Drug-induced mitochondrial dysfunction and cardiotoxicity.

Mitochondria has an essential role in myocardial tissue homeostasis; thus deterioration in mitochondrial function eventually leads to cardiomyocyte and endothelial cell death and consequent cardiovascular dysfunction. Several chemical compounds and drugs have been known to directly or indirectly modulate cardiac mitochondrial function, which can account both for the toxicological and pharmacological properties of these substances. In many cases, toxicity problems appear only in the presence of additional cardiovascular disease conditions or develop months/years following the exposure, making the diagnosis difficult. Cardiotoxic agents affecting mitochondria include several widely used anticancer drugs [anthracyclines (Doxorubicin/Adriamycin), cisplatin, trastuzumab (Herceptin), arsenic trioxide (Trisenox), mitoxantrone (Novantrone), imatinib (Gleevec), bevacizumab (Avastin), sunitinib (Sutent), and sorafenib (Nevaxar)], antiviral compound azidothymidine (AZT, Zidovudine) and several oral antidiabetics [e.g., rosiglitazone (Avandia)]. Illicit drugs such as alcohol, cocaine, methamphetamine, ecstasy, and synthetic cannabinoids (spice, K2) may also induce mitochondria-related cardiotoxicity. Mitochondrial toxicity develops due to various mechanisms involving interference with the mitochondrial respiratory chain (e.g., uncoupling) or inhibition of the important mitochondrial enzymes (oxidative phosphorylation, Szent-Györgyi-Krebs cycle, mitochondrial DNA replication, ADP/ATP translocator). The final phase of mitochondrial dysfunction induces loss of mitochondrial membrane potential and an increase in mitochondrial oxidative/nitrative stress, eventually culminating into cell death. This review aims to discuss the mechanisms of mitochondrion-mediated cardiotoxicity of commonly used drugs and some potential cardioprotective strategies to prevent these toxicities.

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.

Large scale identification of protein SUMOylation by mass spectrometry in HEK293 cells

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Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 mu M 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 mu M ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five mu M ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5X), perifosine (3X), and arsenic trioxide (8.5X). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019661, 1898-1912, 2012.

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 mu mol/mL and < 6.0 mu mol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL. (Blood. 2012;120(1):207-213)

Molecular mechanisms of apoptosis induction in chronic lymphocytic leukemia

CLL is the most common adult leukemia in the Western World, yet very little is known about the biology of this disease. CLL cells have very high levels of NF-κB activity. Factors such as CD40 ligation and phorbol ester treatment induce NF-κB activity and also prevent apoptosis. Previous data from our laboratory demonstrated that MG-132, a proteasome inhibitor, blocked NF-κB activation and promoted apoptosis in CLL cells. These data suggested to us that NF-κB mediates survival in CLL. We examined NF-κB activity using two different chemotherapeutic agents, PS-341 and arsenic trioxide. PS-341, a proteasome inhibitor blocked NF-κB in CLL cells. This however, did not correlate with cell death. Resistant patient isolates displayed delayed Smac/DIABLO release in comparison to cytochrome c release. This suggests that IAPs are contributing to CLL cell survival and drug-resistance. Arsenic trioxide did not block NF-κB activity at therapeutic doses. However it was a potent inducer of apoptosis in CLL cells. We identified a novel mechanism by which arsenic induces increases in mitochondrial calcium to induce cytochrome c release and initiate apoptosis. Both PS-341 and arsenic trioxide are currently in Phase II clinical trials at M.D. Anderson Cancer Center. We conclude that NF-κB is not critical for PS-341 or arsenic trioxide-mediated cell death. ^

Anti-proliferative effects of novel Glyco-lipid-arsenicals (III) on MCF-7 human breast cancer cells

Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)2 group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 μM and 10 μM compound (11) and 10 μM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/ or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness. © 2006 Bentham Science Publishers Ltd.

Studies on the behaviour of Reticulitermes santonensis (Feytaud) in laboratory colonies and its implications for some methods of termite control

An investigation of behavioural patterns that form a basis for termite control in the Australasian region was undertaken using laboratory colonies of the subterranean termite Reticulitermes santonensis (Feytaud). The study attempted to build a picture of the behavioural elements of individuals in a colony and based on this, trophallaxis, aggression and cannibalism were investigated in detail. Preliminary study of food transmission showed that 'workers' played a major role in the distribution of food. It was found, that among factors responsible for release of trophallactic behaviour the presence of 'right odour' between participants was important. It also appeared that the role taken by individuals depended on whether they were hungry or fully fed. Antennal palpation was shown by donors and acceptors alike and this seemed to be excitatory in function. Introduction of aliens into nests elicited aggression and these aliens were often killed. Factors eliciting aggression were investigated and colony odour was found to be important. Further investigations revealed that development of colony odour was governed by genetical and environmental mechanisms. Termite response to injury and death was also governed by odour. In the case of injury either the fresh haemolymph from the wound or some component of the haemolymph evoked cannibalism. Necrophagic behaviour was found to be released by fatty acids found in the corpses. Finally, the response of colonies to nestmates carrying arsenic trioxide was investigated. It was found that living and freshly dead arsenic-carrying nestmates were treated like normal nestmates, resulting in high initial mortality. However, poisoned cadavers soon became repellant and were buried thus preventing further spread of the poison to the rest of the colony. This suggested that complete control of subterranean termites by arsenic trioxide is unlikely to be fully effective, especially in those species which are capable of developing secondary reproductives from survivors and thus rebuilding the community.

Presence of arsenic in different types of MTA and white and gray Portland cement

Objective. The presence of arsenic in various types of mineral trioxide aggregate (MTA) and Portland cements were evaluated to verify if they comply with the ISO-recommended limit for water-based cements of 2 mg arsenic/kg material. Study design. An amount of 5 mL of hydrochloric acid was added to 2 g each of MTA and Portland cement to be analyzed. After 15 minutes, the material was filtered and the volume of supernatant was diluted with reagent-grade water up to 40 mL. Atomic absorption spectrophotometry readings were performed in triplicate. Results. The following mean values were obtained: CPM (Egeo, Buenos Aires, Argentina) 11.06 mg/kg; CPM sealer (Egeo) 10.30 mg/kg; MTA- Obtura (Angelus, Londrina, PR, Brazil) 0.39 mg/kg; Experimental MTA: 10.30 mg/kg; White MTA- Angelus (Angelus) 1.03 mg/kg; Gray MTA- Angelus (Angelus) 5.91 mg/kg; ProRoot-MTA (Dentsply/Tulsa Dental Specialties, Tulsa, OK) 5.25 mg/kg; Gray Portland cement (Votorantim Cimentos, Cubatao, SP, Brazil): 34.27 mg/kg; and White Portland cement (Cimento Rio Branco, Rio de Janeiro, RJ, Brazil) 0.52 mg/kg. Conclusion. All tested materials presented arsenic in their composition. The form of arsenic was not analyzed nor the toxicity of the arsenic found. Only MTA- Obtura, White MTA- Angelus, and White Portland cement presented arsenic levels below the limit set in the ISO 9917-1 standard. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106: 909-913)

Evaluation of physico-chemical properties of Portland cements and MTA

The purpose of this study was to evaluate the hydrogenionic potential and electrical conductivity of Portland cements and MTA, as well as the amount of arsenic and calcium released from these materials. In Teflon molds, samples of each material were agitated and added to plastic flasks containing distilled water for 3, 24, 72 and 168 h. The results were analyzed with a Kruskal-Wallis non-parametric test for global comparisons and a Dunn-Tukey test for pairwise comparisons. The results revealed no significant differences in the pH of the materials (p > 0.05). The electrical conductivity of the cements were not statistically different (p > 0.05). White non-structural cement and MTA BIO released the largest amount of calcium ions into solution (p < 0.05), while arsenic release was insignificant in all of the materials (p > 0.05). The results indicated that the physico-chemical properties of Portland cements and MTA were similar. Furthermore, all materials produced an alkaline environment and can be considered safe for clinical use because arsenic was not released. The electrical conductivity and the amount of calcium ions released into solution increased over time.

Arsenic and Chromium in Brazilian Agricultural Supplies

Arsenic (As) and chromium (Cr) contents were measured in agricultural supplies used at different farms in Sao Paulo State, Brazil. The highest mass fractions of As were found in thermophosphates, reaching levels of 4 mg/kg. The highest mass fractions of Cr (21 g/kg) were found in calcium magnesium silicate, while the thermophosphates also presented high values reaching approximately 1 g/kg. The levels of As were within Brazilian guidelines, but the values of Cr in thermophosphates exceeded the levels permitted in Brazil. The As content in fertilizers may be considered safe (5 mg/kg) in terms of environmental pollution. However, the Cr content in calcium magnesium silicate following continuous use may constitute a significant problem in Brazil due to potentially increasing levels of this metal in soils.

Sequential Injection Analysis (SIA) for Arsenic Speciation by Capillary Electrophoresis Hyphenated to Inductively Coupled Plasma Sector Field Mass Spectrometry (CE-ICP-SFMS)

Sequential injection analysis (SIA) is proposed for managing microvolumes of sample and arsenic species solutions for speciation analysis by capillary electrophoresis focusing on the reduction of hazardous waste residues. An electronically controlled hydrodynamic injector was projected to introduce microvolumes of solutions prepared by SIA into the CE capillary with precision better than 2%. The determination of arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine was performed from 50 mu L volumes of lyophilized urine and extract of shrimp with the system hyphenated to inductively coupled plasma mass spectrometry (CE-ICP-SFMS).

Influence of Redox Environments on the Solubility of Arsenic in Soils

Arsenic (As) is a semimetallic element that is notorious for its toxicity and carcinogenicity. Arsenic can be removed by some ferns. The objectives of this study were to investigate the ability of Pteris vittata L. (Pteridophyta) and Phlebodium aureum (L.) J. Sm. (Polypodiaceae) to absorb inorganic As, in the form of arsenate and arsenite. The removal of As by ferns was observed at varying anion concentrations and As solubility in the absorbing plant. Results obtained with ferns on As-contaminated soil indicate that redox potential and iron (Fe) presence affected the solubility of As and the absorption capacity of ferns. Upon reduction to -200mV, the soluble As content increased to 400mV. The results indicate that Fe oxides and the influence of redox potential strongly affect As absorption. Under nonreducing conditions, Phlebodium aureum did not remove As as well as Pteris vittata. Under more reducing conditions (-200 to 0mV) and under similar soil conditions, the results show that the both ferns remove As.