Palmitate-induced Activation of Mitochondrial Metabolism Promotes Oxidative Stress and Apoptosis in H4IIEC3 Rat Hepatocytes

Objective

Hepatic lipotoxicity is characterized by reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and excessive apoptosis, but the precise sequence of biochemical events leading to oxidative damage and cell death remains unclear. The goal of this study was to delineate the role of mitochondrial metabolism in mediating hepatocyte lipotoxicity.

Materials/Methods

We treated H4IIEC3 rat hepatoma cells with free fatty acids in combination with antioxidants and mitochondrial inhibitors designed to block key events in the progression toward apoptosis. We then applied ¹³C metabolic flux analysis (MFA) to quantify mitochondrial pathway alterations associated with these treatments.

Results

Treatment with palmitate alone led to a doubling in oxygen uptake rate and in most mitochondrial fluxes. Supplementing culture media with the antioxidant N-acetyl-cysteine (NAC) reduced ROS accumulation and caspase activation and partially restored cell viability. However, ¹³C MFA revealed that treatment with NAC did not normalize palmitate-induced metabolic alterations, indicating that neither elevated ROS nor downstream apoptotic events contributed to mitochondrial activation. To directly limit mitochondrial metabolism, the complex I inhibitor phenformin was added to cells treated with palmitate. Phenformin addition eliminated abnormal ROS accumulation, prevented the appearance of apoptotic markers, and normalized mitochondrial carbon flow. Further studies revealed that glutamine provided the primary fuel for elevated mitochondrial metabolism in the presence of palmitate, rather than fatty acid beta-oxidation, and that glutamine consumption could be reduced through co-treatment with phenformin but not NAC.

Conclusion

Our results indicate that ROS accumulation in palmitate-treated H4IIEC3 cells occurs downstream of altered mitochondrial oxidative metabolism, which is independent of beta-oxidation and precedes apoptosis initiation.     

Inhibition of acetylcholinesterase by two genistein derivatives: kinetic analysis,molecular docking and molecular dynamics simulation

In this study two genistein derivatives (G1 and G2) are reported as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and differences in the inhibition of AChE are described. Although they differ in structure by a single methyl group, the inhibitory effect of G1 (IC₅₀=264 nmol/L) on AChE was 80 times stronger than that of G2 (IC₅₀=21,210 nmol/L). Enzyme-kinetic analysis, molecular docking and molecular dynamics (MD) simulations were conducted to better understand the molecular basis for this difference. The results obtained by kinetic analysis demonstrated that G1 can interact with both the catalytic active site and peripheral anionic site of AChE. The predicted binding free energies of two complexes calculated by the molecular mechanics/generalized born surface area (MM/GBSA) method were consistent with the experimental data. The analysis of the individual energy terms suggested that a difference between the net electrostatic contributions (ΔE_ele+ΔG_GB) was responsible for the binding affinities of these two inhibitors. Additionally, analysis of the molecular mechanics and MM/GBSA free energy decomposition revealed that the difference between G1 and G2 originated from interactions with Tyr124, Glu292, Val294 and Phe338 of AChE. In conclusion, the results reveal significant differences at the molecular level in the mechanism of inhibition of AChE by these structurally related compounds.KEY WORDS: Genistein derivatives, Acetylcholinesterase (AChE), Kinetics analysis, Molecular docking, Molecular dynamics simulation, MM/GBSAAbbreviations: ACh, acetylcholine; AChEIs, acetylcholinesterase inhibitors; AChE, acetylcholinesterase; AD, Alzheimer׳s disease; BuChE, butyrylcholinesterase; BuSCh, S-butyrylthiocholine chloride; CAS, catalytic active site; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); GAFF, generalized AMBER force field; G1, 3-(4-methoxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one; G2, (S)-3-(4-methoxyphenyl)-7-(2-(2-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one; iso-OMPA, tetraisopropyl pyrophosphoramide; MD, molecular dynamics; MM/GBSA, molecular mechanics/generalized born surface area; PAS, peripheral anionic site; PDB, protein data bank; PME, particle mesh Ewald; RMSD, root-mean-square deviation; S-ACh, acetylthiocholine iodide; ΔE_ele, electrostatic energy contribution; ΔE_MM, gas-phase interaction energy between receptor and ligand; ΔE_vdw, van der Waals energy contribution; SASA, solvent accessible surface area; ΔG_exp, experimental binding free energy; ΔG_GB, polar desolvation energy term; ΔG_pred, total binding free energy; ΔG_SA, nonpolar desolvation energy term; ΔS, conformational entropy contribution     

A rapid flow cytometry assay for the assessment of calcium mobilization in human neutrophils in a small volume of lysed whole-blood

Flow cytometry-based methods have been developed to measure most neutrophil responses. The assessment of the mobilization of calcium, however, is routinely performed on neutrophils isolated from whole blood. This report describes a flow cytometry-based assay to measure the mobilization of calcium in neutrophils directly in whole blood. This method requires minimal sample manipulation, small volumes of blood and is performed in a short period of time. Both clinical and research laboratories will be able to assess neutrophil function and the quality of granulocyte preparations using a more time and cost effective calcium mobilization test.     

Subcutaneous zygomycosis: report of 10 cases from two institutions in North India

Background Subcutaneous zygomycosis is an uncommon condition observed in tropics. Few series have been published, particularly from the northern regions of India. Objectives The aim of this study was to describe clinical, investigative and therapeutic details in subcutaneous zygomycosis observed in two teaching hospitals in Delhi. Patients and methods Ten patients seen over a period of 10 years (1999–2009) form the material for this report. Results There were four children and six adults. In four children, the presentation was a subcutaneous localized mass or gradually spreading plaque. In the others, it was observed over nasal region of face, spreading inward into mucosal sites and paranasal sinuses, and outward to the contiguous areas. Regional lymphadenopathy was present in two with facial lesions. Majority showed a granulomatous infiltrate with admixture of other cells, mainly eosinophils. Aseptate or poorly septate hyphae were observed in seven. In one patient in whom no hyphae were observed, there was dense perivascular inflammation. Organisms were cultured from four patients, Basidiobolus ranarum in two and Syncephalastrum racemosum in two. The main therapy used was a saturated solution of potassium iodide (KI). Four received only KI of which two attained cure after 3 months and 9 months respectively, and the other two showed signs of regression. In one boy subsidence was associated with reduced circumference of thigh. Ketoconazole or itraconazole was given with KI to hasten regression when response was slow or there were side‐effects to KI. Conclusion Awareness and early recognition will prevent disfigurement produced by advanced disease, misdiagnosis and unnecessary surgical intervention.     

碘化N-正丁基氟哌啶醇对大鼠心脏缺血和再灌注室性心律失常的拮抗作用

目的:研究碘化N-正丁基氟哌啶醇(F2)对心肌缺血和再灌注引发的室性心律失常的拮抗作用。方法:采用大鼠Langendorff灌流心脏模型,通过结扎左冠脉前降支缺血20min,解除结扎再灌注,引出室性心律失常。缺血前5min用含不同浓度F2的台氏液灌流,观察其对缺血和再灌注期室性心律失常发生率的影响,同时观察F2对心电图上PR、QT、RR间期的影响。用心房起搏(5Hz)防止心率变慢,观察1μmol/LF2对缺血和再灌注室性心律失常、PR间期的影响。结果:F2可浓度依赖地降低缺血和再灌注期室性心律失常发生率,并减慢心率,延长PR间期。在心房起搏控制心律下,仍具有拮抗缺血和再灌注室性心律失常、延长PR间期的作用。结论:F2对大鼠心脏缺血和再灌注性心律失常具有直接的抑制作用。     

Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines

Saffron (dried stigmas of Crocus sativus L.) has been used as a spice, food colorant and medicinal plant for millennia. In this study cytotoxic effect of saffron extract was evaluated in HepG2 and HeLa cell lines. Meanwhile role of apoptosis and ROS were explored. Malignant and non-malignant cells (L929) were cultured in DMEM medium and incubated with different concentrations of ethanolic saffron extract. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). ROS was measured using DCF-DA by flow cytometry analysis. Saffron could decrease cell viability in malignant cells as a concentration and time-dependent manner. The IC50 values against HeLa and HepG2 were determined 800 and 950 μg/ml after 48 h, respectively. Saffron induced a sub-G1 peak in flow cytometry histogram of treated cells compared to control indicating apoptotic cell death is involved in saffron toxicity. This toxicity was also independent of ROS production. It might be concluded that saffron could cause cell death in HeLa and HepG2 cells, in which apoptosis or programmed cell death plays an important role. Saffron could be also considered as a promising chemotherapeutic agent in cancer treatment in future.     

Effects of Epigallocatechin-3-gallate on lead-induced oxidative damage

Recent studies have shown that lead (Pb) could disrupt the prooxidant/antioxidant balance of tissue which leads to biochemical and physiological dysfunction. Epigallocatechin-3-gallate (EGCG), a catechin polyphenols component, is found to be an effective antioxidant. The present study investigated whether EGCG administration could reverse the changes on redox states in rat hippocampus caused by lead exposure. The association between redox status changes and long-term potentiation (LTP) in CA1 area of hippocampus were also examined. Wistar rats exposed to lead from postnatal day 1 were followed by 10 days of EGCG (10, 25 and 50 mg/kg) administration through intraperitoneally (ip), and the rats were sacrificed for experiments at the age of 21–23 days. The experimental results showed that glutathione (GSH) and superoxide dismutase (SOD) activity decreased accompanied with LTP amplitude decrease in CA1 area of hippocampus in the lead-exposed group. EGCG supplementation following lead intoxication resulted in increases in the GSH and SOD levels and increases in the LTP amplitude. Malondialdehyde (MDA) levels, a major lipid peroxidation byproduct, increased following lead exposure and decreased following EGCG treatment. In hippocampal neuron culture model, lead exposure (20 μM) significantly inhibited the viability of neurons which was followed by an accumulation of ROS and a decrease of mitochondrial membrane potential (ΔΨ_m). Treatment by EGCG (10–50 μM) effectively increased cell viability, decreased ROS formation and improved ΔΨ_m in hippocampal neurons exposed to lead. These observations suggest that EGCG is a potential complementary agent in the treatment of chronic lead intoxication through its antioxidative character.     

The changes of antioxidant defense system caused by quercetin administration do not lead to DNA damage and apoptosis in the spleen and bone marrow cells of rats.

Quercetin may have the opposite effect, namely anti- as well as pro-oxidant. The aim of this study was to assess the results of quercetin anti- and/or pro-oxidant activity in the bone marrow and spleen cells of rats. The experimental rats were treated daily, with quercetin in a dose of 8 or 80mg/kg b.w. by gavage for 40 days. The intracellular redox state in cells were assessed by measuring the ferric ion reducing antioxidant power (FRAP) level and malonodialdehyde concentration. HO-1 mRNA expression was examined with real-time PCR. The extent of DNA damage was determined by the alkaline-labile comet assay. A potential pro-apoptotic quercetin action was determined using the FITC-Annexin V kit. The quercetin and isorhamnetin concentrations in serum were analyzed by HPLC-ECD. MDA concentration and FRAP values, were significantly decreased in the spleen and bone marrow cells of rats treated with quercetin, in a dose of 80mg/kg b.w. in comparison with the control rats; no significant changes were observed after quercetin was administered in a dose ten times as low. Treatment with quercetin dose-dependently upregulated the expression of HO-1 mRNA in the bone marrow cells. Quercetin administration to the rats did not induce either DNA damage or apoptosis in the examined cells. The results of our study prove that changes in the antioxidant state, caused by quercetin, do not lead to DNA damage or exert any pro-apoptotic activity in vivo.     

人甲状腺钠/碘转运体基因全长的克隆及其重组表达质粒的构建

目的:利用基因工程的方法制备甲状腺钠/碘同向转运体(hNIS)cDNA,为今后的转基因治疗奠定物质基础。方法:采用TRIzol一步法,从甲状腺组织中提取总RNA,再应用RT-PCR扩增出hNIS基因全长,然后采用TOPO克隆技术构建重组表达质粒pcDNA3.1D/FLhNIS,并进行酶切和测序鉴定。结果:成功制备了hNIScDNA并构建了其重组表达质粒。结论:为最终实现131I治疗不摄碘的肿瘤提供了分子生物学基础。     

Contribution of apoptosis in the cytotoxicity of the oxaliplatin-irinotecan combination in the HT29 human colon adenocarcinoma cell line

Interactions between the topoisomerase I inhibitor irinotecan (CPT-11) and the platinum derivative oxaliplatin (L-OHP) were investigated in HT29 colon cancer cell line. Synergism was observed when cells were simultaneously exposed to drugs or when cells were first exposed to CPT-11. Flow cytometric studies showed a G(2)/M accumulation when cells were exposed to the simultaneous and CPT-11-->L-OHP combinations whereas a persistent S phase delay was observed when cells were first exposed to L-OHP. We characterised the cytotoxic effect by assessing the induction of apoptosis. Irinotecan induced substantial DEVDase activity and poly(ADP-ribose) polymerase cleavage while this activity was moderate and delayed after exposure to L-OHP. Combination experiments showed a sequence-dependent onset of apoptosis, the CPT-11-->L-OHP schedule being the earliest and the most effective; on the other hand the apoptotic signaling generated by CPT-11 was partly inhibited in the simultaneous combination and in the L-OHP-->CPT-11 sequence. Cell death studies using a dual staining technique showed a shift from apoptosis to necrosis when combining these drugs at high concentrations. Synergistic interactions observed using CPT-11 before L-OHP may be linked to an early apoptotic signaling while the L-OHP-induced S phase block could account for the observed additive effect in the reverse sequence. An additional phenomenon might work towards synergism for the simultaneous combination.