Mediating effect of ROS on mtDNA damage and low ATP content induced by arsenic trioxide in mouse oocytes

Mitochondria provide most of the adenosine triphosphates (ATP) necessary for the maturation of oocytes. Various environmental toxicants would lead damage to mitochondrial DNA (mtDNA) and hence interfere with oocytes development. In the current study, the effect of arsenic trioxide (As₂O₃) on the common 3867 bp deletion and the copy number of mtDNA in mitochondria of mouse oocytes in vivo or in vitro, as well as the molecular pathway leading to the damage were investigated. PCR strategy was used to detect the damage of mtDNA. Reactive oxygen species (ROS) and ATP content in oocytes were checked to determine the influence of As₂O₃ on oxidative stress and activity of mitochondria. The results showed that As₂O₃ could obviously decrease the copy number of mtDNA and cause severe 3867 bp deletion in mitochondria together with elevated ROS level, while ATP content was decreased. Co-treatment with N-Acetyl-Cysteine (NAC) efficiently eliminated ROS induced by As₂O₃, lessened the mtDNA damage and enhanced ATP content in mouse oocytes both in vivo and in vitro. Taken together, the present study revealed that As₂O₃ could cause severe mtDNA damage and decrease ATP content by inducing excessive ROS, and this damage would then probably restrain the further development of mouse oocytes.     

Preparation, characterization, in vivo and in vitro studies of arsenic trioxide Mg-Fe ferrite magnetic nanoparticles

Aim:

MgFe₂O₄ magnetic nanoparticle composed of As₂O₃ (As₂O₃-MNPs) were prepared and their in vitro and in vivo characteristics were studied.

Methods:

The solvent-displacement method was applied for preparation of the nanoparticle using Poly-D,L-lactic-co-glycolic acid(PLGA). The characteristics studies of the products included magnetic response, morphology (transmission electron microscopy and scanning electron microscopy), entrapment efficiency, drug loading, particle sizes, zeta potential, in vitro drug release and tissue magnetic targeting. Nanoparticle cytotoxicity to Saos-2 cells was investigated using the MTT assay. To guide the external magnetic field in the liver, the concentration of As₂O₃ in the liver and kidney was measured using an atomic fluorescence spectrometer after injecting As₂O₃-MNPs into the caudal veins of mice.

Results:

The As₂O₃-MNPs were approximately spherical. The average diameter, drug loading, entrapment efficiency and zeta potential of As₂O₃-MNPs were 109.9 nm, 10.08%, 82.16%, and −14.33 mV, respectively. The specific saturation magnetism was 8.65 emu/g. In vivo, the concentration of As₂O₃ in the liver was significantly higher than that in the non-magnetic group. While the concentration of As₂O₃ in the kidney was lower than that in the non-magnetic group. The C_max in liver tissue in the magnetic group was 30.65 μg/g, which was 4.17 times the drug concentration in the same group in kidney tissue (7.35 μg/g) and 2.88 times the concentration of drug (10.66 μg/g) in the liver tissue of the non-magnetic group.

Conclusion:

The PLGA polymer-loaded magnetic nanoparticle composed of arsenic trioxide can be magnetically targeted well and applied in biomedicine.     

Arachidonic Acid Activates K+-Cl--cotransport in HepG2 Human Hepatoblastoma Cells

K⁺-Cl^--cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A₂ (PLA₂)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K⁺ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator-induced K⁺ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium-independent PLA₂ (iPLA₂), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF₃) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA₂ (cPLA₂) and the secretory PLA₂ (sPLA₂), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF₃ and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA₂-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.     

Effect of As2O3 on gluconeogenesis

1) The effect of As₂O₃ and As₂O₅ on gluconeogenesis from various substrates in the liver and kidney of rats was investigated. 2) A concentration-dependent inhibition by As₂O₃ was found. The effect was not dependent on the amount of investigated material (hepatocytes or kidney tubules). For either hepatocytes or kidney tubules the extent of inhibition depended strongly on the substrate used. The highest degree of inhibition was observed in incubations with pyruvate. The inhibition of glucose formation was accompanied to a lesser extent by a diminution in O₂ consumption and ATP content. The effect was also dependent on the substrate used. Maximum effect was found in incubations with pyruvate. 3) Oleate, 0.5 mmol/l, increased gluconeogenesis from pyruvate. The effect was not abolished by As₂O₃. 4) A decrease in the content of acetyl-CoA, 3-hydroxybutyrate, and reduced glutathione was found in suspensions of isolated rat kidney tubules or hepatocytes incubated with As₂O₃. 5) About 10 times higher concentrations of As₂O₅ were necessary to induce a similar extent of inhibition of gluconeogenesis, decrease in O₂ consumption, and in ATP content as compared with As₂O₃. The extent of the As₂O₅ effect depended on the concentration of the toxicant and on the substrate used. Gluconeogenesis from pyruvate exhibited the highest sensitivity to As₂O₅. 6) All findings can be largely explained by inhibition of pyruvate dehydrogenase as the central target for arsenicals. The subsequent depletion of acetyl CoA results in impaired formation of reducing equivalents in the citric acid cycle, decrease in high energy phosphates and, acetyl CoA being a strong positive modulator of pyruvate carboxylase, in gluconeogenesis inhibition. Carbohydrate depletion, resulting mainly from gluconeogenesis inhibition, is proposed to be a major problem in poisoning with trivalent arsenicals. In accordance with this proposal, starved rats were shown to be much more sensitive to As₂O₃ than animals with free access to food.     

Arsenic trioxide (As₂O₃) inhibits murine WEHI-3 leukemia in BALB/c mice in vivo

Arsenic trioxide (As₂O₃) is used clinically to treat acute promyelocytic leukemia (APL) and has activity in vitro for induction of apoptosis in several solid tumor cell lines. To investigate the potential therapeutic application of As₂O₃ for leukemia, we analyzed the effects of As₂O₃ on the WEHI‐3 cells‐induced orthotopic leukemia animal model in vivo in this study. We established the WEHI‐3 cells leukemia mice through the injection of murine WEHI‐3 cells into BALB/c mice, and they were then treated with As₂O₃ (0.9 and 4.5 mg kg^?1; p.o.) and/or combined with all‐trans‐retinoic acid (ATRA), (30 mg kg^?1; i.p.). The results indicated that (1) As₂O₃ alone or As₂O₃ combined with ATRA promoted the total survival rate of leukemia mice and these effects are dose‐dependent; (2) As₂O₃ did not affect the body weight but decreased the spleen weight; however, it did not affect liver weight; (3) As₂O₃ alone or As₂O₃ combined with ATRA increased the levels of CD3 and CD19, indicating that the differentiation of T and B cells were promoted; and (4) As₂O₃ alone or As₂O₃ combined with ATRA did not change the levels of Mac‐3 and CD11b markers, indicating that the differentiation of the precursor of macrophage were not inhibited. Based on these observations, As₂O₃ alone or As₂O₃ combined with ATRA have efficacious antileukemia activity in WEHI‐3 cells leukemia in vivo. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Inhibition of TGF-β/SMAD3/NF-κB signaling by microRNA-491 is involved in arsenic trioxide-induced anti-angiogenesis in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Current standard practices for treatment of HCC are less than satisfactory because of metastasis and recurrence, which are primarily attributed to the angiogenesis. So, the anti-angiogenesis treatment has become the new approach for HCC therapy. In addition to treating leukemia, arsenic trioxide (As₂O₃) also suppresses other solid tumors, including HCC. However, the roles of As₂O₃ in the angiogenesis potential of HCC cells remain unclear. In our present study, As₂O₃ attenuated the angiogenic ability by the microRNA-491 (miR-491)-mediated inhibition of TGF-β/SMAD3/NF-κB signal pathway in MHCC97H and MHCC97L cells. Briefly, in these cells, As₂O₃ improved the expression of miR-491 via DNA-demethylation; miR-491, which targeted the SMAD3-3′-UTR, decreased the expression/function of SMAD3, leading to the inactivation of NF-κB/IL-6/STAT-3 signaling; knockdown of miR-491 abolished the As₂O₃-induced inhibitions of the TGF-β/SMAD3/NF-κB pathway, the VEGF secretion, and the angiogenesis. By understanding a novel mechanism whereby As₂O₃ inhibits the angiogenic potential in HCC cells, our study would help in the design of future strategies of developing As₂O₃ as a potential chemopreventive agent when used alone or in combination with other current anticancer drugs.     

Differential mechanistic investigation of protective effects from imperatorin and sec-O-glucosylhamaudol against arsenic trioxide-induced cytotoxicity in vitro

Background and purposeThe clinical use of arsenic trioxide (As₂O₃) for treating acute promyelocytic leukemia (APL) is limited due to its severe cardiotoxicity. The possible mechanisms of As₂O₃-induced cardiotoxicity include DNA fragmentation, reactive oxygen species (ROS) generation, cardiac ion channel changes and apoptosis. The present study is designed to investigate the protective effects of imperatorin and sec-O-glucosylhamaudol and to explore their mechanistic involvement in As₂O₃-induced cytotoxicity.Experimental methodsCell viability assay, Lactate dehydrogenase (LDH) release, Acridine orange/ethidium bromide (AO/EB) double staining, Caspase-3 activity assay, ROS generation, cellular calcium levels, mRNA expression levels by qRT-PCR and protein expression levels by Western blotting were measured in H9c2 cells in combination with As₂O₃ and imperatorin or sec-O-glucosylhamaudol.Key resultsWe observed that H9c2 cells treated with imperatorin or sec-O-glucosylhamaudol were more resistant to As₂O₃-induced cell death. Both imperatorin and sec-O-glucosylhamaudol reduced H9c2 cell apoptosis, but both imperatorin and sec-O-glucosylhamaudol had no effects on Caspase-3 activity and intracellular calcium accumulation. Furthermore, imperatorin was capable of suppressing ROS generation, while sec-O-glucosylhamaudol did not show this effect. Moreover, imperatorin and sec-O-glucosylhamaudol triggered Nrf2 activation, which resulted in upregulation of downstream phase II metabolic enzymes and antioxidant protein/enzyme, probably offering cellular protection to As₂O₃-induced cardiotoxicity via the Nrf2 signal pathway.Conclusions and implicationsImperatorin and sec-O-glucosylhamaudol can ameliorate As₂O₃-induced cytotoxicity and apoptosis in H9c2 cells, the mechanisms probably related to antioxidation. As₂O₃ in combination with imperatorin or sec-O-glucosylhamaudol could be considered as a novel strategy to expand the clinical application of As₂O₃.