Changes In Mitochondrial Permeability Research Articles

DON induced DNA damage triggers absence of p53-mediated G2 arrest and apoptosis in IPEC-1 cells

Deoxynivalenol (DON) stands among the prevalent mycotoxins, and usually contaminates cereal foods and animal feed, leading to human and animal clinical poisoning symptoms such as abdominal pain, diarrhea, and vomiting. To date, the mechanism of toxicity of DON in different mammalian cells is not fully elucidated. In this study, we explored the detrimental impacts of DON on porcine intestinal epithelial cells (IPEC-1), serving as a representative model for porcine intestinal epithelial cells. After treating cells with DON for 24 h, DON can significantly inhibit the activity of cells, induce the production of reactive oxygen species (ROS), significantly reduce the content of glutathione and the activity of catalase, and increase the activity of superoxide dismutase and malondialdehyde, leading to an imbalance in intracellular redox status. In addition, DON can induce DNA double-strand breaks, and decrease mitochondrial membrane potential. Furthermore, DON can promote the release of Cyt C through changes in mitochondrial permeability through inhibit the expression of B-cell lymphoma 2 (Bcl-2) proteins, leading to apoptosis through the mitochondrial pathway. On the other hand, we found that DON can cause IPEC-1 cells G2 phase cycle arrest. Different with our pervious study, DON induces cell cycle arrest in the G2 phase only by activating the ATM-Chk2-Cdc 25 C pathway, but cannot regulate the cell cycle arrest via the ATM-p53 pathway. These results indicate that DON can induce the same toxic phenotype in different cells, but its toxic mechanism is different. All these provide a rationale for revealing DON induced cytotoxicity and intestinal diseases.

Abstract A009: Therapeutic targeting of P2 × 4 receptor and mitochondrial metabolism in clear cell renal carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Large scale metabolomic data have associated metabolic alterations with the pathogenesis and progression of renal carcinoma and have correlated mitochondrial activity with poor survival in a subset of patients. Our data suggest that oxo-phosphorylation is the main source of tumor-derived ATP in ccRCC expressing P2XR4, which exerts a critical impact on tumor energy metabolism and mitochondrial activity. Seahorse experiments showed that P2XR4 inhibition by 5-BDBD, a potent selective antagonist, caused a rapid dose-dependent reduction of mitochondrial activity and lysosome damage. Moreover, prolonged mitochondrial failure induced by 5-BDBD or P2XR4 silencing was associated with increased oxygen radical species, changes in mitochondrial permeability (i.e. opening of the transition pore complex, dissipation of membrane potential and calcium overload), and cell death via both necrosis and apoptosis. P2XR4 pharmacological inhibition was associated with a significant anti-tumor effect both in vitro and in vivo, as shown in ccRCC cell lines, patient-derived organoids and in murine xenografts. Interestingly, higher mitochondrial activity was associated with greater sensitivity to P2XR4 inhibition. Overall, our results suggest that the perturbed balance between lysosomal integrity and mitochondrial activity induced by P2XR4 inhibition may represent a new therapeutic strategy for a subset of renal carcinoma patients. Citation Format: Sabrina Orsi, Christopher Rupert, Roberto Pili, Filomena De Nigris. Therapeutic targeting of P2 × 4 receptor and mitochondrial metabolism in clear cell renal carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A009.

Therapeutic targeting of P2X4 receptor and mitochondrial metabolism in clear cell renal carcinoma models

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer. Large-scale metabolomic data have associated metabolic alterations with the pathogenesis and progression of renal carcinoma and have correlated mitochondrial activity with poor survival in a subset of patients. The aim of this study was to determine whether targeting mitochondria-lysosome interaction could be a novel therapeutic approach using patient-derived organoids as avatar for drug response.MethodsRNAseq data analysis and immunohistochemistry were used to show overexpression of Purinergic receptor 4 (P2XR4) in clear cell carcinomas. Seahorse experiments, immunofluorescence and fluorescence cell sorting were used to demonstrate that P2XR4 regulates mitochondrial activity and the balance of radical oxygen species. Pharmacological inhibitors and genetic silencing promoted lysosomal damage, calcium overload in mitochondria and cell death via both necrosis and apoptosis. Finally, we established patient-derived organoids and murine xenograft models to investigate the antitumor effect of P2XR4 inhibition using imaging drug screening, viability assay and immunohistochemistry.ResultsOur data suggest that oxo-phosphorylation is the main source of tumor-derived ATP in a subset of ccRCC cells expressing P2XR4, which exerts a critical impact on tumor energy metabolism and mitochondrial activity. Prolonged mitochondrial failure induced by pharmacological inhibition or P2XR4 silencing was associated with increased oxygen radical species, changes in mitochondrial permeability (i.e., opening of the transition pore complex, dissipation of membrane potential, and calcium overload). Interestingly, higher mitochondrial activity in patient derived organoids was associated with greater sensitivity to P2XR4 inhibition and tumor reduction in a xenograft model.ConclusionOverall, our results suggest that the perturbed balance between lysosomal integrity and mitochondrial activity induced by P2XR4 inhibition may represent a new therapeutic strategy for a subset of patients with renal carcinoma and that individualized organoids may be help to predict drug efficacy.

Protective effect of vitamin E against plumbagin-induced liver injury and oxidative stress: biochemical, redox, and mitochondrial permeability changes

Protective effect of vitamin E against plumbagin-induced liver injury and oxidative stress: biochemical, redox, and mitochondrial permeability changes

Rates of oxidative ATP synthesis are not augmented beyond the pH threshold in human vastus lateralis muscles during a stepwise contraction protocol.

The oxygen cost of high-intensity exercise at power outputs above an individual's lactate threshold (LT) is greater than would be predicted by the linear oxygen consumption-power relationship observed below the LT. However, whether these augmentations are caused by an increased ATP cost of force generation (ATPCOST ) or an increased oxygen cost of ATP synthesis is unclear. We used 31 P-MRS to measure changes in cytosolic [ADP] (intramyocellular marker of oxidative metabolism), oxidative ATP synthesis (ATPOX ) and ATPCOST during a 6-stage, stepwise knee extension protocol. ATPCOST was unchanged across stages. The relationship between [ADP] and muscle power output was augmented at workloads above the pH threshold (pHT ; proxy for LT), whereas increases in ATPOX were attenuated. These results suggest the greater oxygen cost of contractions at workloads beyond the pHT is not caused by mechanisms that increase ATPCOST , but rather mechanisms that alter intrinsic mitochondrial function or capacity. Increases in skeletal muscle metabolism and oxygen consumption are linearly related to muscle power output for workloads below the lactate threshold (LT), but are augmented (i.e. greater rate of increase relative to workload) thereafter. Presently, it is unclear whether these metabolic augmentations are caused by increases in the ATP cost of force generation (ATPCOST ) or changes in the efficiency of mitochondrial oxygen consumption and oxidative ATP synthesis (ATPOX ). To partition these two hypotheses in vivo, we used 31 P-MRS to calculate slopes relating step-changes in muscle work to concurrent changes in cytosolic phosphates and ATPOX before and after the pH threshold (pHT ; used here as a proxy for LT) within the vastus lateralis muscle of eight young adults during a stepwise knee extension test. Changes in muscle phosphates and ATPOX were linearly related to workload below the pHT . However, slopes above the pHT were greater for muscle phosphates (P<0.05) and lower for ATPOX (P<0.05) than were the slopes observed below the pHT . The maximal capacity for ATPOX ( ) and ADP-specific ATPOX also declined beyond the pHT (P<0.05), whereas ATPCOST was unchanged (P=0.10). These results oppose the hypothesis that high-intensity contractions increase ATPCOST and suggest that greater oxidative metabolism at workloads beyond the pHT is caused by mechanisms that affect intrinsic mitochondrial function or capacity, such as alterations in substrate selection or electron entry into the electron transport chain, temperature-mediated changes in mitochondrial permeability to protons, or stimulation of mitochondrial uncoupling by reactive oxygen species generation.

Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

Photoreceptor death contributes to 50% of irreversible vision loss in the western world. Pro23His (P23H) transgenic albino rat strains are widely used models for the most common rhodopsin gene mutation associated with the autosomal dominant form of retinitis pigmentosa. However, the mechanism(s) by which photoreceptor death occurs are not well understood and were the principal aim of this study. We first used electroretinogram recording and optical coherence tomography to confirm the time course of functional and structural loss. Electroretinogram analyses revealed significantly decreased rod photoreceptor (a-wave), bipolar cell (b-wave) and amacrine cell responses (oscillatory potentials) from P30 onward. The cone-mediated b-wave was also decreased from P30. TUNEL analysis showed extensive cell death at P18, with continued labeling detected until P30. Focused gene expression arrays indicated activation of, apoptosis, autophagy and necroptosis in whole retina from P14-18. However, analysis of mitochondrial permeability changes (ΔΨm) using JC-1 dye, combined with immunofluorescence markers for caspase-dependent (cleaved caspase-3) and caspase-independent (AIF) cell death pathways, indicated mitochondrial-mediated cell death was not a major contributor to photoreceptor death. By contrast, reverse-phase protein array data combined with RIPK3 and phospho-MLKL immunofluorescence indicated widespread necroptosis as the predominant mechanism of photoreceptor death. These findings highlight the complexity of mechanisms involved in photoreceptor death in the Pro23His rat model of degeneration and suggest therapies that target necroptosis should be considered for their potential to reduce photoreceptor death.

CIGARETTES THAT BROKE HER HEART: COPD-INDUCED TAKOTSUBU CARDIOMYOPATHY

SESSION TITLE: Medical Student/Resident Obstructive Lung Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Takotsubo cardiomyopathy (TCM) is a form of sudden transient LV dysfunction often associated with severe emotional and physical stressors; that mimics ACS. Currently, there are limited studies showing the relationship between COPD and TCM. We present a case of Takotsubo cardiomyopathy in a post-menopausal female secondary to AECOPD. CASE PRESENTATION: A 70-year-old female with a history of COPD, HTN, and smoking presented with severe midsternal chest pain and SOB associated with increased cough and sputum production. On arrival, she was tachycardic, tachypneic, and hypoxic. She had decreased air entry in bilateral lung fields with expiratory wheeze, respiratory acidosis, and hyperinflated lungs on CXR. She received multiple Ipratropium-Albuterol nebulizations for AECOPD resulting in symptomatic improvement. Further workup revealed EKG findings of new hyperacute T waves in V3-V6, TWI in V1 and V2; and up-trending Troponin I which peaked at 7.13. She was managed as Type I NSTEMI with Aspirin, Clopidogrel & IV Heparin. Emergent LHC showed 60% occlusion of RCA. TTE revealed a hyperkinetic base with hypokinetic apical and mid anterior wall, new-onset reduced LVEF of 40 to 45%, consistent with TCM. Her noncritical coronary artery disease was not considered to be contributing to her symptom burden and did not correspond to her EKG and echocardiographic findings. Follow up TTE in 6 months revealed improvement in LVEF to 55 to 60% with a resolution of apical hypokinesis. Given the history of no further psychosocial stressors most likely etiology for her TCM was stress related to acute COPD. DISCUSSION: AECOPD related respiratory failure leading to hypoxemia and hypercapnia has been identified as a potential physical trigger of TCM. Catecholamine excess plays a central role in stress-related apical myocardial stunning and coronary vasospasm; reducing the viability of myocytes via cAMP-mediated calcium overload resulting in contraction band necrosis. Hypoxia with coexisting acidosis causes cardiomyocyte damage through activation of BCL-2 family of proapoptotic proteins leading to changes in mitochondrial permeability and DNA fragmentation. Potential mechanisms include sympathetic overstimulation due to the administration of beta-agonist in the setting of endogenous catecholamine excess from the stress of respiratory failure and concurrent administration of ipratropium resulting in inhibition of parasympathetic system, tachycardia, and rarely paradoxical bronchospasm. It is unclear whether respiratory failure from COPD leading to hypoxia and hypercapnia or repeated use of Albuterol-Ipratropium caused TCM in our patient. CONCLUSIONS: In our case, we highlight that by early recognition of COPD induced TCM; complications could be minimized by limiting the use of beta-agonists and treating ongoing cardiac dysfunction. Further studies are required to establish the exact mechanism of COPD induced TCM. Reference #1: Sharkey S, Windenburg D, et al. "Natural History and Expansive Clinical Profile of Stress (Tako-Tsubo) Cardiomyopathy.” J Am Coll Cardiol., 2010 Jan 26; 55(4):333-341. DOI: https://doi.org/10.1016/j.jacc.2009.08.057 Reference #2: Wittstein I, Thiemann D, et al. "Neurohumoral features of myocardial stunning due to sudden emotional stress.” N Engl J Med. 2005 Feb 10; 352:539-548 https://doi.org/10.1056/NEJMoa043046 Reference #3: Kubasiak, Lori A. and Hernandez, Olga M. and Bishopric, Nanette H. and Webster, Keith A., "Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3.” Proceedings of the National Academy of Science, 2002 Oct; 99(20): 12825-12830. DOI = 10.1073/pnas.202474099 DISCLOSURES: No relevant relationships by Tahreem Ahmad, source=Web Response No relevant relationships by Khubaib Ahmad, source=Web Response No relevant relationships by Waqas Ali, source=Web Response No relevant relationships by Pranali Santhoshini Pachika, source=Web Response

Varying effects of tyrosine kinase inhibitors on platelet function-A need for individualized CML treatment to minimize the risk for hemostatic and thrombotic complications?

Since their introduction, tyrosine kinase inhibitors (TKIs, eg, imatinib, nilotinib, dasatinib, bosutinib, ponatinib) have revolutionized the treatment of chronic myeloid leukemia (CML). However, long‐term treatment with TKIs is associated with serious adverse events including both bleeding and thromboembolism. Experimental studies have shown that TKIs can cause platelet dysfunction. Herein, we present the first side‐by‐side investigation comparing the effects of currently used TKIs on platelet function and thrombin generation when used in clinically relevant concentrations. A flow cytometry multiparameter protocol was used to study a range of significant platelet activation events (fibrinogen receptor activation, alpha granule, and lysosomal exocytosis, procoagulant membrane exposure, and mitochondrial permeability changes). In addition, thrombin generation was measured in the presence of TKIs to assess the effects on global hemostasis. Results show that dasatinib generally inhibited platelet function, while bosutinib, nilotinib, and ponatinib showed less consistent effects. In addition to these general trends for each TKI, we observed a large degree of interindividual variability in the effects of the different TKIs. Interindividual variation was also observed when blood from CML patients was studied ex vivo with whole blood platelet aggregometry, free oscillation rheometry (FOR), and flow cytometry. Based on the donor responses in the side‐by‐side TKI study, a TKI sensitivity map was developed. We propose that such a sensitivity map could potentially become a valuable tool to help in decision‐making regarding the choice of suitable TKIs for a CML patient with a history of bleeding or atherothrombotic disease.

Mechanistic insights into functional characteristics of native crotamine

The chemical composition of snake venoms is a complex mixture of proteins and peptides that can be pharmacologically active. Crotamine, a cell-penetrating peptide, has been described to have antimicrobial properties and it exerts its effects by interacting selectively with different structures, inducing changes in the ion flow pattern and cellular responses. However, its real therapeutic potential is not yet fully known. Bearing in mind that crotamine is a promising molecule in therapeutics, this study investigated the action of purified molecule in three aspects: I) antibacterial action on different species of clinical interest, II) the effect of two different concentrations of the molecule on platelet aggregation, and III) its effects on isolated mitochondria. Crotamine was purified to homogeneity in a single step procedure using Heparin Sepharose. The molecular mass of the purified enzyme was 4881.4 Da, as determined by mass spectrometry. To assess antibacterial action, changes in the parameters of bacterial oxidative stress were determined. The peptide showed antibacterial activity on Escherichia coli (MIC: 2.0 μg/μL), Staphylococcus aureus (MIC: 8–16 μg/μL) and methicillin-resistant Staphylococcus aureus (MIC: 4.0–8.0 μg/μL), inducing bacterial death by lipid peroxidation and oxidation of target proteins, determined by thiobarbituric acid reactive substances and sulfhydryl groups, respectively. Crotamine induced increased platelet aggregation (IPA) at the two concentrations analyzed (0.1 and 1.4 μg/μL) compared to ADP-induced aggregation of PRP. Mitochondrial respiratory parameters and organelle structure assays were used to elucidate the action of the compound in this organelle. The exposure of mitochondria to crotamine caused a decrease in oxidative phosphorylation and changes in mitochondrial permeability, without causing damage in the mitochondrial redox state. Together, these results support the hypothesis that, besides the antimicrobial potential, crotamine acts on different molecular targets, inducing platelet aggregation and mitochondrial dysfunction.

Tebufenozide induces G1/S cell cycle arrest and apoptosis in human cells.

Tebufenozide is a non-steroidal insect growth regulator and is extensively used to control pests, although it is considered to be safe for mammals and environmentally friendly. However, previous studies have found that tebufenozide is cytotoxic to man, although the exact mechanism remains elusive. This study will investigate the apoptotic molecular mechanisms which result from tebufenozide-induced DNA damage in HeLa cells. Our results demonstrate that tebufenozide could trigger arrest in G1/S phase related to a downregulation of cyclin E and cyclin-dependent kinase (CDK) 2 protein. In addition, Western blotting showed apoptosis was associated with the upregulation of p53, Bax and cleaved-PARP, as well as downregulation of Bcl-2 and PARP. Tebufenozide also regulated changes in mitochondrial permeability and reduced mitochondrial number and intracellular ATP production. Briefly, these results suggest that tebufenozide- induces cell cycle arrest and apoptosis through activating p53 protein in a Bax- and Bcl-2-triggered mitochondrial pathway. This work provides some scientific context for the safe use of tebufenozide in agriculture.

Alcohol hangover induces mitochondrial dysfunction and free radical production in mouse cerebellum.

Alcohol hangover (AH) is defined as the temporary state after alcohol binge-like drinking, starting when ethanol (EtOH) is absent in plasma. Previous data indicate that AH induces mitochondrial dysfunction and free radical production in mouse brain cortex. The aim of this work was to study mitochondrial function and reactive oxygen species production in mouse cerebellum at the onset of AH. Male mice received a single i.p. injection of EtOH (3.8g/kg BW) or saline solution. Mitochondrial function was evaluated 6h after injection (AH onset). At the onset of AH, malate-glutamate and succinate-supported state 4 oxygen uptake was 2.3 and 1.9-fold increased leading to a reduction in respiratory control of 55% and 48% respectively, as compared with controls. Decreases of 38% and 16% were found in Complex I-III and IV activities. Complex II-III activity was not affected by AH. Mitochondrial membrane potential and mitochondrial permeability changes were evaluated by flow cytometry. Mitochondrial membrane potential and permeability were decreased by AH in cerebellum mitochondria. Together with this, AH induced a 25% increase in superoxide anion and a 92% increase in hydrogen peroxide production in cerebellum mitochondria. Related to nitric oxide (NO) metabolism, neuronal nitric oxide synthase (nNOS) protein expression was 52% decreased by the hangover condition compared with control group. No differences were found in cerebellum NO production between control and treated mice. The present work demonstrates that the physiopathological state of AH involves mitochondrial dysfunction in mouse cerebellum showing the long-lasting effects of acute EtOH exposure in the central nervous system.

The effects of baicalein on canine osteosarcoma cell proliferation and death

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti-inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage-independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.

Characterization and Solution Structure of Mouse Myristoylated Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.

Abstract 1345: Bioactivation of luteolin as a prodrug by tyrosinase inhibits human glutathione S-transferase and induces toxicity in SK-MEL-28 melanoma cells

Abstract Glutathione S-transferase (GST) play significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in their efficacy against melanoma. In the present study, we investigated: i) the selective inhibition of GST by luteolin in the presence of tyrosinase, an abundant enzyme found in melanoma, and ii) the induction of apoptosis, change in mitochondrial permeability, cell viability, cell migration and GSH depletion by luteolin in human melanoma SK-MEL-28 cell culture. The inhibition of GST was investigated using CDNB method. DTNB method was used to measure GSH depletion. MTT assay, Annexin V apoptosis assay and TMRM fluorescence were used in the investigation of biochemical mechanism of luteolin toxicity in melanoma cells. Luteolin (40 µM) induced toxicity and death in 50% of cells after 48 h incubation. Cell treatment with luteolin (40 µM) resulted in apoptotic cell death in 70% of cells and caused 45% decrease in TMRM fluorescence indicating change in mitochondrial membrane permeability. At 1 h incubation, luteolin was bioactivated by tyrosinase to luteoline-quinone for 71% as measured by GSH depletion. The luteolin is effective in preventing metastasis of cancerous cells as evident by cell migration assay. At 48 h incubation, luteolin as low as 5 µM effectively prevented the cell migration. In the presence of tyrosinase, luteolin (10 µM) showed about 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. Both luteolin-SG conjugate and luteolin-quinone (10 µM) inhibited ≥90% of GST activity via reversible and irreversible competitive mechanisms with Ki of 0.9 and 6.4 µM with respect to GSH, respectively. The luteolin-SG conjugate inhibited GST activity reversibly and non-competitively with Ki of 1.8, whereas luteolin-quinone showed irreversible competitive inhibition of GST activity with Ki of 1.0 µM with respect to CDNB. While luteolin is not a substrate for GST, Luteolin (25 µM) non-competitively inhibited GST with Ki of 39 µM with respect to GSH and competitively with Ki of 61 µM with respect to CDNB. Luteolin, at the concentration range of 5-80 µM, exhibited 78-99% GST inhibition in human SK-MEL-28 cell homogenate. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a quinone and luteolin-glutathione conjugate, which played major role in the inhibition of GST. For the first time, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1345. doi:10.1158/1538-7445.AM2011-1345

Titanium dioxide nanoparticles impair lung mitochondrial function

Titanium dioxide nanoparticles (TiO 2 NPs) are used in an increasing number of human products such as cosmetics, sunscreen, toothpaste and paints. However, there is clear evidence about effects associated to TiO 2 NPs exposure, which include lung inflammation and tumor formation and these effects are related to reactive oxygen species (ROS) formation. The ROS generation could be attributed to a mitochondrial dysfunction. Even though, it has been shown that TiO 2 NPs exposure can induce some alterations in mitochondria including cytochrome c release to cytosol, change in mitochondrial permeability and decrease of mitochondrial membrane potential (Δ Ψ m), there is no information about the changes in mitochondrial function induced by TiO 2 NPs. We hypothesized that TiO 2 NPs effects are associated with mitochondrial dysfunction and redox unbalance. To test our hypothesis we isolated mitochondria from lung tissue of rats and exposed them to 10 (g TiO 2 NPs (particle size < 25 nm)/mg protein for 1 h. Our results showed that TiO 2 NPs decreases NADH levels and impairs Δ Ψ m and mitochondrial function accompanied by ROS generation during mitochondrial respiration.

Changes In Mitochondrial Permeability Initiate Platelet Phosphatidylserine Exposure In Response to Elevated Cytoplasmic Calcium Levels

AbstractAbstract 2024Platelet phosphatidylserine (PS) exposure and procoagulant activity occur in a subset of activated platelets following stimulation with multiple or high levels of agonists. Key among the upstream regulators of agonist-initiated PS exposure is elevated cytoplasmic calcium levels. Additionally, the importance of mitochondrial permeability transition pore (mPTP) formation in regulating PS exposure has been highlighted by recent studies using cyclophilin D null (CypD -/-) platelets. CypD is a critical regulatory protein in the initiation of mPTP formation, and in the absence of CypD agonist-initiated PS exposure is markedly impaired. An alternative, slower pathway of PS exposure, initiated by the BH3 mimetic ABT-737, is regulated by members of the Bcl-2 protein pathway.It is currently unclear how mitochondrial and calcium-signaling pathways interact to regulate PS exposure in platelets. mPTP formation has been reported as both a consequence and a cause of elevated intracellular calcium levels in other cells. To clarify the relative roles of these two upstream signals, we examined the sequence of events that occur upon platelet activation using calcium and mitochondrial transmembrane potential (ΔΨm) sensitive fluorescent probes. In isolated platelets studied by flow cytometry and among adherent platelets evaluated by confocal microscopy, elevated intracellular calcium levels were observed prior to mPTP formation (as determined by loss of ΔΨm). A rise in mitochondrial calcium levels as measured by rhod-2 occurred coincidentally with elevations in cytoplasmic calcium levels. Similar increases in intracellular calcium levels were observed in CypD -/- and CypD +/+ platelets, despite the almost complete absence of mPTP formation and PS exposure in the CypD -/- platelets. These results are consistent with calcium acting as an upstream signal regulating mPTP formation.Ionomycin (3 μM) stimulation can overcome the defect in PS exposure in CypD -/- platelets. This result has been interpreted to indicate that calcium activates effector pathways downstream of the mPTP. However, an alternative explanation is that ionomycin raises cytoplasmic calcium to an elevated level that is sufficient to initiate mPTP formation even in CypD's absence. Dose response studies indicated that the calcium sensitivity of the mPTP was decreased in CypD -/- platelets, but ionomycin (3uM) caused mPTP opening and PS externalization in both CypD +/+ and CypD -/- platelets. Furthermore, in both CypD +/+ and CypD -/- platelets, mPTP formation and PS exposure were closely correlated at all doses of ionomycin utilized. These results indicate that high doses of ionomycin overcome the CypD -/- defect in PS exposure by initiating non-CypD-dependent mPTP formation.The roles of the mitochondrial and calcium pathways in regulating ABT-737-mediated PS exposure were examined. Extracellular calcium was not required for PS exposure initiated by ABT-737, distinct from its importance in agonist-initiated PS exposure. In contrast to previously reported results (Schoenwaelder et al, Blood 2009) we observed that ABT-737 stimulation causes a loss of ΔΨm coincident with PS exposure. Since disruptions in oxidative metabolism could also cause the observed effect on ΔΨm, ABT-737's effects on mitochondrial permeability were confirmed using a calcein-cobalt quenching assay. Unlike agonist-initiated PS exposure, both increased mitochondrial permeability and PS exposure were not dependent on CypD.Together these results indicate a central role for alterations in mitochondrial membrane permeability in mediating PS exposure. In agonist-stimulated platelets increases in cytoplasmic calcium result in mPTP formation and mitochondrial permeabilization, triggering downstream events that result in PS exposure. In ABT-737 stimulated platelets non-CypD-dependent mitochondrial permeability changes mediated by Bcl-2 proteins may also act as an important initiator of the events that result in PS exposure. Disclosures:No relevant conflicts of interest to declare.

Phase I Trial of Flavopiridol In Relapsed Myeloma: Brief Response In t(4;14) with Significant Neutropenia

AbstractAbstract 1933 Introduction:A pharmacokinetically derived schedule of flavopiridol administered as a 30 min intravenous bolus followed by 4-hour continuous intravenous infusion is active in fludarabine-refractory chronic lymphocytic leukemia, but no studies examining the feasibility and maximum tolerated dose of this schedule have been reported in relapsed multiple myeloma. Flavopiridol is a novel anti-cancer agent that targets cyclin dependent kinases (CDK) including the CDK9/cyclin T complex (preventing activation of RNA polymerase II), downregulates Mcl-1 and other anti-apoptotic proteins, and induces mitochondrial permeability changes. Flavopiridol is highly protein bound when in human serum, compared to protein binding seen in fetal bovine serum. This difference helps to explain the previous lack of clinical activity of flavopiridol (Dispenzieri et al, Haematologica, 2006) that targeted plasma concentrations based on in vitro cytotoxicity. A novel schedule of administration was designed to achieve and maintain target plasma levels predicted to be active in chronic lymphocytic leukemia from preclinical studies performed in human serum: 30-minute intravenous bolus (IVB) followed by 4-hour intravenous infusion (IVB/CIVI). This schedule, given for four of six weeks, is highly active in fludarabine refractory chronic lymphocytic leukemia. Methods:This was a phase I 3×3 single arm (standard method) study for relapsed myeloma patients having received at least two prior therapies. Adequate organ function was required with creatinine < 1.5 mg/dL and total bilirubin < 2x IULN. Adequate hematologic parameters were required with Hb > 9 g/dL, ANC>1500, and platelets > 50K during screening unless attributable to the patient's underlying myeloma. Results:15 patients (ages 49–81 y.o.) with relapsed myeloma were consented. Median number of prior therapies was 7 (3-12). At the time of study entry, 8 patients displayed a complex karyotype, 2 patients with 17p deletion by CD138-selected FISH, and one patient with t(4;14); 3 patients with karyotypic chr 13 deletion and 9 patients by FISH. At study entry, 7 patients had ISS stage 3 disease, 4 with stage 2, and 4 with stage 1. 5 patients were treated in cohort 1 (30 mg/m2 bolus/30 mg/m2 CIV), 3 patients in cohort 2 (30 mg/m2 bolus/50 mg/m2 CIV), and 7 patients in cohort 3 (50 mg/m2 bolus/50 mg/m2 CIV). Median number of cycles received was 1. No patients achieved a confirmed PR – two patients achieved a minor response by IMWG criteria (see figure). The one patient with near 50% response in his IgA myeloma was the only patient with a t(4;14), 13-, and tetraploid cytogenetics by FISH. Grade 3/4 toxicities were significant with grade 4 neutropenia (10 patients), diarrhea (6 patients), transaminitis (4 patients), thrombocytopenia (3 patients), and anemia (5 patients). The most common toxicities included neutropenia, diarrhea, and AST elevation. Two patients in the first cohort and 1 patient in the final cohort were replaced due to inability to complete the first cycle. Pharmacokinetic results and immunohistochemical staining results for cyclin-D1 and pRb will be presented at the meeting; cyclin-D1 overexpression has been linked to CDK-inhibitor response (Dai Y et al, Cell Cycle 2006). One patient is undergoing screening to complete the final cohort and complete response and toxicity data will be reported. Discussion:Flavopiridol as a single agent given by intravenous bolus/continuous intravenous infusion causes significant neutropenia and diarrhea but objective clinical responses were uncommon. (ClinicalTrials.gov Identifier: NCT00112723). [Display omitted] Disclosures:Jones:Glaxo Smith-Kline: Consultancy; Abbott: Research Funding.

A Red Wine Extract Supplement Protects against Apoptosis and Inhibits Changes in Mitochondrial Permeability and Membrane Potential

The effects of a red wine extract supplement (Super S.O.D) on hypoxia/reoxygenation-induced apoptosis were examined in H9c2 cardiomyocytes. Super S.O.D pretreatment dose-dependently protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated with increases in cellular reduced glutathione level and mitochondrial membrane potential, as well as the decrease in the extent of Ca2+-induced mitochondrial permeability transition (MPT) pore opening in both unchallenged and challenged cells, as compared with the unpretreated control. The results indicated that Super S.O.D pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes, possibly mediated by the inhibition of MPT triggered by hypoxia/reoxygenation challenge.

Oxidative stress causes reversible changes in mitochondrial permeability and structure

Mitochondria are a primary source as well a principal target of reactive oxygen species within cells. Using immunofluorescence microscopy, we have found that a number of mitochondrial matrix proteins are normally undetectable in formaldehyde-fixed cells permeabilized with the cholesterol-binding detergent saponin. However, exogenous or endogenous oxidative stress applied prior to fixation altered the permeability of mitochondria, rendering these matrix proteins accessible to antibodies. Electron microscopy revealed a loss of matrix density and disorganization of inner membrane cristae upon oxidative stress. Notably, the changes in permeability and in structure were rapidly reversed when the oxidative stress was relieved. The ability of reactive oxygen species to reversibly alter the permeability of the mitochondrial membrane provides a potential mechanism for communication within the cell such as between nucleus and mitochondria.

Spatial and Temporal Dynamics of Mitochondrial Membrane Permeability Waves during Apoptosis

Change in the permeability of the mitochondrial membrane to proteins (cytochrome c and Smac) and protons is a critical step in apoptosis. Although the time from the induction of apoptosis to the change of mitochondrial permeability is variable over a period of hours, the release of proteins is an “all or none” phenomenon that is completed in an individual cell within minutes. Here, using single-cell fluorescence microscopy, we show that the release of cytochrome c from a single mitochondrion occurs in a single step. However, this increased permeability of the outer membrane to cytochrome c propagates throughout the cell as a slower, spatially coordinated wave. The permeability of the outer membrane to Smac propagates with the same spatial pattern but lagging in time. This is followed by a wave of increased permeability of the inner membrane to protons. Only afterward do the mitochondria fission. The spatial dependence of the permeability wave was inhibited by thapsigargin, an inhibitor of the endoplasmic reticulum calcium pumps, but buffering cytosolic calcium had no effect. These results show that the trigger for apoptosis is spatially localized, initiating at one or only a few mitochondria preceding the loss of mitochondrial energetics, and the subsequent temporal propagation of mitochondrial membrane permeability is calcium-dependent.