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Chemical Research in Toxicology Nov 2022Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the...
Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the medium () can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log -0.07-6.84), in the culture medium () and cells () after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished mass balance model that combined relevant equations from similar models published by others. The total for all chemicals except tamoxifen (TAM) were similar to the . This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) for the low/no protein binding chemicals were similar to the , whereas values of all moderately to highly protein-bound chemicals were less than 30% of the . Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the was the same as the . The for all other chemicals tended to increase over time and were all 2- to 274-fold higher than . Measurements of , using a digitonin method to release cytosol, compared well with (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total within 30% of the measured values for 11 chemicals. The free of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between and total and free and , which were also relatively well predicted by the mass balance model.
Topics: Mice; Animals; Acetaminophen; Hydrophobic and Hydrophilic Interactions; Protein Binding; Cell Culture Techniques
PubMed: 36264934
DOI: 10.1021/acs.chemrestox.2c00128 -
Cells Feb 2022Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase...
Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.
Topics: Biological Assay; Cell Line; Holography; Microscopy; Nanoparticles
PubMed: 35203295
DOI: 10.3390/cells11040644 -
Current Research in Microbial Sciences 2022Chagas disease (CD), caused by , occurs in several countries in Latin America and non-endemic countries. Heterogeneity among population has been the Achilles' heel to...
Chagas disease (CD), caused by , occurs in several countries in Latin America and non-endemic countries. Heterogeneity among population has been the Achilles' heel to find a better treatment for CD. In this study, we characterized the biochemical parameters and mitochondrial bioenergetics of epimastigotes differentiated from eight isolates (I1-I8) obtained from Brazilian CD patients. Molecular analysis of parasites DTUs grouped all of them as TcII. The profile of the growth curves in axenic cultures was distinct among them, except for I1 and I3 and I2 and I4. Doubling times, growth rates, cell body length, and resistance to benznidazole were also significantly different among them. All the isolates were more glucose-dependent than other strains adapted to grow in axenic culture. Mitochondrial bioenergetics analysis showed that each isolate behaved differently regarding oxygen consumption rates in non-permeabilized and in digitonin-permeabilized cells in the presence of a complex II-linked substrate. When complex IV-linked respiratory chain substrate was used to provide electrons to the mitochondrial respiratory chain (MRC), similarity among the isolates was higher. Our findings show that TcII epimastigotes derived from patients' trypomastigotes displayed their own characteristics , highlighting the intra-TcII diversity, especially regarding the functionality of mitochondrial respiratory complexes II and IV. Understanding intraspecific biological features help us to move a step further on our comprehension regarding parasite's survival and adaptability offering clues to improve the development of new therapies for CD.
PubMed: 35199071
DOI: 10.1016/j.crmicr.2022.100110 -
Scientific Reports Apr 2019The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells....
The plasma membrane of eukaryotic cells forms the essential barrier to the extracellular environment, and thus plasma membrane disruptions pose a fatal threat to cells. Here, using invasive breast cancer cells we show that the Ca - and phospholipid-binding protein annexin A7 is part of the plasma membrane repair response by enabling assembly of the endosomal sorting complex required for transport (ESCRT) III. Following injury to the plasma membrane and Ca flux into the cytoplasm, annexin A7 forms a complex with apoptosis linked gene-2 (ALG-2) to facilitate proper recruitment and binding of ALG-2 and ALG-2-interacting protein X (ALIX) to the damaged membrane. ALG-2 and ALIX assemble the ESCRT III complex, which helps excise and shed the damaged portion of the plasma membrane during wound healing. Our results reveal a novel function of annexin A7 - enabling plasma membrane repair by regulating ESCRT III-mediated shedding of injured plasma membrane.
Topics: Annexin A7; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Membrane; Digitonin; Endosomal Sorting Complexes Required for Transport; Female; HeLa Cells; Humans; MCF-7 Cells
PubMed: 31040365
DOI: 10.1038/s41598-019-43143-4 -
Journal of Neurochemistry Apr 2020Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function...
Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na and Ca sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na and Ca signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na and Ca signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na and Ca waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na and Ca signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.
Topics: Acid Sensing Ion Channels; Animals; Cerebral Cortex; Energy Metabolism; Homeostasis; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Neurons; Signal Transduction
PubMed: 31976561
DOI: 10.1111/jnc.14971 -
Journal of Fungi (Basel, Switzerland) Jan 2021Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in...
Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in under diverse energy conditions. Supercomplexes were obtained by solubilization of mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I:IV), 1400 kDa (I:III), 1600 kDa (I:III:IV), and 1800 kDa (I:III:IV). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.
PubMed: 33440829
DOI: 10.3390/jof7010042 -
The Journal of Biological Chemistry Feb 2016To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this...
To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.
Topics: Animals; Cattle; Digitonin; Electron Transport Chain Complex Proteins; Mitochondria, Heart; Muscle Proteins; Myocardium; Polymers; Propylamines
PubMed: 26698328
DOI: 10.1074/jbc.M115.680553 -
International Journal of Molecular... Oct 2020Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact...
Conformational conversion of the cellular prion protein, PrP, into the abnormally folded isoform, PrP, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrP, designated Tg(PrP∆91-106)/ mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/ mice were resistant to RML, 22L and FK-1 prions, neither producing PrP∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrP∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrP∆91-104 after incubation with BSE-PrP-prions but not with RML- and 22L-PrP-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrP∆91-104 even after incubation with RML- and 22L-PrP-prions. These results suggest that residues 91-106 or 91-104 of PrP are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrP into PrP.
Topics: Animals; Baculoviridae; Base Sequence; Brain; Cattle; Cloning, Molecular; Disease Susceptibility; Encephalopathy, Bovine Spongiform; Gene Expression; Injections, Intraventricular; Mice; Mice, Transgenic; PrPC Proteins; PrPSc Proteins; Proteostasis Deficiencies; Recombinant Proteins; Scrapie; Sequence Deletion; Species Specificity
PubMed: 33019549
DOI: 10.3390/ijms21197260 -
International Journal of Molecular... Mar 2021This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and...
This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.
Topics: Cell Membrane; Cell Membrane Permeability; Dose-Response Relationship, Drug; Ion Channel Gating; Ion Channels; Lipid Bilayers; Membrane Lipids; Membrane Potentials; Molecular Structure; Phase Transition; Saponins
PubMed: 33804648
DOI: 10.3390/ijms22063167 -
Frontiers in Physiology 2021In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca, mitochondria undergo permeability transition (PT) leading to the...
In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.
PubMed: 34950052
DOI: 10.3389/fphys.2021.773839