Decrease In ATP Content Research Articles

Hydrogen-rich medium ameliorates lipopolysaccharides-induced mitochondrial fission and dysfunction in human umbilical vein endothelial cells (HUVECs) via up-regulating HO-1 expression

BackgroundSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It has been showed that the change of mitochondrial dynamics has been proved to be one of the main causes of death in patients with severe sepsis. And hydrogen has been proved to exert its protective effects against sepsis via heme oxygenase-1 (HO-1). This study was designed to demonstrate that whether the benefit effects of hydrogen can maintain the dynamic process of mitochondrial fusion/fission to mitigate human umbilical vein endothelial cells (HUVECs) injury exposed to endotoxin through HO-1. MethodsHUVECs cells cultured with medium which contained Lipopolysaccharides (LPS), Saline, hydrogen, Mdivi-1 (a dynamin-related protein 1 [Drp1] inhibitor) or zinc protoporphyrin IX (Znpp) (a HO-1 inhibitor) were also used in the research. Cell death and apoptosis were assessed using FITC annexin V and PI. Mitochondria were stained with Mitotracker orange and observed by confocal microscope. Oxygen consumption rate was assessed by seahorse xf24 extracellular analyzer. Mitochondrial membrane potential monitored by JC-1 dye. The expressions of Drp1 and HO-1 were tested by Western blot. The co-localization of Drp1 and mitochondria was determined by immunofluorescence. ResultsLPS caused a decrease in ATP content, mitochondrial membrane potential, and maximal respiration rate. At the same time, increased expression of Drp1 were observed in LPS-stimulated HUVECs, concomitantly with excessive mitochondrial fission. We found that hydrogen-rich medium can increase ATP content, mitochondrial membrane potential and maximal respiration rate, and decrease the expression of Drp1 in LPS-treated HUVECs. Meanwhile, hydrogen can ameliorate excessive mitochondrial fission caused by LPS. Furthermore, hydrogen-rich medium had a similar effect to Mdivi-1, a mitochondrial fission blocker. Both of them rescued the up-regulation of Drp1 and mitochondrial fission induced by LPS, then normalized mitochondrial shape after LPS stimulation. But after Znpp pretreatment, HO-1 expression was inhibited and the protective effects of hydrogen were abrogated. ConclusionsHydrogen-rich medium can alleviate the LPS-induced mitochondrial fusion/fission and dysfunction in HUVECs via HO-1 up-regulation.

Abstract 2374: Early metabolic rewiring of prostate cancer cells triggered by bone progenitors defines survival of metastatic prostate cancer

Abstract Bone hosts the 88% of Prostate Cancer (PCa) metastases and no curative therapy is currently available for this stage. Arrival of PCa cells to the bone homing organ is accompanied by a metabolic adaptation, which may be mediated by bone secreted factors. Herein, we sought to identify key metabolic genes fueling PCa bone metastasis and soluble factors secreted by bone cells leading to the metabolic rewiring of tumoral cells. By an indirect transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3, pre-osteoblasts; or Raw264.7, pre-osteoclasts) we analyzed the transcriptome (RNA-seq) of PC3 cells modulated by soluble factors released from bone precursors. GSEA showed a strong activation of lipid metabolism, including PPAR and PI3K-Akt pathways, fat absorption and digestion. We then performed a Principal Component Analysis using transcriptomic data from human PCa and bone metastasis samples (GSE74685), showcasing that those metabolic genes that appeared significantly dysregulated in the co-culture model could accurately cluster samples by their tissue of origin in two defined groups: primary PCa and bone metastasis. This result was confirmed by an unsupervised clustering analysis, highlighting that the transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastasis samples. Interestingly, when performing a survival analysis for those genes in the SU2C-PCF dataset, we observed that 4 lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, correlated with a shorter overall survival time, and appeared as independent risk-predictors of death (HR: 4.96, 2.85, 3.93 and 3.67, respectively; P<0.05). These results prompted us to evaluate the communication axis by which the expression of the lipid transcriptomic signature is regulated in PC3 cells co-cultured with bone progenitor cells. We performed an Ingenuity Pathway Analysis (QIAGEN) showing that the tumoral Protein Kinase A (PKA) appears as a master regulator of this signature. Accordingly, when we treated PC3 cells with the conditioned media (CM) of PC3 grown alone or the co-culture, we observed a decreased ATP content in the latter compared with controls, which is restored upon PKA inhibition, confirming the role of this kinase in the metabolic phenotype of co-cultured cells. Finally, secretome analysis (ESI-MS/MS) of CM from the co-cultures displayed relevant soluble factors secreted by bone progenitors (Col1a1, Fn1) which are directly linked to PKA activity. Overall, we identified an early lipid-related gene signature in PCa cells triggered by the dialogue with bone cells, enough to discriminate metastatic human PCa from primary tumors and critical for PCa survival. This signature may respond to released soluble bone factors through tumoral cell PKA activation. Our findings pinpoint new attractive metabolic druggable targets to halt disease progression. Citation Format: Pablo Sanchis, Nicolas Anselmino, Rosario Lavignolle, Agustina Sabater, Estefania Labanca, Juan Bizzotto, Sofia Lage-Vickers, Gaston Pascual, Rocio Seniuk, Ayelen Toro, Nora Navone, Javier Cotignla, Elba Vazquez, Geraldine Gueron. Early metabolic rewiring of prostate cancer cells triggered by bone progenitors defines survival of metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2374.

Protein Kinase A (PKA) as a Master Regulator of the Early Metabolic Reprogramming in Bone Metastatic Prostate Cancer Cells

PURPOSE The arrival of metastatic prostate cancer (PCa) tumor cells to the bone niche requires a metabolic adaptation. We sought to identify metabolic dysregulations fueling PCa metastasis, modulated by bone secreted factors. METHODS By an indirect co-culture system of PCa (PC3) and bone progenitors (MC3T3, pre-osteoblasts, or Raw 264.7, pre-osteoclasts) we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. We validated the transcriptional profile of metabolic genes in open-access transcriptomic datasets. We performed an Ingenuity Pathway Analysis (IPA) to delineate the regulators of these metabolic genes. Bone secretome was profiled on the conditioned media (CM) by ESI-MS/MS. RESULTS PC3 cells co-cultured with bone progenitors displayed an activation of lipidic categories, including PPAR-signaling and fat absorption/digestion. Principal Component and Unsupervised Clustering analyses using transcriptomic data from human PCa and bone metastatic samples (GSE74685) showed that the metabolic genes deregulated in PC3 accurately clustered samples in primary tumor or bone metastasis. Moreover, four lipid-associated genes, PPARA, VDR, SLC16A1 and GPX1, were associated with a shorter survival time (SU2C-PCF dataset), and were independent risk-predictors of death ( P < .05). An IPA revealed that these genes are regulated by the Protein Kinase A (PKA). Accordingly, PC3 cells treated with the CM of the co-culture presented a decreased ATP content compared to the treatment with the CM of PC3 grown alone, which was restored upon PKA inhibition. Finally, the secretome analysis revealed soluble factors secreted by bone progenitors (Col1a1, Fn1) which could regulate PKA activity. CONCLUSION We identified a novel lipid-associated gene signature important for metastatic PCa, triggered by the dialogue with bone cells. Moreover, PKA could regulate this signature in response to bone-secreted factors reprogramming the metabolic phenotype of metastatic cells, emerging as a potential druggable target for this disease.

Polystyrene microplastics induce mitochondrial damage in mouse GC-2 cells

Microplastics are widely distributed, such as oceans, rivers and the atmosphere, with many opportunities for human exposure and potential health risks. Polystyrene microplastic (PS-MPS) exposure has been found to cause sperm damage to mice; however, the mechanism by which this happens remains unclear. Here, GC-2 cells, a mouse spermatocyte line, were exposed to 5 µm PS-MPS to investigate mitochondrial damage. The results showed that 5 µm PS-MPS decreased ATP content, reduced the mitochondrial membrane potential, damaged the integrity of the mitochondrial genome, and caused an imbalance of homoeostasis between mitochondrial division and fusion. The mitochondrial PINK1/Parkin autophagy pathway was activated. Time-series analysis revealed that PS-MPS damaged the mitochondrial structure through cellular oxidative stress, and mitochondrial function was maintained to some extent after PS-MPS damage. This study revealed the mitochondrial toxicity of polystyrene microplastics, thus providing a basis for understanding the causes of sperm damage by polystyrene microplastics.

Deltamethrin induces apoptosis in cerebrum neurons of quail via promoting endoplasmic reticulum stress and mitochondrial dysfunction

Deltamethrin (DLM) is a widely used and highly effective insecticide. DLM exposure is harmful to animal and human. Quail, as a bird model, has been widely used in the field of toxicology. However, there is little information available in the literature about quail cerebrum damage caused by DLM. Here, we investigated the effect of DLM on quail cerebrum neurons. Four groups of healthy quails were assigned (10 quails in each group), respectively given 0, 15, 30, and 45 mg/kg DLM by gavage for 12 weeks. Through the measurements of quail cerebrum, it was found that DLM exposure induced obvious histological changes, oxidative stress, and neurons apoptosis. To further explore the possible molecular mechanisms, we performed real-time quantitative PCR to detect the expression of endoplasmic reticulum (ER) stress-related mRNA such as glucose regulated protein 78 kD, activating transcription factor 6, inositol requiring enzyme, and protein kinase RNA (PKR)-like ER kinase. In addition, we detected ATP content in quail cerebrum to evaluate the functional status of mitochondria. The study showed that DLM exposure significantly increased the expression of ER stress-related mRNA and decreased ATP content in quail cerebrum tissues. These results suggest that chronic exposure to DLM induces apoptosis of quail cerebrum neurons via promoting ER stress and mitochondrial dysfunction. Furthermore, our results provide a novel explanation for DLM-induced apoptosis of avian cerebrum neurons.

Influence of physical load of maximum aerobic power on hemodynamics and morpho-biochemical changes in erythrocytes in female volleyball players

Purpose: to study the types of cardiohemodynamic response of the organism and morpho-biochemical changes of erythrocytes of peripheral blood of volleyball players to physical activity of maximum aerobic capacity.
 Material and methods In 18 highly qualified volleyball players aged 22.0±0.60 years, cardiohemodynamics (by functional methods), functional changes (by biochemical methods) and the structure of erythrocytes (using scanning electron microscopy) were studied before and after the maximum (3.5 W/kg of body weight) of physical activity.
 Results. It was found that maximal physical activity causes significant changes in cardiohemodynamics, which result in morphological rearrangement of peripheral blood erythrocytes (increase in erythrocyte deformation index) and closely correlate with biochemical changes (decrease in ATP concentration and increase in magnesium concentration in erythrocytes). Possible mechanisms of realization of features of reaction of an organism of volleyball players to physical activity of the maximum aerobic power depending on type of regulation of blood circulation and morpho-biochemical changes of erythrocytes of peripheral blood are discussed.
 Conclusions. Physical activity of maximum aerobic power in highly qualified female volleyball players reveals the typological features of the cardiohemodynamic response of the body of volleyball players, which is expressed by conformational changes in peripheral blood erythrocytes and depends on the concentration of individual macroelements in these cells and on the type of cardiohemodynamics.

Molybdenum and Cadmium Co-induce Mitochondrial Quality Control Disorder via FUNDC1-Mediated Mitophagy in Sheep Kidney.

Molybdenum (Mo), fundamental trace mineral for animals and plants, but undue Mo damages animal health. Cadmium (Cd) is a toxic heavy metal that exists in the environment. Nevertheless, the mechanism of Mo and Cd on mitochondrial quality control are still indistinct. The objective of this research was to explore the effects of mitophagy on mitochondrial quality control via the FUNDC1-mediated by Mo and Cd in sheep kidney. Forty-eight 2-month-old sheep were stochastically divided into four groups, as shown below: control group, Mo [45 mg/kg body weight (BW)] group, Cd (1 mg/kg BW) group and Mo (45 mg/kg BW)+Cd (1 mg/kg BW) group, with 50 days feed technique. The results showed that Mo or/and Cd attract an unbalance of trace minerals and vacuoles and granular degeneration of renal tubular epithelial cells, and increase the number of mitophagosomes and vacuole-mitochondria and LC3 puncta and MDA and H2O2 contents, and decrease ATP content in the kidney. Moreover, Mo or/and Cd treatment could upregulate the mRNA levels of FUNDC1, LC3A, LC3B, PGAM5, DRP1, FIS1 and MFF, and the protein levels of FUNDC1, p-FUNDC1, LC3II/LC3I, DRP1, MFF and FIS1, downregulate the mRNA levels of MFN1, MFN2, OPA1, PGC-1α, SIRT1, SIRT3, FOXO1 and FOXO3, and the protein levels of MFN1, MFN2, OPA1 and PGC-1α. Notably, variations of above-mentioned factors in Mo and Cd group were more obvious than in Mo or Cd groups. Taken together, these results displayed that Mo and Cd co-treatment might induce mitochondrial quality control disorder via FUNDC1-mediated mitophagy in sheep kidney.

Crocetin ameliorates non-alcoholic fatty liver disease by modulating mitochondrial dysfunction in L02 cells and zebrafish model

Ethnopharmacological relevanceTraditional Chinese medicine considers that the etiology and pathogenesis of non-alcoholic fatty liver disease (NAFLD) are related to liver depression and qi stagnation. Saffron and its active ingredient, crocetin (CCT), are used for the treatment of metabolic diseases owing to their “Liver deobstruent” and “Liver tonic” effects. However, the effect of CCT on NAFLD has not been fully elucidated. In the present study, the effect and potential molecular mechanism of CCT were explored in both in vivo and in vitro models of NAFLD. Materials and methodsCCT was isolated from saffron and purity and structure characterization were performed using HPLC, MS, 1H-NMR, and 13C-NMR. The effect of CCT on the viability of L02 cells and its maximum tolerable concentration (MTC) in zebrafish were investigated. Free fatty acids (FFA) and thioacetamide (TAA) were used to induce lipid accumulation in L02 cells and steatosis in zebrafish, respectively. The effects of CCT on indexes related to lipid metabolism, oxidative stress, and mitochondrial function in NAFLD models were explored using biochemical assay kits, Western blot analysis, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), histopathology analysis, and determination of mitochondrial membrane potential (ΔΨm). Morphological analysis of mitochondria was performed using transmission electron microscopy (TEM). ResultsThe levels of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), and alanine/aspartate aminotransferases (ALT/AST) activities in FFA treated L02 cells were significantly reduced after CCT treatment. CCT treatment significantly increased ATP concentration, ΔΨm, and activities of superoxide dismutase (SOD), catalase (CAT), and cytochrome c oxidase (COX IV) in FFA treated L02 cells. TEM images showed restoration of mitochondrial morphology. CCT decreased ATP concentration and upregulated expression of B-cell lymphoma-2 (Bcl-2) and COX IV, whereas, CCT downregulated expression of BCL2-Associated X (Bax) and cleaved caspase-3 in TAA treated zebrafish. These findings indicated that mitochondrial dysfunction was alleviated after CCT treatment. Oil Red O staining of L02 cells and zebrafish showed that CCT treatment reversed the accumulation of lipid droplets. ConclusionIn summary, CCT treatment effectively alleviated the symptoms of NAFLD and restored mitochondrial function in L02 cells and zebrafish NAFLD model.

Trial in Progress: A Phase 2, Open-Label Study Evaluating the Safety and Efficacy of the PKR Activator Etavopivat (FT-4202) in Patients with Thalassemia or Sickle Cell Disease

Trial in Progress: A Phase 2, Open-Label Study Evaluating the Safety and Efficacy of the PKR Activator Etavopivat (FT-4202) in Patients with Thalassemia or Sickle Cell Disease

Yiqi Huoxue preserves heart function by upregulating the Sigma-1 receptor in rats with myocardial infarction.

Yiqi Huoxue (YQHX) is widely used in traditional Chinese medical practice due to its reported cardioprotective effects. The aim of the present study was to investigate the mechanism underlying these effects of YQHX via the regulation of the Sigma-1 receptor. The Sigma-1 receptor is a chaperone protein located on the mitochondrion-associated endoplasmic reticulum (ER) membrane. It serves an important role in heart function by regulating intracellular Ca2+ homeostasis and enhancing cellular bioenergetics. In the present study, male Sprague Dawley rats with myocardial infarction (MI)-induced heart failure were used. MI rats were administered different treatments, including normal saline, YQHX and fluvoxamine, an agonist of the Sigma-1 receptor. Following four weeks of treatment, YQHX was revealed to improve heart function and attenuate myocardial hypertrophy in MI rats. Additionally, YQHX increased the ATP content and improved the mitochondrial ultrastructure in the heart tissues of MI rats in comparison with acontrol. Treatment was revealed to attenuate the decreased expression of the Sigma-1 receptor and increase the expression of inositol triphosphate type 2 receptors (IP3R2) in MI rats. By exposing H9c2 cells to angiotensin II (Ang II), YQHX prevented cell hypertrophy and normalized the decreased ATP content. However, these positive effects were partially inhibited when the Sigma-1 receptor was knocked down via small interfering RNA transfection. The results of the present study suggested that the Sigma-1 receptor serves an important role in the cardioprotective efficacy of YQHX by increasing ATP content and attenuating cardiomyocyte hypertrophy.

Glucose Metabolism Reprogramming of Regulatory T Cells in Concanavalin A-Induced Hepatitis.

Autoimmune hepatitis (AIH) is an inflammatory liver disease caused by a dysregulated immune response. Although the pathogenesis of AIH remains unclear, impaired regulatory T cells (Tregs) have been considered a driver of AIH development. Unlike autoreactive T cells, Tregs mainly utilize oxidative phosphorylation (OXPHOS) as their energy supply. Elevated glycolysis has been reported to limit the suppressive functions of Tregs. However, whether glucose metabolism reprogramming in Tregs is involved in AIH etiology remains unknown. The aim of this study was to examine alternations in Treg numbers and functions in AIH patients and concanavalin A (Con A)-induced hepatitis, while exploring associations between impaired Tregs and glucose metabolism. The frequency of Tregs was decreased in the peripheral blood but increased in liver biopsies of AIH patients. Moreover, immunosuppressive therapy rescued circulating Tregs in AIH. In Con A-induced immune hepatitis, enhanced intrahepatic Treg accumulation was observed over time, accompanied by reduced splenic Treg numbers. To investigate whether functional impairment of Tregs occurs in AIH, Tregs were isolated from experimental AIH (EAH) model mice and normal controls and the former displayed downregulated mRNA levels of FOXP3, CTLA4, CD103, TIGIT, CD39, and CD73. EAH model-derived Tregs also produced fewer anti-inflammatory mediators (TGF-β and IL-35) than control Tregs. Moreover, enhanced glycolysis and reduced OXPHOS were found in Tregs from EAH model mice, as reflected by elevated levels of key glycolytic enzymes (HK2, PK-M2, and LDH-A) and a decreased ATP concentration. This study revealed a decreased peripheral Treg frequency and abnormal intrahepatic Treg infiltration in AIH. It is first reported that glucose metabolism reprogramming is associated with decreases and functional impairments in the Treg population, promoting AIH development. Targeting glucose metabolism may provide novel insights for the treatment of AIH.

All-trans retinoic acid impairs glucose-stimulated insulin secretion by activating the RXR/SREBP-1c/UCP2 pathway.

Diabetes is often associated with vitamin A disorders. All-trans retinoic acid (ATRA) is the main active constituent of vitamin A. We aimed to investigate whether ATRA influences diabetic progression and its mechanisms using both Goto-Kazizazi (GK) rats and INS-1 cells. Rat experiments demonstrated that ATRA treatment worsened diabetes symptoms, as evidenced by an increase in fasting blood glucose (FBG) levels and impairment of glucose homeostasis. Importantly, ATRA impaired glucose-stimulated insulin secretion (GSIS) and increased the expression of sterol regulatory element-binding protein 1c (SREBP-1c) and uncoupling protein 2 (UCP2) in the rat pancreas. Data from INS-1 cells also showed that ATRA upregulated SREBP-1c and UCP2 expression and impaired GSIS at 23 mM glucose. Srebp-1c or Ucp2 silencing attenuated GSIS impairment by reversing the ATRA-induced increase in UCP2 expression and decrease in ATP content. ATRA and the retinoid X receptor (RXR) agonists 9-cis RA and LG100268 induced the gene expression of Srebp-1c, which was almost completely abolished by the RXR antagonist HX531. RXRα-LBD luciferase reporter plasmid experiments also demonstrated that ATRA concentration-dependently activated RXRα, the EC50 of which was 1.37 μM, which was lower than the ATRA concentration in the pancreas of GK rats treated with a high dose of ATRA (approximately 3 μM), inferring that ATRA can upregulate Srebp-1c expression in the pancreas by activating RXR. In conclusion, ATRA impaired GSIS partly by activating the RXR/SREBP-1c/UCP2 pathway, thus worsening diabetic symptoms. The results highlight the roles of ATRA in diabetic progression and establish new strategies for diabetes treatment.

Increased Synthesis of a Magnesium Transporter MgtA During Recombinant Autotransporter Expression in Escherichia coli

Overproduction of the membrane proteins in Escherichia coli cells is a common approach to obtain sufficient material for their functional and structural studies. However, the efficiency of this process can be limited by toxic effects which decrease the viability of the host and lead to low yield of the product. During the expression of the esterase autotransporter AT877 from Psychrobacter cryohalolentis K5T, we observed significant growth inhibition of the C41(DE3) cells in comparison with the same cells producing other recombinant proteins. Induction of AT877 synthesis also resulted in the elevated expression of a magnesium transporter MgtA and decreased ATP content of the cells. To characterize the response to overexpression of the autotransporter in bacterial cells, we performed a comparative analysis of their proteomic profile by mass spectrometry. According to the obtained data, E. coli cells which synthesize AT877 experience complex stress condition presumably associated with secretion apparatus overloading and improper localization of the recombinant protein. Several response pathways were shown to be activated by AT877 overproduction including Cpx, PhoP/PhoQ, Psp, and σE The obtained results open new opportunities for optimization of the recombinant membrane protein expression in E. coli for structural studies and biotechnological applications.

Вміст сіалових кислот, АТФ і супероксидного аніон-радикала у зародках в’юна за впливу флуренізиду

It is known that flurenizide is a newly synthesized drug with pronounced antimicrobial, immunomodulatory functions. However, its effect on the functional properties of cells, in particular germ cells, is unknown. The aim of this study was to determine the presence of sialic acids as the final components of glycoconjugates, the main macroergic compound - ATP, to evaluate the generation of O2¯ in embryos of lobster (Misgurnus fossilis L.) under the influence of the antibiotic flurenizide. Studies have been performed on embryos of Misgurnus fossilis L. After fertilization, the zygotes were placed in Petri dishes with solutions of flurenizide in concentrations of 0.01; 0.05; 0.15; 1.0; 5.0; 15.0 mM, where allowed to develop. At the development stages, 2 blastomeres, 16 blastomeres, 64 blastomeres, VIII (256 blastomeres) and X divisions (1024 blastomeres) were sampled. In parallel, control studies were performed, where flureniside was not added to the samples. The content of sialic acids, ATP, superoxide anion radical was determined in the selected samples. We found that flureniside at the lowest concentration of 0.01 mM does not cause changes in the content of superoxide anion radical during early embryogenesis. The antibiotic in the maximum concentration causes changes in the content of free radical from the stage of development of 16 blastomeres to 1024 blastomeres, and from the stage of 64 blastomeres there is an increase in its number. Probably flurenizide at a concentration of 15.0 mM is the most reactive. It is known that flurenizide has antioxidant properties, but in its structure there are groups that have a toxic effect, which is most pronounced when exposed to high concentrations. In general, at the stage of development of 16 blastomeres there is a decrease in the amount of О2¯ under the influence of the studied antibiotic. At this time, the content of this free radical in the control increases, compared with other stages of development. Flureniside leads to an increase in the content of superoxide anion radical at development stage 2, 64 blastomeres and is particularly pronounced at concentrations from 0.05 to 15.0 mM at development stages 256 and 1024 blastomeres. 1024 blastomeres are the 10th stage of separation, where desynchronization occurs and the mitotic index decreases. Probably, the changes that take place at this stage of development are related to this. The antibiotic causes an increase in the content of sialic acids in the first stage of crushing (2 blastomeres). In step 16 of the blastomere, flureniside in low concentrations leads to a decrease in the content of sialic acids. However, already at the stage of separation of 64 and 256 blastomeres, the test substance in all concentrations causes a predominant decrease in the amount of sialic acids. At the last stage of synchronous crushing (1024 blastomeres) flurenizide in the maximum investigated concentration (15.0 mM) causes a significant accumulation of sialic acid content. Flureniside at concentrations of 0.15, 5.0 and 15.0 mМ causes a decrease in ATP content in loach embryos at the stage of development of 256 blastomeres by 28, 67 and 38 %, respectively. An increase in ATP content by 33 % occurs under the influence of flurenizide at a concentration of 1.0 mM. The ATP content also increases at the stage of development of embryos of 1024 blastomeres under the influence of flurenizide of all studied concentrations. Analysis of variance has shown that a significant contribution to the growth of ATP, sialic acid and superoxide anion radical, during the early embryogenesis of lobster embryos, is made by the factor of embryo development, while the factor of flureniside has a smaller contribution. Therefore, studies have shown that flurenizide causes increased generation of superoxide anion radical, decreased sialic acid content, as well as changes in ATP content in embryonic cells during early embryogenesis.

Abstract 2468: The role of autophagy in lymphangiosarcoma development

Abstract Lymphangiosarcoma is a rare malignant vascular tumor which is formed from aberrant proliferation of endothelial cells (ECs). We previously established an endogenous mouse model of lymphangiosarcoma by conditional knockout of Tsc1 gene in ECs and revealed that hyper-activated mTORC1 is required for the vascular tumor development and progression through increased VEGF autocrine signaling. Using this model, we investigated the role of an essential autophagy gene FIP200 (FAK family interacting protein of 200 kDa) and its canonical autophagy function in lymphangiosarcoma development and progression by generating a FIP200 knockout (designated as Tsc1/Fip200 2cKO mice) or introducing a knockin allele that specifically blocks its canonical autophagy function (designated as Tsc1/Fip200 cKI mice) in combination with Tsc1 cKO in this study. We found that while both Tsc1/Fip200 cKO and Tsc1/Fip200 cKI mice exhibited lymph edema phenotype as Tsc1 cKO mice, they did not develop lymphangiosarcoma, suggesting that autophagy is required for lymphangiosarcom formation. Consistent with the above results, we also found that CRISPR-Cas9 mediated Fip200 KO in an immortalized vascular tumor cell line Tsc1ΔEC from Tsc1 cKO mice blocked their ability to grow tumors in xenograft transplants. Mechanistically, we found that autophagy was required for maintaining mTORC1 hyper-activation under nutrient stress condition. Moreover, the impaired lipophagy by Fip200 knockout affected lipid catabolism under glucose free condition and thus reduced the substrate for fatty acid oxidation (FAO). Our data showed that Fip200, Atg5 or Atg7 ablation in Tsc1ΔEC inhibited mitochondria respiration and decreased ATP contents as measured by Seahorse assays. Either lipase inhibitor Orlistat or carnitine palmitoyltransferase-1 (Cpt1) inhibitor Etomoxir treatment decreased Tsc1ΔEC mitochondria respiration and ATP production. Collectively, our data suggested that autophagy is required for lymphangiosarcoma initiation and progression by modulating FAO to supply cellular energy under stress for sustaining mTORC1 hyper-activation for tumor growth. Citation Format: Fuchun Yang, Michael Haas, Jun-Lin Guan. The role of autophagy in lymphangiosarcoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2468.

DMEP induces mitochondrial damage regulated by inhibiting Nrf2 and SIRT1/PGC-1α signaling pathways in HepG2 cells

Dimethoxyethyl phthalate (DMEP) is an environmental endocrine disruptor. However, research into the underlying mechanisms of DMEP mitochondrial toxicity is still in its infancy. We therefore expect to understand whether DMEP induced mitochondrial damage in HepG2 cells and the associated signaling pathways. DMEP (0.125, 0.25, 0.5, 1 and 2 mM) exposure for 48 h induced a notable increment in reactive oxygen species (ROS), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate transaminase (AST) and 8-hydroxydeoxyguanosine (8-OHdG) in hepG2 cells, resulting in cellular oxidative stress. Low doses of DMEP upregulated nuclear factor E2-related factor 2 (Nrf2) and downstream protein haeme oxygenase-1 (HO-1) levels and high doses down-regulated their levels. Nrf2 levels increased after ROS scavenging by N-acetyl-L-cysteine (NAC), which indicated that the Nrf2 pathway may be affected by oxidative stress. We also found that DMEP decreased ATP content, mitochondrial copy number (mtDNA), translocase of the outer membrane subunit 20 (TOM20) expression, mitochondria-encoded genes CO1, CO2, CO3, ATP6, ATP8 expression, inhibited mitochondrial biogenesis pathway, down-regulated sirtuin 1(SIRT1), PPAR gamma co-activator 1 alpha (PGC-1α), Nuclear respiratory factor 1(Nrf1), Mitochondrial transcription factor A (TFAM) content and activated PINK1/Parkin autophagy pathway. DMEP also activated the mitochondrial apoptotic pathway, causing cytochrome c cytoplasmic translocation and caspase 3 cleavage. What’s more, DMEP activated the Nuclear factor-κB (NF-κB) pathway and levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly upregulated, causing an inflammatory response. In summary, DMEP can cause inflammatory response and oxidative stress in HepG2 cells, inhibited the Nrf2 pathway and mitochondrial biogenesis, and induced autophagy and apoptosis. And oxidative stress at least partially affected the Nrf2 pathway and mitochondrial biogenesis SIRT1/PGC-1α pathway.

Phenolic acid metabolites of polyphenols act as inductors for hormesis in C. elegans

IntroductionAging represents a major risk factors for metabolic diseases, such as diabetes, obesity, or neurodegeneration. Polyphenols and their metabolites, especially simple phenolic acids, gained growing attention as a preventive strategy against age-related, non-communicable diseases, due to their hormetic potential. Using Caenorhabditis elegans (C. elegans) we investigate the effect of protocatechuic, gallic, and vanillic acid on mitochondrial function, health parameters, and the induction of potential hormetic pathways. MethodsLifespan, heat-stress resistance and chemotaxis of C. elegans strain P X 627, a specific model for aging, were assessed in 2-day and 10-day old nematodes. Mitochondrial membrane potential (ΔΨm) and ATP generation were measured. mRNA expression levels of longevity and energy metabolism-related genes were determined using qRT-PCR. ResultsAll phenolic acids were able to significantly increase the nematodes lifespan, heat-stress resistance and chemotaxis at micromolar concentrations. While ΔΨm was only affected by age, vanillic acid (VA) significantly decreased ATP concentrations in aged nematodes. Longevity pathways, were activated by all phenolic acids, while VA also induced glycolytic activity and response to cold. ConclusionWhile life- and health span parameters are positively affected by the investigated phenolic acids, the concentrations applied were unable to affect mitochondrial performance. Therefore we suggest a hormetic mode of action, especially by activation of the sirtuin-pathway.

Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury.

The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B1 (AFB1 ) on human renal cells. Hek293 cells were exposed to AFB1 (0-100 μM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50 ) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50 ) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1 -treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated renal cells. The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.

Nuclei DNA and mitochondria dual damages induced by thiosemicarbazone tripyridyl copper complexes with potential anti-tumor activity

In this study, we synthesised and characterised a di-nuclear complex, [Cu2(dppco)Br3] (1), and a mononuclear complex, [Cu(dppc)Br] (2), with the thiosemicarbazone tripyridyl ligand dppc = 2-(di(pyridin-2)-yl)methylene)-N-(pyridin-2-yl)hydrazine-1-carbothio-amide. The S atom of the ligand in complex 1 was replaced by an O atom in the solvothermal reaction, while only the ligand in complex 2 contains an S tom (it is therefore a thiosemicarbazone). Compared to complex 1, complex 2 has a stronger inhibitory effect on the colon cancer cell line (HCT116), human breast cancer cell line (CAOV-3) and liver cancer cell line (SMMC7721), especially being more sensitive to HCT116 cells. Fluorescence, circular dichroism and DNA cleavage experiments showed that complexes 1 and 2 have an excellent DNA binding ability, mostly binding to DNA by the intercalation mode. In contrast, the thiosemicarbazone-based complex 2 has a stronger cell uptake ability, which triggered more prominent cellular apoptosis and cell cycle arrest. Further mechanism studies revealed that complex 2 caused more serious mitochondrial damage than that for complex 1, including an increased level of reactive oxygen species (ROS) and Ca2+, decreased ATP content and mitochondrial membrane potential (Δψm), and significantly transformed the mitochondrial morphology.

Mitochondrial localization of ORF346 causes pollen abortion in alloplasmic male sterility

The Nsa cytoplasmic male sterility (CMS) system confers stable male sterility and offers great potential for production of hybrid seeds in oilseed rape. However, genes responsible for male sterility in Nsa CMS have not been identified. By mitochondrial genome sequencing of Nsa CMS and its maintainer line, we identified in an Nsa CMS line several chimeric genes encoding hypothetical proteins harboring transmembrane domains. One novel chimeric gene orf346 showed high identity with cox1 at the 5′ terminal region and was co-transcribed with nad3 and rps12 genes. Transgenic plants of orf346 fused with or without mitochondrial targeting peptide conferred complete male sterility in Arabidopsis. ORF346 was mitochondrion-localized. Expression of orf346 in Escherichia coli inhibited bacterial growth, with excessive accumulation of reactive oxygen species and decreased ATP content. These results reveal a link between the newly identified mitochondrial gene orf346 and the abortion of Nsa CMS. Inadequate energy supply and excessive accumulation of reactive oxygen species may account for pollen abortion in Nsa CMS plants.