An Evaluation of Boar Spermatozoa as a Biosensor for the Detection of Sublethal and Lethal Toxicity.

A novel, objective, and rapid computed motility inhibition (CMI) assay was developed to identify and assess sublethal injury in toxin-exposed boar spermatozoa and compared with a subjective visual motility inhibition (VMI) assay. The CMI values were calculated from digital micrographic videos using a custom MATLAB® script by contrasting the motility index values of each experiment with those of the background and control experiments. Following a comparison of the CMI and VMI assays results, it was determined that their agreement depended on the shape of the dose-response curve. Toxins that exhibited a steep slope were indicative of good agreement between the assays. Those depicted by a gentle decline in the slope of the dose-response curve, the CMI assay were shown to be two times more sensitive than the VMI assay. The CMI assay was highly sensitive to the inhibition of mitochondrial function and glucose transport activity by sublethal doses of toxins and to disruption of cellular cation homeostasis by carrier ionophoric toxins, when compared to the cytotoxicity and lethal toxicity assays (i.e., that evaluated the inhibition of cell proliferation in somatic cell lines (FL, PK-15, and MNA cells)) and disruption to spermatozoa membrane integrity. The CMI assay recognized subtle sublethal toxicity changes in metabolism, manifested as a decrease in boar spermatozoa motility. Thus, it was feasible to effectively compare the objectively-measured numerical values for motility inhibition using the CMI assay against those reflecting lethal damage in the spermatozoa cells and somatic cell lines using a cytotoxicity assay.

Virtual Cell Based Assay simulations of intra-mitochondrial concentrations in hepatocytes and cardiomyocytes.

In order to replace the use of animals in toxicity testing, there is a need to predict human in vivo toxic doses from concentrations that cause adverse effects in in vitro test systems. The virtual cell based assay (VCBA) has been developed to simulate intracellular concentrations as a function of time, and can be used to interpret in vitro concentration-response curves. In this study we refine and extend the VCBA model by including additional target-organ cell models and by simulating the fate and effects of chemicals at the organelle level. In particular, we describe the extension of the original VCBA to simulate chemical fate in liver (HepaRG) cells and cardiomyocytes (ICell cardiomyocytes), and we explore the effects of chemicals at the mitochondrial level. This includes a comparison of: a) in vitro results on cell viability and mitochondrial membrane potential (mmp) from two cell models (HepaRG cells and ICell cardiomyocytes); and b) VCBA simulations, including the cell and mitochondrial compartment, simulating the mmp for both cell types. This proof of concept study illustrates how the relationship between intra cellular, intra mitochondrial concentration, mmp and cell toxicity can be obtained by using the VCBA.

Multiscale modelling approaches for assessing cosmetic ingredients safety.

The European Union's ban on animal testing for cosmetic ingredients and products has generated a strong momentum for the development of in silico and in vitro alternative methods. One of the focus of the COSMOS project was ab initio prediction of kinetics and toxic effects through multiscale pharmacokinetic modeling and in vitro data integration. In our experience, mathematical or computer modeling and in vitro experiments are complementary. We present here a summary of the main models and results obtained within the framework of the project on these topics. A first section presents our work at the organelle and cellular level. We then go toward modeling cell levels effects (monitored continuously), multiscale physiologically based pharmacokinetic and effect models, and route to route extrapolation. We follow with a short presentation of the automated KNIME workflows developed for dissemination and easy use of the models. We end with a discussion of two challenges to the field: our limited ability to deal with massive data and complex computations.

Mitochondrial Membrane Potential Assay.

Mitochondrial function, a key indicator of cell health, can be assessed by monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in HepG2 cells. A homogenous cell-based MMP assay was optimized and performed in a 1536-well plate format to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on MMP.

Reduction of Mitochondrial Function by FCCP During Mouse Cleavage Stage Embryo Culture Reduces Birth Weight and Impairs the Metabolic Health of Offspring.

The periconceptual environment represents a critical window for programming fetal growth trajectories and susceptibility to disease; however, the underlying mechanism responsible for programming remains elusive. This study demonstrates a causal link between reduction of precompaction embryonic mitochondrial function and perturbed offspring growth trajectories and subsequent metabolic dysfunction. Incubation of embryos with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial oxidative phosphorylation, significantly reduced mitochondrial membrane potential and ATP production in 8-cell embryos and the number of inner cell mass cells within blastocysts; however, blastocyst development was unchanged. This perturbed embryonic mitochondrial function was concomitant with reduced birth weight in female offspring following embryo transfer, which persisted until weaning. FCCP-treated females also exhibited increased adiposity at 4 wk, increased adiposity gain between 4 and 14 wk, glucose intolerance at 8 wk, and insulin resistance at 14 wk. Although FCCP-treated males also exhibited reduced glucose tolerance, but their insulin sensitivity and adiposity gain between 4 and 14 wk was unchanged. To our knowledge, this is one of the first studies to demonstrate that reducing mitochondrial function and, thus, decreasing ATP output in the precompacting embryo can influence offspring phenotype. This is of great significance as a large proportion of patients requiring assisted reproductive technologies are of advanced maternal age or have a high body mass index, both of which have been independently linked with perturbed early embryonic mitochondrial function.

MITOsym®: A Mechanistic, Mathematical Model of Hepatocellular Respiration and Bioenergetics.

PURPOSE: MITOsym, a new mathematical model of hepatocellular respiration and bioenergetics, has been developed in partnership with the DILIsym® model with the purpose of translating in vitro compound screening data into predictions of drug induced liver injury (DILI) risk for patients. The combined efforts of these two models should increase the efficiency of evaluating compounds in drug development in addition to enhancing patient care. METHODS: MITOsym includes the basic, essential biochemical pathways associated with hepatocellular respiration and bioenergetics, including mitochondrial oxidative phosphorylation, electron transport chain activity, mitochondrial membrane potential, and glycolysis; also included are dynamic feedback signals based on perturbation of these pathways. The quantitative relationships included in MITOsym are based primarily on published data; additional new experiments were also performed in HepG2 cells to determine the effects on oxygen consumption rate as media glucose concentrations or oligomycin concentrations were varied. The effects of varying concentrations of FCCP on the mitochondrial proton gradient were also measured in HepG2 cells. RESULTS: MITOsym simulates and recapitulates the reported dynamic changes to hepatocellular oxygen consumption rates, extracellular acidification rates, the mitochondrial proton gradient, and ATP concentrations in the presence of classic mitochondrial toxins such as rotenone, FCCP, and oligomycin. CONCLUSIONS: MITOsym can be used to simulate hepatocellular respiration and bioenergetics and provide mechanistic hypotheses to facilitate the translation of in vitro data collection to predictions of in vivo human hepatotoxicity risk for novel compounds.

Knockdown of NYGGF4 (PID1) rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

NYGGF4 is a recently identified gene that is involved in obesity-associated insulin resistance. Previous data from this laboratory have demonstrated that NYGGF4 overexpression might contribute to the development of insulin resistance (IR) and to mitochondrial dysfunction. Additionally, NYGGF4 knockdown enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We designed this study to determine whether silencing of NYGGF4 in 3T3-L1 adipocytes could rescue the effect of insulin sensitivity and mitochondrial function induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to ascertain further the mechanism of NYGGF4 involvement in obesity-associated insulin resistance. We found that 3T3-L1 adipocytes, incubated with 5µM FCCP for 12h, had decreased levels of insulin-stimulated glucose uptake and had impaired insulin-stimulated GLUT4 translocation. Silencing also diminished insulin-stimulated tyrosinephosphorylation of IRS-1 and serine phosphorylation of Akt. This phenomenon contrasts with the effect of NYGGF4 knockdown on insulin sensitivity and describes the regulatory function of NYGGF4 in adipocytes insulin sensitivity. We next analyzed the mitochondrial function in NYGGF4-silenced adipocytes incubated with FCCP. NYGGF4 knockdown partly rescued the dissipation of mitochondrial mass, mitochondrial DNA, intracellular ATP synthesis, and intracellular reactive oxygen species (ROS) production occurred following the addition of FCCP, as well as inhibition of mitochondrial transmembrane potential (ΔΨm) in 3T3-L1 adipocytes incubated with FCCP. Collectively, our results suggested that addition of silencing NYGGF4 partly rescued the effect of insulin resistance and mitochondrial dysfunction in NYGGF4 silenced 3T3-L1 adipocytes incubated with FCCP, which might explain the involvement of NYGGF4-induced IR and the development of NYGGF4 in mitochondrial function.

Factors affecting SOCE activation in mammalian skeletal muscle fibers.

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca(2+) entry, SOCE. Sarcoplasmic reticulum (SR) Ca(2+) stores were depleted by repetitive exposures to high K(+) or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca(2+). SR Ca(2+) store replenishment was substantially reduced using 5 microM cyclopiazonic acid (CPA). Readmission of external Ca(2+) (5 mM) increased basal [Ca(2+)](i) under two modalities. In mode 1 [Ca(2+)](i) initially increased at a rate of 0.8 +/- 0.1 nM/s and later at a rate of 12.3 +/- 2.6 nM/s, reaching a final value of 477.8 +/- 36.8 nM in 215.7 +/- 25.9 s. In mode 2, [Ca(2+)](i) increased at a rate of 0.8 +/- 0.1 nM/s to a value of 204.9 +/- 20.6 nM in 185.4 +/- 21.1 s. FCCP, 2 microM, reduced this Ca(2+) entry. In nine FCCP-poisoned fibers, the initial rate of Ca(2+) increase was 0.34 +/- 0.1 nM/s (mean +/- SEM), reaching a plateau of 149.2 +/- 14.1 nM in 217 +/- 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca(2+), we show that during depletion [Ca(2+)](mito) initially increases and later diminishes. Finally, we show that the increase in basal [Ca(2+)](i), associated with SOCE activation, diminishes upon external Na(+) withdrawal. Na(+) entry through the SOCE pathway and activation of the reversal of Na(+)/Ca(2+) exchanger could explain this SOCE modulation by Na(+).

Knockdown of JNK rescues 3T3-L1 adipocytes from insulin resistance induced by mitochondrial dysfunction.

Mitochondrial dysfunction has been linked to etiology of insulin resistance, however the mechanism remains unknown. In this study we investigated whether mitochondrial dysfunction induced by cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) alters insulin sensitivity in 3T3-L1 adipocytes and which cellular signaling molecules might be involved. Fully differentiated 3T3-L1 adipocytes were treated with 10 microM FCCP for 1h, resulting in increased serine-307 phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation, association of p85alpha subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with IRS-1, decreased insulin-stimulated PI 3-kinase activity and H(3)-2-deoxyglucose (2DOG) uptake. A partial (46%) knockdown of JNK1 blocked FCCP-induced serine phosphorylation of IRS-1 and restored insulin-stimulated tyrosine phosphorylation of IRS-1, association of p85alpha subunit of PI 3-kinase with IRS-1, activation of PI 3-kinase, and stimulation of 2DOG uptake. Thus, FCCP-induced mitochondrial dysfunction may cause insulin resistance that is ameliorated by reduction of JNK1 expression.

Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death.

Phosphine is the most widely used fumigant for the protection of stored commodities against insect pests, especially food products such as grain. However, pest insects are developing resistance to phosphine and thereby threatening its future use. As phosphine inhibits cytochrome c oxidase (complex IV) of the mitochondrial respiratory chain and reduces the strength of the mitochondrial membrane potential (DeltaPsi(m)), we reasoned that mitochondrial uncouplers should act synergistically with phosphine. The mitochondrial uncouplers FCCP and PCP caused complete mortality in populations of both wild-type and phosphine-resistant lines of Caenorhabditis elegans simultaneously exposed to uncoupler and phosphine at concentrations that were individually nonlethal. Strong synergism was also observed with a third uncoupler DNP. We have also tested an alternative complex IV inhibitor, azide, with FCCP and found that this also caused a synergistic enhancement of toxicity in C. elegans. To investigate potential causes of the synergism, we measured DeltaPsi(m), ATP content, and oxidative damage (lipid hydroperoxides) in nematodes subjected to phosphine-FCCP treatment and found that neither an observed 50% depletion in ATP nor oxidative stress accounted for the synergistic effect. Instead, a synergistic reduction in DeltaPsi(m) was observed upon phosphine-FCCP co-treatment suggesting that this is directly responsible for the subsequent mortality. These results support the hypothesis that phosphine-induced mortality results from the in vivo disruption of normal mitochondrial activity. Furthermore, we have identified a novel pathway that can be targeted to overcome genetic resistance to phosphine.

Effect of mitochondria poisoning by FCCP on Ca2+ signaling in mouse skeletal muscle fibers.

We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca(2+) signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca(2+)](i) and MagFluo-4AM to measure Ca(2+) transients. Exposure to FCCP (2 microM, 2 min) caused a continuous increase in [Ca(2+)](i) at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca(2+) transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca(2+)](i) = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca(2+) release. FCCP diminished the rate but not the amount of Ca(2+) released, indicating that depletion of Ca(2+) stores did not cause the decrease in Ca(2+) transient amplitude. Ca(2+) transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca(2+)](i) induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca(2+) in the FCCP effect on transient amplitude.

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum.

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.

Effects of minimally toxic levels of carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone (FCCP), elucidated through differential gene expression with biochemical and morphological correlations.

Uncouplers of oxidative phosphorylation have relevance to bioenergetics and obesity. The mechanisms of action of chemical uncouplers of oxidative phosphorylation on biological systems were evaluated using differential gene expression. The transcriptional response in human rhabdomyosarcoma cell line (RD), was elucidated following treatment with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a classical uncoupling agent. Changes in mitochondrial membrane potential were used as the biological dosimeter. There was an increase in membrane depolarization with increasing concentrations of FCCP. The concentration at 75% uncoupling (20 microM) was chosen to study gene expression changes, using cDNA-based large-scale differential gene expression (LSDGE) platforms. At the above concentration, subtle light microscopic and clear gene expression changes were observed at 1, 2, and 10 h. Statistically significant transcriptional changes were largely associated with protein synthesis, cell cycle regulation, cytoskeletal proteins, energy metabolism, apoptosis, and inflammatory mediators. Bromodeoxyuridine (BrdU) and propidium iodide (PI) assays revealed cell cycle arrest to occur in the G1 and S phases. There was a significant initial decrease in the intracellular adenosine triphosphate (ATP) concentrations. The following seven genes were selected as potential molecular markers for chemical uncouplers: seryl-tRNA synthetase (Ser-tRS), glutamine-hydrolyzing asparagine synthetase (Glut-HAS), mitochondrial bifunctional methylenetetrahydrofolate dehydrogenase (Mit BMD), mitochondrial heat shock 10-kDa protein (Mit HSP 10), proliferating cyclic nuclear antigen (PCNA), cytoplasmic beta-actin (Act B), and growth arrest and DNA damage-inducible protein 153 (GADD153). Transcriptional changes of all seven genes were later confirmed with reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that gene expression changes may provide a sensitive indicator of uncoupling in response to chemical exposure.

Extracellular acidosis and chloride channel inhibitors act in the late phase of cellular injury to prevent death.

Extracellular acidosis is cytoprotective in several models against anoxia/hypoxia and a variety of toxicants. The goal of this study was to determine the temporal relationships among toxicant exposure, the initiation of extracellular acidosis, Cl. Influx, Cl- channel inhibition and the onset of cellular death in rabbit renal proximal tubule suspensions. Extracellular acidosis was produced by adding HCl or H2SO4 to renal proximal tubule suspensions to decrease the extracellular buffer pH to 6.4 or by resuspending renal proximal tubules in a pH 6.4 buffer. The initiation of extracellular acidosis 15 min after the mitochondrial inhibitors antimycin A or carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone addition, a time point in which adenosine triphosphate levels are depleted and intracellular K+ is decreased, ameliorated lactate dehydrogenase release, a marker of necrotic cellular death. The initiation of extracellular acidosis 120 min after the addition of the toxicants tetrafluoroethyl-L-cysteine or t-butyl hydroperoxide decreased lactate dehydrogenase release 120 min later. Increased Cl- influx is an important step during the late phase of toxicant-induced cellular injury. Therefore, we determined if extracellular acidosis cytoprotection was associated with inhibition of Cl- influx and whether the Cl- channel inhibitors indanyloxyacetic acid (1.0 mM). niflumic acid (100 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (100 microM) decreased Cl- influx and cellular death in renal proximal tubules exposed to antimycin A. Indeed, all three Cl- channel inhibitors significantly decreased 38Cl- influx and cellular death. In contrast, extracellular acidosis did not decrease 38Cl- influx but did prevent lactate dehydrogenase release. These results demonstrate that extracellular acidosis cytoprotection occurs during the late phase of cellular injury at a site distal to Cl- influx. Furthermore, the Cl- influx that occurs during the late phase of cellular injury and is critical for cellular swelling and lysis is sensitive to 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, niflumic acid and indanyloxyacetic acid.

Continuous monitoring of mitochondrial membrane potential in hepatocyte cell suspensions.

We report a simple fluorometric method for the continuous monitoring of mitochondrial membrane potential and cell viability in suspensions of hepatocytes exposed in vitro to cytotoxic agents. Suspensions of freshly isolated hepatocytes (10(6) cells/mL) preloaded with rhodamine 123 (Rh 123, 100 mumol/L) are transferred to a thermostatically controlled mixed cuvette to which the desired cytotoxic agent is added. Rh 123 is a cationic fluorophore that is actively accumulated by cells in direct proportion to the mitochondrial membrane potential. Cell viability was estimated by monitoring propidium iodide (PI) fluorescence. Exposure of cell suspensions to the mitochondrial uncoupling agent FCCP caused an immediate and titratable increase in Rh 123 fluorescence. Subsequent treatment with digitonin did not change Rh 123 fluorescence, suggeseting that Rh 123 equilibrates rapidly across the intact cell membrane. Likewise, treatment of hepatocyte suspensions with inhibitors of mitochondrial respiration (rotenone, cyanide, or menadione) caused an immediate increase in Rh 123 fluorescence. This was accompanied by a progressive increase in PI fluorescence, suggesting a causal relationship between mitochondrial depolarization and cell injury. In contrast, 1,4-benzoquinone caused a time-dependent and linear increase in PI fluorescence that paralleled changes in Rh 123 fluorescence. Comparing the time courses for changes in PI and Rh 123 fluorescence suggests that for benzoquinone, the depolarization of the mitochondria is a consequence rather than a cause of the cell injury. This modified procedure provides a simple and specific technique for continuously monitoring mitochondrial membrane potential and cell viability in suspensions of freshly isolated hepatocytes. The advantage is that there is no need to separate cells from the incubation medium, making it possible to record real-time changes in mitochondrial membrane potential and cell viability throughout the in vitro exposure period.

Protective actions of L-carnitine and acetyl-L-carnitine on the neurotoxicity evoked by mitochondrial uncoupling or inhibitors.

The mechanism for the pathological increase in cell death in various disease states e.g. HIV immunodefficiency or even ageing or Alzheimer's disease, occurs by complex and as yet undefined mechanism(s) related to immunological, virological or biochemical disturbances (i.e. energy depletion, oxidative stress, increased protein degradation). We have studied mitochondrial uncoupling or inhibitor toxicity on neurones at the cellular level and at the mitochondrial level using rhodamine (Rh123) and 10-nonylacridine orange (NAO) fluorescence with confocal microscopy. Blockade of the mitochondrial chain complexes at various points was studied. The possible protective effects of the compound L-carnitine, which plays a central role in mitochondrial function, was tested in this form of neurotoxicity. It appears that L-carnitine and its acetylated form, acetyl-L-carnitine, can attenuate the cell damage, as assessed by lactate dehydrogenase (LDH) release, evoked by the uncoupler, p-(trifluoromethoxy)phenylhdyrazone (FCCP), or by the inhibitors, 3-nitropropionic acid (3-NPA) or rotenone. Further, the FCCP-induced inhibition of Rh123 uptake was antagonized by the preincubation of cells with L-carnitine. Since such neurotoxic mechanisms may be operating in the various pathological forms of myotoxicity and neurotoxicity, these observations suggest potential for a therapeutic approach.

Intracellular acidification is not a prerequisite for glutamate-triggered death of cultured hippocampal neurons.

Glutamate decreased intracellular pH (pHi) in cultured rat hippocampal neurons. The protonophore, FCCP (1 microM), produced an acidification comparable to that produced by glutamate. Application of glutamate to FCCP-treated cells, returned pHi to resting levels. This alkaline shift resulted from a glutamate-induced membrane depolarization that removed the driving force across the plasmalemma for H+ entry via FCCP. The endogenous protonophore, arachidonic acid (10 microM), produced pHi changes similar to those elicited by FCCP. Because application of glutamate and FCCP in combination did not change pHi, this treatment was used to determine the role of glutamate-induced acidification in neurotoxicity. FCCP (1 microM, 5 min) did not affect neuronal viability, either alone or in combination with various concentrations of glutamate, as indicated by the release of lactate dehydrogenase into the bathing medium. Thus, acidification was not the cause of glutamate-induced cell death although, it may be symptomatic of neurotoxic processes.