation and reuse. • Under glucose-deprived condition, lactic acidosis could keep cell growth and maintain ATP content to resist cell death through glycolysis/ OXPHOS inter-conversion in U251 cell. • Different region of tumor tissue has diverse glucose metabolic way to adapt adverse microenvironment and maintain proliferation and progression in GMB. The Warburg effect is a dominant phenotype of most tumor cells. Recent reports have shown that the Warburg effect can be reprogrammed by the tumor microenvironment. Lactic acidosis and glucose deprivation are the common adverse microenvironments in solid tumor. The metabolic reprogramming induced by lactic acid and glucose deprivation remains to be elucidated in glioblastoma. Here, we show that, under glucose deprivation, lactic acid can preserve high ATP levels and resist cell death in U251 cells. At the same time, we find that MCT1 and MCT4 are significantly highly expressed. The metabolic regulation factor HIF-1α decreased and C-MYC increased. Nuclear respiratory factor 1 (NRF1) and oxidative phosphorylation (OXPHOS)-related proteins (NDUFB8, ND1) are all distinctly increased. Therefore, lactic acid can induce lactate transport and convert the dominant Warburg effect to OXPHOS. Through bioinformatics analysis, the high expression of HIF-1α, MCT1 or MCT4 indicate a poor prognosis in glioblastoma. In addition, in glioblastoma tissue, HIF-1α, MCT4 and LDH are highly expressed in the interior region, and their expression is decreased in the lateral region. MCT1 can not be detected in the interior region and is highly expressed in the lateral region. Hence, different regions of glioblastoma have diverse energy metabolic pathways. Glycolysis occurs mainly in the interior region and OXPHOS in the lateral region. In general, lactic acid can induce regional energy metabolic reprogramming and assist tumor cells to adapt and resist adverse microenvironments. This study provides new ideas for furthering understanding of the metabolic features of glioblastoma. It may promote the development of new therapeutic strategies in GBM.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

TMEM16F/ANO6, a Ca2+-activated anion channel, is negatively regulated by the actin cytoskeleton and intracellular MgATP

Highlights: • ANO6 current shows sluggish activation even with high [Ca{sup 2+}]{sub i}, and inactivation. • Actin cytoskeleton disruption accelerate both activation and inactivation of ANO6. • Intracellular MgATP further delays ANO6 activation, and prevent inactivation. • The effects of MgATP on ANO6 were overcome by actin inhibitor, cytochalasin-D. • State actin cytoskeleton is crucial for determining ANO6 activity in intact cells. Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca{sup 2+}]{sub i}. In addition to the low sensitivity to Ca{sup 2+}, the activation of ANO6 Cl{sup −} conductance is very slow (>3–5 min to reach peak level at 10 μM [Ca{sup 2+}]{sub i}), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (I{sub ANO6,w-c}), disruption of the actin cytoskeleton with cytochalasin-D (cytoD) significantly accelerated the activation kinetics, while actin filament-stabilizing agents (phalloidin and jasplakinolide) commonly inhibited I{sub ANO6,w-c}. Inside-out patch clamp recording of ANO6 (I{sub ANO6,i-o}) showed immediate activation by raising [Ca{sup 2+}]{sub i}. We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of I{sub ANO6,w-c}, and also prevented the inactivation of I{sub ANO6,w-c}. However, the addition of cytoD still accelerated both activation and inactivation of I{sub ANO6,w-c}. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca{sup 2+}-dependent activation and inactivation of I{sub ANO6,w-c}, respectively.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a distinctive mode of engaging poly(ADP-ribose)

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

STK40 Is a Pseudokinase that Binds the E3 Ubiquitin Ligase COP1

Serine/threonine kinase 40 (STK40) was originally identified as a distant homolog of Tribbles-family proteins. Despite accumulating data attesting to the importance of STK40 in a variety of different physiologic processes, little is known about its biological activity or mechanism of action.In this work, we show that STK40 interacts with Constitutive Photomorphogenic Protein 1 (COP1), relying primarily on a C-terminal sequence analogous to the motif found in Tribbles proteins. In order to further elucidate structure-function relationships in STK40, we determined the crystal structure of the STK40 kinase homology domain at 2.5 Å resolution. The structure, together with ATP-binding assay results, show that STK40 is a pseudokinase, in which substitutions of conserved residues within the kinase domain prevent ATP binding. Although the structure of the kinase homology domain diverges from the analogous region of Trib1, the results reported here suggest functional parallels between STK40 and Tribbles-family proteins as COP1 adaptors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Plasmatic concentration of organochlorine lindane acts as metabolic disruptors in HepG2 liver cell line by inducing mitochondrial disorder

Lindane (LD) is a persistent environmental pollutant that has been the subject of several toxicological studies. However, concentrations used in most of the reported studies were relatively higher than those found in the blood of the contaminated area residents and effects of low concentrations remain poorly investigated. Moreover, effects on cell metabolism and mitochondrial function of exposure to LD have received little attention. This study was designed to explore the effects of low concentrations of LD on cellular metabolism and mitochondrial function, using the hepatocarcinoma cell line HepG2. Cells were exposed to LD for 24, 48 and 72 h and different parameters linked with mitochondrial regulation and energy metabolism were analyzed. Despite having any impact on cellular viability, exposure to LD at plasmatic concentrations led to an increase of maximal respiratory capacity, complex I activity, intracellular ATP and NO release but decreased uncoupled respiration to ATP synthesis and medium lactate levels. In addition, LD exposure resulted in the upregulation of mitochondrial biogenesis genes. We suggest that, at plasmatic concentrations, LD acts as a metabolic disruptor through impaired mitochondrial function and regulation with an impact on cellular energetic metabolism. In addition, we propose that a cellular assay based on the analysis of mitochondria function, such as described here for LD, may be applicable for larger studies on the effects of low concentrations of xenobiotics, because of the exquisite sensitivity of this organelle. - Highlights: Our data clearly demonstrated in HepG2 cells that exposure at plasmatic low concentrations of LD were able to: • Impair mitochondrial function • Caused alteration on nucleo-mitochondrial cross-talk • Increase nitric oxide release and protein nitration • Impair cellular energetic metabolism and lipid accumulation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP-treated embryo exhibits a Warburg-like effect in tumor cell.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Characterizing the mechanism of thiazolidinedione-induced hepatotoxicity: An in vitro model in mitochondria

Objective: To characterize the mechanism of action of thiazolidinedione (TZD)-induced liver mitochondrial toxicity caused by troglitazone, rosiglitazone, and pioglitazone in HepaRG cells. Methods: Human hepatoma cells (HepaRG) were treated with troglitazone, rosiglitazone, or pioglitazone (12.5, 25, and 50 μM) for 48 h. The Seahorse Biosciences XF24 Flux Analyzer was used to measure mitochondrial oxygen consumption. The effect of TZDs on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. The mitochondrial ultrastructure of HepaRG cells was observed under a transmission electrical microscope (TEM). mtDNA content was evaluated by real-time PCR, and ATP content and mitochondrial respiratory chain (MRC) complex I, II, III, IV activity were measured via chemiluminescence. Results were considered statistically significant at p < 0.05. Results: Among the three drugs, troglitazone exhibited the highest potency, followed by rosiglitazone, and then pioglitazone. The TZDs caused varying degrees of mitochondrial respiratory function disorders including decreases in oxygen consumption, MRC activity, and ATP level, and an elevation in ROS level. TZD treatment resulted in mtDNA content decline, reduction in MMP, and alterations of mitochondrial structure. Conclusion: All investigated TZDs show a certain degree of mitochondrial toxicity, with troglitazone exhibiting the highest potency. The underlying mechanism of TZD-induced hepatotoxicity may be associated with alterations in mitochondrial respiratory function disorders, oxidative stress, and changes in membrane permeability. These parameters may be used early in drug development to further optimize risk:benefit profiles. - Highlights: • We compared three TZD mitochondrial toxicity characteristics in HepaRG cells. • TZD induced respiratory disorders and mitochondrial structural damage. • Mitochondrial toxicity evaluation presents guidance value for hepatotoxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Toluene diisocyanate: Induction of the autotaxin-lysophosphatidic acid axis and its association with airways symptoms

Diisocyanates are industrial chemicals which have a wide range of applications in developed and developing countries. They are notorious lung toxicants and respiratory sensitizers. However, the mechanisms behind their adverse effects are not adequately characterized. Autotaxin (ATX) is an enzyme producing lysophosphatidic acid (LPA), and the ATX-LPA axis has been implicated in lung related inflammatory conditions and diseases, including allergic asthma, but not to toxicity of environmental low-molecular-weight chemicals. We investigated effects of toluene diisocyanate (TDI) on ATX induction in human lung epithelial cell models, and we correlated LPA-levels in plasma to biomarkers of TDI exposure in urine collected from workers exposed to < 5 ppb (parts per billion). Information on workers' symptoms was collected through interviews. One nanomolar TDI robustly induced ATX release within 10 min in vitro. A P2X7- and P2X4-dependent microvesicle formation was implicated in a rapid ATX release and a subsequent protein synthesis. Co-localization between purinergic receptors and ATX was documented by immunofluorescence and confocal microscopy. The release was modulated by monocyte chemoattractant protein-1 (MCP-1) and by extracellular ATP. In workers, we found a dose–response relationship between TDI exposure biomarkers in urine and LPA levels in plasma. Among symptomatic workers reporting “sneezing”, the LPA levels were higher than among non-symptomatic workers. This is the first report indicating induction of the ATX-LPA axis by an environmental low-molecular-weight chemical, and our data suggest a role for the ATX-LPA axis in TDI toxicity. - Highlights: • Human epithelial cells release autotaxin in response to 1 nM toluene diisocyanate (TDI). • The release involves P2X4 and P2X7 receptors and is modulated by ATP and MCP-1. • Lysophosphatidic acid (LPA) was measured in workers exposed to < 5 ppb TDI. • LPA in plasma correlated to TDI exposure biomarkers in workers. • Symptomatic workers had higher LPA levels than those without symptoms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major determinants of the intracellular concentration of 4HNE. • Higher concentrations of 4HNE promote apoptosis whereas lower promote proliferation. • Stress-mediated signaling can be modulated by the α-class glutathione S-transferase. • Genotoxic effect of 4HNE may be ameliorated by modulating the cellular GSH levels. • RLIP76 (RalBP1) mediates ATP-dependent transport of GSH-conjugate of 4HNE (GSHNE).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen

Obesity and nonalcoholic fatty liver disease (NAFLD) can increase susceptibility to hepatotoxicity induced by some xenobiotics including drugs, but the involved mechanisms are poorly understood. For acetaminophen (APAP), a role of hepatic cytochrome P450 2E1 (CYP2E1) is suspected since the activity of this enzyme is consistently enhanced during NAFLD. The first aim of our study was to set up a cellular model of NAFLD characterized not only by triglyceride accumulation but also by higher CYP2E1 activity. To this end, human HepaRG cells were incubated for one week with stearic acid or oleic acid, in the presence of different concentrations of insulin. Although cellular triglycerides and the expression of lipid-responsive genes were similar with both fatty acids, CYP2E1 activity was significantly increased only by stearic acid. CYP2E1 activity was reduced by insulin and this effect was reproduced in cultured primary human hepatocytes. Next, APAP cytotoxicity was assessed in HepaRG cells with or without lipid accretion and CYP2E1 induction. Experiments with a large range of APAP concentrations showed that the loss of ATP and glutathione was almost always greater in the presence of stearic acid. In cells pretreated with the CYP2E1 inhibitor chlormethiazole, recovery of ATP was significantly higher in the presence of stearate with low (2.5 mM) or high (20 mM) concentrations of APAP. Levels of APAP-glucuronide were significantly enhanced by insulin. Hence, HepaRG cells can be used as a valuable model of NAFLD to unveil important metabolic and hormonal factors which can increase susceptibility to drug-induced hepatotoxicity. - Highlights: • Nonalcoholic fatty liver disease (NAFLD) is frequent in obese individuals. • NAFLD can favor hepatotoxicity induced by some drugs including acetaminophen (APAP). • A model of NAFLD was set up by using HepaRG cells incubated with stearate or oleate. • Stearate-loaded HepaRG cells presented higher cytochrome P450 2E1 (CYP2E1) activity. • APAP cytotoxicity was stronger in steatotic HepaRG cells with higher CYP2E1 activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Destabilization of mitochondrial functions as a target against breast cancer progression: Role of TPP{sup +}-linked-polyhydroxybenzoates

Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP{sup +}). Thus, we evaluated five TPP{sup +}-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having a wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP{sup +}-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP{sup +} and its derivatives warrant future investigation as potential anti-tumor agents. - Highlights: • TPP{sup +}-polyhydroxybenzoates are cytotoxic to various subtypes of breast cancer cells. • Cytotoxicity is not-dependent on the expression of estrogen/growth factor receptors. • Cytotoxicity appears to be triggered by a weak mitochondrial uncoupling effect. • Effects include loss of transmembrane potential and apoptosis was detected. • TPP{sup +}-polyhydroxybenzoates inhibit migration of highly metastatic cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

60 APPLIED LIFE SCIENCES↗