Reducing Cancer Risk Through Primary Prevention Activities Among Children: A Demonstration Project.

CONTEXT: Opportunities to reduce the risk of cancer, including cervical, liver, and skin cancer, start early in life. To encourage adoption of primary prevention activities in childhood to reduce cancer risk later in life, Centers for Disease Control and Prevention conducted a demonstration project with 3 National Comprehensive Cancer Control Program (NCCCP) recipients. PROGRAM: Iowa, Northwest Portland Area Indian Health Board (NPAIHB), and Pennsylvania NCCCP recipients implemented evidence-based primary prevention activities for cervical, liver, and skin cancer among children using health care provider education, patient education, and policy development. IMPLEMENTATION: Iowa implemented an announcement approach to improve provider education on human papillomavirus (HPV) vaccination. Pennsylvania focused on patient education for reducing skin cancer risk and both provider and patient education for liver cancer prevention. NPAIHB created a sun safety intervention for tribal organizations, including a policy guide, media materials, and patient education. RESULTS: In Iowa, health care providers taking the announcement approach reported significantly higher mean scores on a posttest compared with a pretest regarding perceptions about HPV vaccination, self-efficacy, and behavioral intentions related to vaccination. Pennsylvania integrated sun safety education and sunscreen dispenser programs as a health and wellness initiative in 8 state parks and the Pennsylvania Department of Conservation and Natural Resources incorporated the program in its Pennsylvania Outdoor Recreation Plan. Pennsylvania also implemented health care provider education on the primary prevention of liver cancer through hepatitis B and hepatitis C screening and hepatitis B vaccination. The NPAIHB skin cancer policy guide was created and distributed for use to all 43 federally recognized tribes of Oregon, Washington, and Idaho served by NPAIHB. DISCUSSION: The identification, dissemination, and implementation of these efforts can serve as best practices for future childhood primary prevention programs. NCCCP recipients and public health professionals can use health care provider education, patient education, and policy development to reduce future risk for cervical, liver, and skin cancer among children.

Functional role and molecular mechanisms underlying prohibitin 2 in platelet mitophagy and activation.

Platelet mitophagy is a major pathway involved in the clearance of injured mitochondria during hemostasis and thrombosis. Prohibitin 2 (PHB2) has recently emerged as an inner mitochondrial membrane receptor involved in mitophagy. However, the mechanisms underlying PHB2­mediated platelet mitophagy and activation are not completely understood. PHB2 is a highly conserved inner mitochondrial membrane protein that regulates mitochondrial assembly and function due to its unique localization on the mitochondrial membrane. The present study aimed to investigate the role and mechanism underlying PHB2 in platelet mitophagy and activation. Phorbol­12­myristate­13­acetate (PMA) was used to induce MEG­01 cells maturation and differentiate into platelets following PHB2 knockdown. Cell Counting Kit­8 assays were performed to examine platelet viability. Flow cytometry was performed to assess platelet mitochondrial membrane potential. RT­qPCR and western blotting were conducted to measure mRNA and protein expression levels, respectively. Subsequently, platelets were exposed to CCCP and the role of PHB2 was assessed. The results of the present study identified a crucial role for PHB2 in platelet mitophagy and activation, suggesting that PHB2­mediated regulation of mitophagy may serve as a novel strategy for downregulating the expression of platelet activation genes. Although further research into mitophagy is required, the present study suggested that PHB2 may serve as a novel therapeutic target for thrombosis­related diseases due to its unique localization on the mitochondrial membrane.

Carbonyl cyanide 3-chlorophenylhydrazone induced the imbalance of mitochondrial homeostasis in the liver of Megalobrama amblycephala: A dynamic study.

Carbonylcyanide-3-chlorophenylhydrazone (CCCP) is a protonophore, which causes uncoupling of proton gradient in the inner mitochondrial membrane, thus inhibiting the rate of ATP synthesis. However, this information is manly derived from mammals, while its effects on the mitochondrial homeostasis of aquatic animals are largely unknown. In this study, the mitochondrial homeostasis of a carp fish Megalobrama amblycephala was investigated systematically in a time-course manner by using CCCP. Fish was injected intraperitoneally with CCCP (1.8 mg/kg per body weight) and DMSO (control), respectively. The results showed that CCCP treatment induced hepatic mitochondrial oxidative stress, as was evidenced by the significantly increased MDA and PC contents coupled with the decreased SOD and MnSOD activities. Meanwhile, mitochondrial fission was up-regulated remarkably characterized by the increased transcriptions of Drp-1, Fis-1 and Mff. However, the opposite was true for mitochondrial fusion, as was indicative of the decreased transcriptions of Mfn-1, Mfn-2 and Opa-1. This consequently triggered mitophagy, as was supported by the accumulated mitochondrial autophagosomes and the increased protein levels of PINK1, Parkin, LC3-II and P62 accompanied by the increased LC3-II/LC3-I ratio. Mitochondrial biogenesis and function both decreased significantly addressed by the decreased activities of CS, SDH and complex I, IV and V, as well as the protein levels of PGC-1ß coupled with the decreased transcriptions of TFAM, COX-1, COX-2 and ATP-6. Unlikely, DMSO treatment exerted little influence. Overall, CCCP treatment resulted in the imbalance of mitochondrial homeostasis in Megalobrama amblycephala by promoting mitochondrial oxidative stress, fission and mitophagy, but depressing mitochondrial fusion, biogenesis and function.

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler.

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1-2 µM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.

Glycolysis and mitochondrial function regulate the radical oxygen species production induced by platelet-activating factor in bovine polymorphonuclear leukocytes.

Dairy cows undergo metabolic disturbances in the peripartum period, during which infectious inflammatory diseases and detrimental polymorphonuclear leukocytes (PMN) functions, such as radical oxygen species (ROS) production, are observed. Platelet-activating factor (PAF) is a key pro-inflammatory mediator that increases PMN ROS production. To date, the role of glycolysis and mitochondria in PAF-induced ROS production in bovine PMN has not been known. The aim of this study was to assess whether inhibition of glycolysis and disruption of mitochondrial function alter the oxidative response induced by PAF. We isolated PMN from non-pregnant Holstein Friesian heifers and pre-incubated them with 2-deoxy-d-glucose (2-DG; 2 mM, 30 min), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 5 µM, 5 min), oligomycin (10 µM, 30 min) or rotenone (10 µM, 30 min). Respiratory burst was measured by luminol-chemiluminescence assay, while mitochondrial ROS (mtROS) were evaluated by MitoSOX probe and flow cytometry. Also, we detected the presence of mitochondria by MitoTracker Deep Red FM probe and changes in mitochondrial membrane potential (Δψm) were assessed by JC-1 probe and flow cytometry. We observed that all inhibitors separately were able to reduce PAF-induced ROS production. Presence of mitochondria was detected and PAF increased the Δψm, while CCCP reduced it. 2-DG and rotenone reduced the mtROS production induced by PAF. CCCP did not alter the mtROS and oligomycin administered independently increased mtROS production. We concluded that PAF-induced ROS production is glycolysis- and mitochondria-dependent. Bovine PMN have a functional mitochondrion and PAF induced mtROS via glycolysis and mitochondrial complex-I activity. Our results highlight an important modulation of cellular metabolism in the oxidative response induced by proinflammatory agents, which could contribute to PMN disfunction during peripartum in cattle.

Stress-induced phospho-ubiquitin formation causes parkin degradation.

Mutations in the E3 ubiquitin ligase parkin are the most common known cause of autosomal recessive Parkinson's disease (PD), and parkin depletion may play a role in sporadic PD. Here, we sought to elucidate the mechanisms by which stress decreases parkin protein levels using cultured neuronal cells and the PD-relevant stressor, L-DOPA. We find that L-DOPA causes parkin loss through both oxidative stress-independent and oxidative stress-dependent pathways. Characterization of the latter reveals that it requires both the kinase PINK1 and parkin's interaction with phosphorylated ubiquitin (phospho-Ub) and is mediated by proteasomal degradation. Surprisingly, autoubiquitination and mitophagy do not appear to be required for such loss. In response to stress induced by hydrogen peroxide or CCCP, parkin degradation also requires its association with phospho-Ub, indicating that this mechanism is broadly generalizable. As oxidative stress, metabolic dysfunction and phospho-Ub levels are all elevated in PD, we suggest that these changes may contribute to a loss of parkin expression.

Structure and Function of a Bacterial Gap Junction Analog.

Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease.

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

Cleaved PGAM5 is released from mitochondria depending on proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy.

PGAM5 is a unique type of protein phosphatase that exists in mitochondria. It has been shown to exist in the inner mitochondrial membrane through its transmembrane domain and to be cleaved within the transmembrane domain upon mitochondrial dysfunction. However, its submitochondrial localization remains controversial; many researchers claim that PGAM5 localizes to the outer mitochondrial membrane based on the findings that PGAM5 associates with many cytoplasmic proteins. Here, we found that cleaved PGAM5 was released from mitochondria during mitophagy, a selective form of autophagy specific for mitochondria, and that the release was inhibited by proteasome inhibitors in HeLa cells stably expressing the E3 ubiquitin ligase Parkin. However, treatment of parental HeLa cells lacking Parkin with mitophagy-inducing agents caused PGAM5 cleavage but did not cause its release from mitochondria. Thus, cleaved PGAM5 appears to be released from mitochondria depending on proteasome-mediated rupture of the outer membrane during mitophagy, which has been previously shown to precede autophagy-mediated degradation of whole mitochondria. This study suggests that PGAM5 senses mitochondrial dysfunction in the inner mitochondrial membrane and serves as a signalling intermediate that regulates the cellular response to mitochondrial stress upon its cleavage and release from mitochondria.

Gene disruption of ribosomal protein L5 (RPL5) decreased the sensitivity of CHO-K1 cells to uncoupler carbonylcyanide-3-chlorophenylhydrazone.

Protonophoric uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) decreases the proton motive force (ΔP) of the mitochondrial inner membrane and results in inhibition of oxidative phosphorylation. In this study, a CCCP-resistant clone was isolated from a random gene trap insertional mutant library of Chinese hamster ovary (CHO)-K1 cells which was constructed by infecting a retrovirus vector, ROSAßgeo. Although we expected the isolation of the mutants defective in nuclear genes responsible for mitochondrial functions, the disrupted gene of the isolated mutant that we named R1 cells was identified as one of the alleles for ribosomal protein 5 of large subunit (RPL5). The R1 cells express as much as 80% RPL5 protein compared with the parental CHO-K1 cells, possibly due to enhanced transcription from a remaining wild-type RPL5 allele in R1 cells. Furthermore, the protein amount is not decreased by CCCP in R1 cells, in contrast to its clear reduction by CCCP in parental cells. Since mutations of RPL5 and other ribosomal proteins are responsible for the ribosomopathies and cancer, the present mutant may be a useful cellular model of such human diseases from a viewpoint of energy metabolism as well as a tool for the study of ribosome biogenesis and extra-ribosomal function of the RPL5 protein.

Pleckstrin homology domain of p210 BCR-ABL interacts with cardiolipin to regulate its mitochondrial translocation and subsequent mitophagy.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain. Arg726, a basic amino acid in the ligand-binding region, was crucial for ligand recognition. A subset of wild-type p210 BCR-ABL that was transiently expressed in HEK293 cells was dramatically translocated from the cytosol to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, which induces mitochondrial depolarization and subsequent externalization of CL to the organelle's outer membrane, whereas an R726A mutant of the protein was not translocated. Furthermore, only wild-type p210 BCR-ABL, but not the R726A mutant, suppressed CCCP-induced mitophagy and subsequently enhanced reactive oxygen species production. Thus, p210 BCR-ABL can change its intracellular localization via interactions between the PH domain and CL to cope with mitochondrial damage. This suggests that p210 BCR-ABL could have beneficial effects for cancer proliferation, providing new insight into the PH domain's contribution to CML pathogenesis.

Effect of Cyanide on Mitochondrial Membrane Depolarization Induced by Uncouplers.

In this work, it was found that the ability of common uncouplers - carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) - to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor - cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.

Mitochondrial membrane potential (MMP) regulates sperm motility.

Sperm motility is the major decisive factor in determining male fertility. The objective of the present study was to analyse the effect of mitochondrial membrane potential (MMP) on the temporal regulation of sperm motility. Observations were recorded in various rodent species and among differentially motile sperm fractions including swim up and leftover layer of human semen sample using JC-1 stain (a marker of the MMP) through FACS. Swim-up sperms having highest motility showed significantly higher MMP as compared to leftover sperms, which had the least motility. Interestingly, infertile patients with compromised motility showed low MMP as compared to the healthy individuals. Further, as per the time lapse, sperm motility goes down, at the same time, it was observed that MMP also decreases in human as well as in rodent sperms. Treatment of known spermicides on human sperms reduced their motility drastically which in turn also reduced its MMP significantly. Treatment of human sperms with oxidative uncoupler also impeded their motility by reducing MMP, indicating a definitive role on MMP on sperm motility and fertility. Based on the results of the study, MMP can be considered as a potential regulator and indicator of sperm motility and hence could be directly related to male fertility.

Seahorse Xfe 24 Extracellular Flux Analyzer-Based Analysis of Cellular Respiration in Caenorhabditis elegans.

Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans.

Memristors: Memory elements in potato tubers.

A memristor is a nonlinear element because its current-voltage characteristic is similar to that of a Lissajous pattern for nonlinear systems. This element was postulated recently and researchers are looking for it in different biosystems. We investigated electrical circuitry of red Irish potato tubers (Solanum tuberosum L.). The goal was to discover if potato tubers might have a new electrical component - a resistor with memory. The analysis was based on a cyclic current-voltage characteristic where the resistor with memory should manifest itself. We found that the electrostimulation by bipolar sinusoidal or triangle periodic waves induces electrical responses in the potato tubers with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in potato tubers. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of potato tubers has properties of a memristor. Uncoupler carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decreases the amplitude of electrical responses at low and high frequencies of bipolar periodic sinusoidal or triangle electrostimulating waves. The discovery of memristors in plants creates a new direction in the understanding of electrical phenomena in plants.

Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.

Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.