Mitochondria are targets of newly synthesized drugs and being tested for the treatment of various diseases caused or accompanied by disruption of cellular bioenergetics. In drug development, it is necessary to test for drug-induced changes in mitochondrial enzyme activity that may be related to therapeutic or adverse drug effects. Measurement of drug effect on mitochondrial oxygen consumption kinetics and/or protective effects of drugs against calcium-induced inhibition of the mitochondrial respiration can be used for the study mitochondrial toxicity and neuroprotective effects of drugs. Supposing that the drug-induced inhibition of the mitochondrial respiratory rate and/or individual mitochondrial complexes is associated with adverse drug effects, the effects of drugs on mitochondrial respiration in isolated mitochondria allow selection of novel molecules that are relatively safe for mitochondrial toxicity.

• MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• mozek cytologie   MeSH
• prasata MeSH
• preklinické hodnocení léčiv přístrojové vybavení   metody   MeSH
• respirační komplex I metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Assessment of drug-induced mitochondrial dysfunctions is important in drug development as well as in the understanding of molecular mechanism of therapeutic or adverse effects of drugs. The aim of this study was to investigate the effects of three typical antipsychotics (APs) and seven atypical APs on mitochondrial bioenergetics. The effects of selected APs on citrate synthase, electron transport chain complexes (ETC), and mitochondrial complex I- or complex II-linked respiratory rate were measured using mitochondria isolated from pig brain. Complex I activity was decreased by chlorpromazine, haloperidol, zotepine, aripiprazole, quetiapine, risperidone, and clozapine. Complex II + III was significantly inhibited by zotepine, aripiprazole, quetiapine, and risperidone. Complex IV was inhibited by zotepine, chlorpromazine, and levomepromazine. Mitochondrial respiratory rate was significantly inhibited by all tested APs, except for olanzapine. Typical APs did not exhibit greater efficacy in altering mitochondrial function compared to atypical APs except for complex I inhibition by chlorpromazine and haloperidol. A comparison of the effects of APs on individual respiratory complexes and on the overall mitochondrial respiration has shown that mitochondrial functions may not fully reflect the disruption of complexes of ETC, which indicates AP-induced modulation of other mitochondrial proteins. Due to the complicated processes associated with mitochondrial activity, it is necessary to measure not only the effect of the drug on individual mitochondrial enzymes but also the respiration rate of the mitochondria or a similar complex process. The experimental approach used in the study can be applied to mitochondrial toxicity testing of newly developed drugs.

• MeSH
• antipsychotika toxicita   MeSH
• energetický metabolismus účinky léků   MeSH
• mitochondrie účinky léků   patologie   MeSH
• mozek účinky léků   metabolismus   MeSH
• prasata MeSH
• respirační komplex I účinky léků   metabolismus   MeSH
• respirační komplex II účinky léků   metabolismus   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Respiration is one of the major functions of mitochondria, whereby these vital organelles use oxygen to produce energy. Many agents that may be of potential clinical relevance act by targeting mitochondria, where they may suppress mitochondrial respiration. It is therefore important to evaluate this process and understand how this is modulated by small molecules. Here, we describe the general methodology to assess respiration in cultured cells, followed by the evaluation of the effect of one anticancer agent targeted to mitochondria on this process, and also how to assess this in tumor tissue.

• MeSH
• buněčné dýchání účinky léků   MeSH
• lidé MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce účinky léků   MeSH
• protinádorové látky farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• spotřeba kyslíku účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Photodynamic therapy (PDT) is gradually becoming an alternative method in the treatment of several diseases. Here, we investigated the role of oxygen in photodynamically treated cervical cancer cells (HeLa). The effect of PDT on HeLa cells was assessed by exposing cultured cells to disulphonated zinc phthalocyanine (ZnPcS2) and tetrasulphonated zinc tetraphenylporphyrin (ZnTPPS4). Fluorescence microscopy revealed their different localizations within the cells. ZnTPPS4 seems to be mostly limited to the cytosol and lysosomes, whereas ZnPcS2 is most likely predominantly attached to membrane structures, including plasmalemma and the mitochondrial membrane. Phototoxicity assays of PDT-treated cells carried out under different partial pressures of oxygen showed dose-dependent responses. Interestingly, ZnPcS2 was also photodynamically effective at a minimal level of oxygen, under a nitrogen atmosphere. On the other hand, hyperbaric oxygenation did not lead to a higher PDT efficiency of either photosensitizer. Although both photosensitizers can induce a significant drop in mitochondrial membrane potential, ZnPcS2 has a markedly higher effect on mitochondrial respiration that was completely blocked after two short light cycles. In conclusion, our observations suggest that PDT can be effective even in hypoxic conditions if a suitable sensitizer is chosen, such as ZnPcS2, which can inhibit mitochondrial respiration.

• MeSH
• fotochemoterapie metody   MeSH
• fotosenzibilizující látky farmakologie   MeSH
• HeLa buňky MeSH
• indoly aplikace a dávkování   farmakologie   MeSH
• kyslík aplikace a dávkování   farmakologie   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metaloporfyriny aplikace a dávkování   farmakologie   MeSH
• mitochondrie účinky léků   MeSH
• organokovové sloučeniny aplikace a dávkování   farmakologie   MeSH
• parciální tlak MeSH
• singletový kyslík analýza   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 μM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 μM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 μM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 μM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 μM fipronil treatments, but there was a ∼40-60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 μM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 μM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 μM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson's disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents.

• MeSH
• dopaminergní neurony účinky léků   metabolismus   MeSH
• insekticidy toxicita   MeSH
• krysa rodu rattus MeSH
• membránový potenciál mitochondrií účinky léků   fyziologie   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• pyrazoly toxicita   MeSH
• transformované buněčné linie MeSH
• transkriptom účinky léků   fyziologie   MeSH
• viabilita buněk účinky léků   fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The identification and quantification of mitochondrial effects of novel antipsychotics (brexpiprazole, cariprazine, loxapine, and lurasidone) were studied in vitro in pig brain mitochondria. Selected parameters of mitochondrial metabolism, electron transport chain (ETC) complexes, citrate synthase (CS), malate dehydrogenase (MDH), monoamine oxidase (MAO), mitochondrial respiration, and total ATP and reactive oxygen species (ROS) production were evaluated and associated with possible adverse effects of drugs. All tested antipsychotics decreased the ETC activities (except for complex IV, which increased in activity after brexpiprazole and loxapine addition). Both complex I- and complex II-linked respiration were dose-dependently inhibited, and significant correlations were found between complex I-linked respiration and both complex I activity (positive correlation) and complex IV activity (negative correlation). All drugs significantly decreased mitochondrial ATP production at higher concentrations. Hydrogen peroxide production was significantly increased at 10 μM brexpiprazole and lurasidone and at 100 μM cariprazine and loxapine. All antipsychotics acted as partial inhibitors of MAO-A, brexpiprazole and loxapine partially inhibited MAO-B. Based on our results, novel antipsychotics probably lacked oxygen uncoupling properties. The mitochondrial effects of novel antipsychotics might contribute on their adverse effects, which are mostly related to decreased ATP production and increased ROS production, while MAO-A inhibition might contribute to their antidepressant effect, and brexpiprazole- and loxapine-induced MAO-B inhibition might likely promote neuroplasticity and neuroprotection. The assessment of drug-induced mitochondrial dysfunctions is important in development of new drugs as well as in the understanding of molecular mechanism of adverse or side drug effects.

• MeSH
• adenosintrifosfát biosyntéza   MeSH
• antipsychotika klasifikace   farmakologie   MeSH
• chinolony farmakologie   MeSH
• elektronový transportní řetězec účinky léků   MeSH
• energetický metabolismus účinky léků   MeSH
• inhibitory MAO farmakologie   MeSH
• loxapin farmakologie   MeSH
• lurasidon hydrochlorid farmakologie   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• piperaziny farmakologie   MeSH
• prasata MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• receptory neurotransmiterů účinky léků   MeSH
• spotřeba kyslíku účinky léků   MeSH
• thiofeny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Effectiveness of L-asparaginase administration in acute lymphoblastic leukemia treatment is mirrored in the overall outcome of patients. Generally, leukemia patients differ in their sensitivity to L-asparaginase; however, the mechanism underlying their inter-individual differences is still not fully understood. We have previously shown that L-asparaginase rewires the biosynthetic and bioenergetic pathways of leukemia cells to activate both anti-leukemic and pro-survival processes. Herein, we investigated the relationship between the metabolic profile of leukemia cells and their sensitivity to currently used cytostatic drugs. METHODS: Altogether, 19 leukemia cell lines, primary leukemia cells from 26 patients and 2 healthy controls were used. Glycolytic function and mitochondrial respiration were measured using Seahorse Bioanalyzer. Sensitivity to cytostatics was measured using MTS assay and/or absolute count and flow cytometry. Mitochondrial membrane potential was determined as TMRE fluorescence. RESULTS: Using cell lines and primary patient samples we characterized the basal metabolic state of cells derived from different leukemia subtypes and assessed their sensitivity to cytostatic drugs. We found that leukemia cells cluster into distinct groups according to their metabolic profile. Lymphoid leukemia cell lines and patients sensitive to L-asparaginase clustered into the low glycolytic cluster. While lymphoid leukemia cells with lower sensitivity to L-asparaginase together with resistant normal mononuclear blood cells gathered into the high glycolytic cluster. Furthermore, we observed a correlation of specific metabolic parameters with the sensitivity to L-asparaginase. Greater ATP-linked respiration and lower basal mitochondrial membrane potential in cells significantly correlated with higher sensitivity to L-asparaginase. No such correlation was found in the other cytostatic drugs tested by us. CONCLUSIONS: These data support that cell metabolism plays a prominent role in the treatment effect of L-asparaginase. Based on these findings, leukemia patients with lower sensitivity to L-asparaginase with no specific genetic characterization could be identified by their metabolic profile.

• MeSH
• akutní lymfatická leukemie krev   farmakoterapie   patologie   MeSH
• asparaginasa farmakologie   terapeutické užití   MeSH
• biosyntetické dráhy účinky léků   MeSH
• chemorezistence MeSH
• dítě MeSH
• glykolýza účinky léků   MeSH
• kojenec MeSH
• kostní dřeň patologie   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metabolom účinky léků   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• mladiství MeSH
• mladý dospělý MeSH
• nádorové buněčné linie MeSH
• oxidativní fosforylace účinky léků   MeSH
• předškolní dítě MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg(-1) of body weight·day(-1), 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial (14)C-glucose and (14)C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 μg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

• MeSH
• analýza přežití MeSH
• glykogen metabolismus   MeSH
• hemodynamika účinky léků   MeSH
• hypoglykemika krev   terapeutické užití   MeSH
• krevní glukóza metabolismus   MeSH
• krysa rodu rattus MeSH
• metabolismus lipidů účinky léků   MeSH
• metformin krev   terapeutické užití   MeSH
• modely nemocí na zvířatech MeSH
• myokard metabolismus   patologie   MeSH
• plíce patologie   MeSH
• potkani Wistar MeSH
• preklinické hodnocení léčiv MeSH
• proteinkinasy metabolismus   MeSH
• srdeční mitochondrie fyziologie   MeSH
• srdeční selhání farmakoterapie   patofyziologie   ultrasonografie   MeSH
• tělesná hmotnost účinky léků   MeSH
• velikost orgánu účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH