Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

Anti-adipogenic action of a novel oxazole derivative through activation of AMPK pathway.

Obesity is a chronic disorder with multifactorial etiology, including genetic, medical, dietary and other environmental factors. Both natural and synthetic heterocyclic compounds, especially oxazoles, represent an interesting group of compounds and have gained much attention due to their remarkable biological activities. Therefore, a library of 3,3-DMAH (3,3-dimethylallylhalfordinol) inspired N-alkylated oxazole bromide salts with varied substitutions were prepared and screened using the 3T3-L1 model of adipogenesis and HFD-induced obesity model in Syrian golden hamsters. Several compounds in the synthesized series displayed remarkable anti-adipogenic potential on the differentiation of 3T3-L1 preadipocytes. Compound 19e, displayed the most potent activity of all and selected for further studies. Compound 19e inhibited mitotic clonal expansion of 3T3-L1 cells and enhanced the mitochondrial oxygen consumption rate of the cells during early phase of differentiation via AMPK activation. 19e also improved the dyslipidaemia in high calorie diet fed Syrian Golden Hamsters. Therefore, compound 19e can serve as a potential lead against adipogenesis and dyslipidaemia models and could be further investigated to affirm its significance as a drug candidate.

Acyl-Carnitines Exert Positive Effects on Mitochondrial Activity under Oxidative Stress in Mouse Oocytes: A Potential Mechanism Underlying Carnitine Efficacy on PCOS.

Carnitines play a key physiological role in oocyte metabolism and redox homeostasis. In clinical and animal studies, carnitine administration alleviated metabolic and reproductive dysfunction associated with polycystic ovarian syndrome (PCOS). Oxidative stress (OS) at systemic, intraovarian, and intrafollicular levels is one of the main factors involved in the pathogenesis of PCOS. We investigated the ability of different acyl-carnitines to act at the oocyte level by counteracting the effects of OS on carnitine shuttle system and mitochondrial activity in mouse oocytes. Germinal vesicle (GV) oocytes were exposed to hydrogen peroxide and propionyl-l-carnitine (PLC) alone or in association with l-carnitine (LC) and acetyl-l-carnitine (ALC) under different conditions. Expression of carnitine palmitoyltransferase-1 (Cpt1) was monitored by RT-PCR. In in vitro matured oocytes, metaphase II (MII) apparatus was assessed by immunofluorescence. Oocyte mitochondrial respiration was evaluated by Seahorse Cell Mito Stress Test. We found that Cpt1a and Cpt1c isoforms increased under prooxidant conditions. PLC alone significantly improved meiosis completion and oocyte quality with a synergistic effect when combined with LC + ALC. Acyl-carnitines prevented Cpt1c increased expression, modifications of oocyte respiration, and ATP production observed upon OS. Specific effects of PLC on spare respiratory capacity were observed. Therefore, carnitine supplementation modulated the intramitochondrial transfer of fatty acids with positive effects on mitochondrial activity under OS. This knowledge contributes to defining molecular mechanism underlying carnitine efficacy on PCOS.

Role of D(-)-Lactic Acid in Prevention of Chlamydia trachomatis Infection in an In Vitro Model of HeLa Cells.

A vaginal microbiota dominated by certain Lactobacillus species may have a protective effect against Chlamydia trachomatis infection. One of the key antimicrobial compounds produced is lactic acid, which is believed to play a central role in host defense. Lactobacillus strains producing the D(-)-lactic acid isomer are known to exert stronger protection. However, the molecular mechanisms underlying this antimicrobial action are not well understood. The aim of this study was to investigate the role of D(-)-lactic acid isomer in the prevention of C. trachomatis infection in an in vitro HeLa cell model. We selected two strains of lactobacilli belonging to different species: a vaginal isolate of Lactobacillus crispatus that releases both D(-) and L(+) isomers and a strain of Lactobacillus reuteri that produces only the L(+) isomer. Initially, we demonstrated that L. crispatus was significantly more effective than L. reuteri in reducing C. trachomatis infectivity. A different pattern of histone acetylation and lactylation was observed when HeLa cells were pretreated for 24 h with supernatants of Lactobacillus crispatus or L. reuteri, resulting in different transcription of genes such as CCND1, CDKN1A, ITAG5 and HER-1. Similarly, distinct transcription patterns were found in HeLa cells treated with 10 mM D(-)- or L(+)-lactic acid isomers. Our findings suggest that D(-) lactic acid significantly affects two non-exclusive mechanisms involved in C. trachomatis infection: regulation of the cell cycle and expression of EGFR and α5ß1-integrin.

Attenuation of PM2.5-induced alveolar epithelial cells and lung injury through regulation of mitochondrial fission and fusion.

BACKGROUND: Exposure to particulate matter (PM) with an aerodynamic diameter less than 2.5 µm (PM2.5) is a risk factor for developing pulmonary diseases and the worsening of ongoing disease. Mitochondrial fission and fusion are essential processes underlying mitochondrial homeostasis in health and disease. We examined the role of mitochondrial fission and fusion in PM2.5-induced alveolar epithelial cell damage and lung injury. Key genes in these processes include dystrophin-related protein 1 (DRP1) and optic atrophy 1 (OPA1) respectively. METHODS: Alveolar epithelial (A549) cells were treated with PM2.5 (32 µg/ml) in the presence and absence of Mdivi-1 (10µM, a DRP1 inhibitor) or BGP-15 (10µM, an OPA1 activator). Results were validated using DRP1-knockdown (KD) and OPA1-overexpression (OE). Mice were injected intraperitoneally with Mdivi-1 (20 mg/kg), BGP-15 (20 mg/kg) or distilled water (control) one hour before intranasal instillation of PM2.5 (7.8 mg/kg) or distilled water for two consecutive days. RESULTS: PM2.5 exposure of A549 cells caused oxidative stress, enhanced inflammation, necroptosis, mitophagy and mitochondrial dysfunction indicated by abnormal mitochondrial morphology, decreased mitochondrial membrane potential (ΔΨm), reduced mitochondrial respiration and disrupted mitochondrial fission and fusion. Regulating mitochondrial fission and fusion pharmacologically using Mdivi-1 and BGP-15 and genetically using DRP1-KD and OPA1-OE prevented PM2.5-induced celluar damage in A549 cells. Mdivi-1 and BGP-15 attenuated PM2.5-induced acute lung injury in mice. CONCLUSION: Increased mitochondrial fission and decreased mitochondrial fusion may underlie PM2.5-induced alveolar epithelial cell damage in vitro and lung injury in vivo.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

Mitochondrial bioenergetic changes in systemic lupus erythematosus immune cell subsets: Contributions to pathogenesis and clinical applications.

The association of dysregulated metabolism in systemic lupus erythematosus (SLE) pathogenesis has prompted investigations into metabolic rewiring and the involvement of mitochondrial metabolism as a driver of disease through NLRP3 inflammasome activation, disruption of mitochondrial DNA maintenance, and pro-inflammatory cytokine release. The use of Agilent Seahorse Technology to gain functional in situ metabolic insights of selected cell types from SLE patients has identified key parameters that are dysregulated during disease. Mitochondrial functional assessments specifically can detect dysfunction through oxygen consumption rate (OCR), spare respiratory capacity, and maximal respiration measurements, which, when coupled with disease activity scores could show potential as markers of disease activity. CD4+ and CD8 + T cells have been assessed in this way and show that oxygen consumption rate, spare respiratory capacity, and maximal respiration are blunted in CD8 + T cells, with results not being as clear cut in CD4 + T cells. Additionally, glutamine, processed by mitochondrial substrate level phosphorylation is emerging as a key role player in the expansion and differentiation of Th1, Th17, ϒδ T cells, and plasmablasts. The role that circulating leukocytes play in acting as bioenergetic biomarkers of diseases such as diabetes suggests that this may also be a tool to detect preclinical SLE. Therefore, the metabolic characterization of immune cell subsets and the collection of metabolic data during interventions is also essential. The delineation of the metabolic tuning of immune cells in this way could lead to novel strategies in treating metabolically demanding processes characteristic of autoimmune diseases such as SLE.

Conditioned place avoidance is associated with a distinct hippocampal phenotype, partly preserved pattern separation, and reduced reactive oxygen species production after stress.

Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.

Measurement of Mitochondrial (Dys)Function in Cellular Systems Using Electron Paramagnetic Resonance (EPR): Oxygen Consumption Rate and Superoxide Production.

The oxygen consumption rate (OCR) and superoxide production are crucial when assessing mitochondrial function and/or dysfunction. EPR spectroscopy allows the measurement of both components either independently or simultaneously in a same cellular or mitochondrial preparation. OCR determination using EPR oximetry is based on the change in EPR linewidth of a paramagnetic oxygen sensing probe (a perdeuterated nitroxide) in the presence of oxygen consuming cells in a closed system. Superoxide production can be monitored by the oxidation of cyclic hydroxylamines into nitroxides. The contribution of superoxide to the nitroxide formation is deduced from experiments in the presence and in the absence of SOD and PEG-SOD as appropriate controls.

Bicalutamide may enhance kidney injury in diabetes by concomitantly damaging energy production from OXPHOS and glycolysis.

Bicalutamide (Bic), frequently used in androgen-deprivation therapy for treating prostate cancer, was demonstrated to induce multiple apoptosis and fibrosis pathways and mitochondrial dysfunction in renal mesangial cells. Whether Bic also damages the glycolytic pathway has never been cited. To investigate this, we performed an in vitro model study with mesangial cells, and at the same time, collected data from an in vivo experiment. Bic induced hypoxia-inducible factor (HIF)-1 which upregulates phosphorylated-5'-AMP-activated protein kinase (p-AMPK) and severely suppresses the rate of adenosine triphosphate (ATP) production in both the oxidative phosphorylation and glycolysis pathways. Bic suppressed the oxygen consumption rate, extracellular acidification rate, and mitochondrial proton efflux rate, downregulated in vivo but upregulated in vitro glucose transporter (GLUT)-1, reduced glucose uptake, inhibited key glycolytic enzymes, including phosphofructokinase (PFK), pyruvate kinase (PK), and pyruvate dehydrogenase (PDH), and upregulated hexokinase II (HKII) and lactic dehydrogenase A (LDHA). In vivo, Bic downregulated renal cubilin levels, thereby disrupting the glomerular reabsorption function. Conclusively, Bic can damage bioenergenesis from both mitochondria and glycolysis. It was suggested that long-term administration of Bic can initiate renal damage depending on the duration and dose of treatment, which requires cautious follow-up.

GRPEL2 Knockdown Exerts Redox Regulation in Glioblastoma.

Malignant brain tumors are responsible for catastrophic morbidity and mortality globally. Among them, glioblastoma multiforme (GBM) bears the worst prognosis. The GrpE-like 2 homolog (GRPEL2) plays a crucial role in regulating mitochondrial protein import and redox homeostasis. However, the role of GRPEL2 in human glioblastoma has yet to be clarified. In this study, we investigated the function of GRPEL2 in glioma. Based on bioinformatics analyses from the Cancer Gene Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), we inferred that GRPEL2 expression positively correlates with WHO tumor grade (p < 0.001), IDH mutation status (p < 0.001), oligodendroglial differentiation (p < 0.001), and overall survival (p < 0.001) in glioma datasets. Functional validation in LN229 and GBM8401 GBM cells showed that GRPEL2 knockdown efficiently inhibited cellular proliferation. Moreover, GRPEL2 suppression induced cell cycle arrest at the sub-G1 phase. Furthermore, GRPEL2 silencing decreased intracellular reactive oxygen species (ROS) without impending mitochondria membrane potential. The cellular oxidative respiration measured with a Seahorse XFp analyzer exhibited a reduction of the oxygen consumption rate (OCR) in GBM cells by siGRPEL2, which subsequently enhanced autophagy and senescence in glioblastoma cells. Taken together, GRPEL2 is a novel redox regulator of mitochondria bioenergetics and a potential target for treating GBM in the future.

Mitochondrial Respiration Defects in Single-Ventricle Congenital Heart Disease.

Background: Congenital heart disease (CHD) with single-ventricle (SV) physiology is now survivable with a three-stage surgical course ending with Fontan palliation. However, 10-year transplant-free survival remains at 39-50%, with ventricular dysfunction progressing to heart failure (HF) being a common sequela. For SV-CHD patients who develop HF, undergoing the surgical course would not be helpful and could even be detrimental. As HF risk cannot be predicted and metabolic defects have been observed in Ohia SV-CHD mice, we hypothesized that respiratory defects in peripheral blood mononuclear cells (PBMCs) may allow HF risk stratification in SV-CHD. Methods: SV-CHD (n = 20), biventricular CHD (BV-CHD; n = 16), or healthy control subjects (n = 22) were recruited, and PBMC oxygen consumption rate (OCR) was measured using the Seahorse Analyzer. Respiration was similarly measured in Ohia mouse heart tissue. Results: Post-Fontan SV-CHD patients with HF showed higher maximal respiratory capacity (p = 0.004) and respiratory reserve (p < 0.0001), parameters important for cell stress adaptation, while the opposite was found for those without HF (reserve p = 0.037; maximal p = 0.05). This was observed in comparison to BV-CHD or healthy controls. However, respiration did not differ between SV patients pre- and post-Fontan or between pre- or post-Fontan SV-CHD patients and BV-CHD. Reminiscent of these findings, heart tissue from Ohia mice with SV-CHD also showed higher OCR, while those without CHD showed lower OCR. Conclusion: Elevated mitochondrial respiration in PBMCs is correlated with HF in post-Fontan SV-CHD, suggesting that PBMC respiration may have utility for prognosticating HF risk in SV-CHD. Whether elevated respiration may reflect maladaptation to altered hemodynamics in SV-CHD warrants further investigation.

The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines.

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

Mitochondria Content and Activity Are Crucial Parameters for Bull Sperm Quality Evaluation.

Standard sperm evaluation parameters do not enable predicting their ability to survive cryopreservation. Mitochondria are highly prone to suffer injuries during freezing, and any abnormalities in their morphology or function are reflected by a decline of sperm quality. Our work focused on describing a link between the number and the activity of mitochondria, with an aim to validate its applicability as a biomarker of bovine sperm quality. Cryopreserved sperm collected from bulls with high (group 1) and low (group 2) semen quality was separated by swim up. The spermatozoa of group 1 overall retained more mitochondria (MitoTrackerGreen) and mtDNA copies, irrespective of the fraction. Regardless of the initial ejaculate quality, the motile sperm contained significantly more mitochondria and mtDNA copies. The same trend was observed for mitochondrial membrane potential (ΔΨm, JC-1), where motile sperm displayed high ΔΨm. These results stay in agreement with transcript-level evaluation (real-time polymerase chain reaction, PCR) of antioxidant enzymes (PRDX1, SOD1, GSS), which protect cells from the reactive oxygen species. An overall higher level of glutathione synthetase (GSS) mRNA was noted in group 1 bulls, suggesting higher ability to counteract free radicals. No differences were noted between basal oxygen consumption rate (OCR) (Seahorse XF Agilent) and ATP-linked respiration for group 1 and 2 bulls. In conclusion, mitochondrial content and activity may be used as reliable markers for bovine sperm quality evaluation.

A versatile EPR toolbox for the simultaneous measurement of oxygen consumption and superoxide production.

In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of 15N-PDT (linked to OCR) and the formation of the nitroxide from 14N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues.

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

High-concentration homocysteine inhibits mitochondrial respiration function and production of reactive oxygen species in neuron cells.

OBJECTIVE: Homocysteine plays critical roles in cellular redox homeostasis, and hyperhomocysteinemia has been associated with multiple diseases, including neurological disorders involving reactive oxygen species-inducing and pro-inflammatory effects of homocysteine that are related to mitochondria. This study investigated the role of homocysteine in regulating mitochondria of neuron cell lines. METHODS: Neuron cells were pre-treated with homocysteine, and then flow cytometry was used to detect reactive oxygen species production and mitochondrial membrane potential, while Seahorse XFp Mito stress assay was used to comprehensively analyze mitochondrial function. RESULTS: The experimental results showed that high-concentration homocysteine diminished carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone-stimulated oxygen consumption rate and mitochondrial spare respiration capacity in a time- and concentration-dependent manner, and homocysteine also reduced reactive oxygen species in cultured neuron cell lines while no changes in mitochondrial membrane potential were observed. CONCLUSION: These results indicate that homocysteine diminished mitochondrial respiration function in neuron cell lines mediated by its reactive oxygen species-reducing effects, which may underlie the association between hyperhomocysteinemia and human diseases.

Bicalutamide Elicits Renal Damage by Causing Mitochondrial Dysfunction via ROS Damage and Upregulation of HIF-1.

Combined androgen blockade using bicalutamide (Bic) is a therapeutic choice for treating prostate cancer (PCa). However, even at regular clinical dosages, Bic frequently shows adverse effects associated with cardiovascular and renal damage. Previously, we found that Bic selectively damaged mesangial cells compared to tubular cells and in an in vivo rat model, we also found renal damage caused by Bic. In the present study, a rat mesangial cell model was used to further the investigation. Results indicated that Bic enhanced lactate dehydrogenase release, reactive oxygen species (ROS) production, lysosome population and kidney injury molecule-1 and decreased N-cadherin. Bic elicited mitochondrial swelling and reduced the mitochondrial potential, resulting in severe suppression of the oxygen consumption rate (OCR), maximum respiration and ATP production. The hypoxia-inducible factor (HIF)-1 transcriptional activity and messenger RNA were significantly upregulated in dose-dependent manners. The HIF-1 protein reached a peak value at 24 h then rapidly decayed. BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 and cleaved caspase-3 were dose-dependently upregulated by Bic (60 M) and that eventually led to cell apoptosis. It is suggested that Bic induces renal damage via ROS and modulates HIF-1 pathway and clinically, some protective agents like antioxidants are recommended for co-treatment.

The continuous physiological changes of zebrafish (Danio rerio) based on metabolism under controlled thallium stress.

In this research, the continuous physiological changes of zebrafish (Danio rerio) in 0.1 µg/L thallium (Tl) in 15 days were investigated. The results showed that Tl(I) stress had a significant positive linear correlation with zebrafish ammonia nitrogen excretion (ANE) (p < 0.001), and the mean value of ANE in Tl(I) treatment (435 ±â€¯227 mg/kg/h) was approximately 2 times higher than in the control group (239 ±â€¯168 mg/kg/h), which suggested that ANE was suitable for Tl(I) stress assessment. A substantial difference based on oxygen consumption rate (OCR) between the control group (587 ±â€¯112 mg/kg/h) and Tl(I) treatment (260 ±â€¯88 mg/kg/h) with a high significance p < 0.001 could be observed, and the results indicated that Tl(I) played a negative role in OCR of zebrafish. The characteristics of both ANE and OCR changes under slight Tl(I) stress could be reflected by the ammonia quotient (AQ). It was noteworthy that AQ increased rapidly in first 6 h from 0.66 to 4.50, which was 3 times higher than 1.2, indicating rapid increase in both anaerobic energy utilization and protein metabolism in 0.1 µg/L Tl(I) exposure. It is concluded that the physiological changes of zebrafish based on metabolism can be regarded as a sensitive biological indicator of Tl(I) pollution, which could work as a substitute of potassium that disrupts the normal biological metabolism in the process of transport.

Optimization of experimental conditions and measurement of oxygen consumption rate (OCR) in zebrafish embryos exposed to organophosphate flame retardants (OPFRs).

The measurement of oxygen consumption rate (OCR) provides a comprehensive understanding of mitochondrial metabolism. However, no study has been conducted to investigate the mitochondrial dysfunction caused by organophosphate flame retardants (OPFRs). The objectives of this study were to optimize the experimental conditions to measure OCR in zebrafish embryos using the Seahorse XFe 24 Extracellular Flux Analyzer, and to investigate the changes of OCR in zebrafish embryos exposed to OPFRs. We first optimized the experimental conditions such as the number of embryos, concentrations of inhibitors, and time points. We determined the factors, i.e., three embryos, 12.5 µM of oligomycin, 8 µM of carbonyl cyanaide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 24 hpf (hours post-fertilization) time point, for obtaining the typical pattern of OCR in dechorinated zebrafish embryos. After confirming the determinants upon exposure of triclosan, the inhibition of OCR was measured in zebrafish embryos exposed to two major OPFRs, triphenyl phosphate (TPHP) and tris (1,3-dichloro-2-propyl) phosphate (TDCIPP). We found that significant inhibition of OCR was observed in basal respiration for TPHP, and in basal and maximal respiration for TDCIPP exposure, respectively. We suggest the optimum conditions of the Seahorse XFe 24 analyzer to better evaluate OCR in zebrafish embryos, and demonstrate the potential of TPHP and TDCIPP to cause the disruption of energy metabolism associated with mitochondrial dysfunction.