Toxic and carcinogenic effects of parenteral and percutaneous ATP and its iron complex

The long term effects of percutaneous, subcutaneous and intraperitoneal administration of sodium-ATP (NaATP) and ferric iron-ATP (FeATP) were studied on an animal model. Both compounds induce a generalized lymphoadenitis which in the case of FeATP led to lymphomas. The analytical study of the involved target tissues showed intracellular composition changes that result from the impairment of the cell membrane permeability. The morbidity and mortality rate were higher with FeATP which seems to be the result of two different, in intensity and duration, interactions with the cell plasma membrane. The influence of the changes in cellular calcium homeostasis, and its relationship with carcinogenesis and immuno response are discussed.     

Pro-apoptotic and microtubule-disassembly effects of ardisiacrispin (A+B), triterpenoid saponins from Ardisia crenata on human hepatoma Bel-7402 cells

Ardisiacrispin (A+B) is a mixture of ardisiacrispins A and B, derived from Ardisia crenata with a fixed proportion (2:1). The present study was conducted to investigate its anticancer activity on human cancer cells and its underlying mechanism of action. The (IC₅₀)s of ardisiacrispin (A+B) on proliferation of several human cancer cell lines were in the range of 0.9–6.5 μg/ml by sulphorhodamine B-based colorimetric assay, in which Bel-7402 was the most sensitive cell line. Moreover, ardisiacrispin (A+B) induced dose-dependent apoptosis in Bel-7402 cells at doses of 1–10 μg/ml by flow cytometry, and resulted in the changes of the mitochondrial membrane depolarization, membrane permeability enhancement, and nuclear condensation in a dose-dependent manner through high-content screening analysis. Furthermore, ardisiacrispin (A+B) could disassemble microtubule in Bel-7402 cells; the fluorescence intensity of microtubules decreased at the concentration of 5–20 μg/ml. These findings suggest that ardisiacrispin (A+B) could inhibit the proliferation of Bel-7402 cells by inducing apoptosis and disassembling microtubule.     

An in vitro comparative study with furyl-1,4-quinones endowed with anticancer activities

We describe the biological activity of some furylbenzo- and naphthoquinones (furylquinones) on hepatocarcinoma cells and healthy rat liver slices. The effects of furylquinones on cancer cells (Transplantable Liver Tumor, TLT) were assessed by measuring cell death (membrane cell lysis); intracellular contents of ATP and GSH and the activity of caspase-3 were used to determine the type of cell death. Most of the furylquinones tested (at a concentration of 25 μg/ml) induced caspase-independent cell death but compound 4 had no cytotoxic effects. The levels of both ATP and GSH were severely affected by quinones 1, 2 and 5, while no effect was observed with compound 4. These cytotoxic properties of quinones are associated with physico-chemical properties as shown by the LUMO energies and lipophilicity. Interestingly, no cytotoxic effects of furylquinones were detected when the in vitro model of precision-cut liver slices (PCLS) was used. Indeed, although CYP2E1 activity was slightly affected, ATP and GSH levels as well as protein synthesis were not modified by furylquinones. Paracetamol, a well-known hepatotoxicant, reduced these parameters by more than 80% compared to control conditions. Taking into account the considerable incidence of adverse-effects induced by most current anticancer drugs, the selective cytotoxicity shown by compounds 1, 2 and 5, in particular that of 1, represents a safety factor that encourages the further development of these quinones as new drugs in cancer therapy.     

Electrophysiological and molecular mechanisms of protection by iron sucrose against phosphine-induced cardiotoxicity: a time course study

Abstract

The present study was designed for determining the exact mechanism of cytotoxic action of aluminum phosphide (AlP) in the presence of iron sucrose as the proposed antidote. Rats received AlP (12?mg/kg) and iron sucrose (5–30?mg/kg) in various sets and were connected to cardiovascular monitoring device. After identification of optimum doses of AlP and iron sucrose, rats taken in 18 groups received AlP (6?mg/kg) and iron sucrose (10?mg/kg), treated at six different time points, and then their hearts were surgically removed and used for evaluating a series of mitochondrial parameters, including cell lipid peroxidation, antioxidant power, mitochondrial complex activity, ADP/ATP ratio and process of apoptosis. ECG changes of AlP poisoning, including QRS, QT, P-R, ST, BP and HR were ameliorated by iron sucrose (10?mg/kg) treatment. AlP initiated its toxicity in the heart mitochondria through reducing mitochondrial complexes (II, IV and V), which was followed by increasing lipid peroxidation and the ADP/ATP ratio and declining mitochondrial membrane integrity that ultimately resulted in cell death. AlP in acute exposure (6?mg/kg) resulted in an increase in hydroxyl radicals and lipid peroxidation in a time-dependent fashion, suggesting an interaction of delivering electrons of phosphine with mitochondrial respiratory chain and oxidative stress. Iron sucrose, as an electron receiver, can compete with mitochondrial respiratory chain complexes and divert electrons to another pathway. The present findings supported the idea that iron sucrose could normalize the activity of mitochondrial electron transfer chain and cellular ATP level as vital factors for cell escaping from AlP poisoning.     

The Effect of Pentoxifylline on Erythrocyte Deformability and on Phosphatide Fatty Acid Distribution in the Erythrocyte Membrane

Summary

Ten maturity-onset diabetics with chronic vascular disease were treated with 400 mg pentoxifylline 3-times daily for 14 days. Erythrocyte deformability (using a filtration technique for whole blood) and phosphatide fatty acid distribution in the erythrocyte membrane were measured before and after the treatment period. Statistical analysis of the data showed that the erythrocyte filtration rate had increased significantly by the end of treatment (2a. = 0.02), and that there were only slight changes in erythrocyte membrane phosphatide fatty acid levels. The drug was well tolerated, and there were no adverse laboratory findings in the parameters measured. On the basis of results described by other investigators, the improvement in erythrocyte deformability was attributed to an increase in erythrocyte ATP levels. The authors discuss the importance of red cell fluidity for capillary perfusion.     

Correlations Among mRNA Expression Levels of ATP7A,Serum Ceruloplasmin Levels,and Neuronal Metabolism in Unmedicated Major Depressive Disorder

BackgroundPrevious studies have found that elevated copper levels induce oxidation, which correlates with the occurrence of major depressive disorder (MDD). However, the mechanism of abnormal cerebral metabolism of MDD patients remains ambiguous. The main function of the enzyme ATPase copper-transporting alpha (ATP7A) is to transport copper across the membrane to retain copper homeostasis, which is closely associated with the onset of mental disorders and cognitive impairment. However, less is known regarding the association of ATP7A expression in MDD patients.MethodsA total of 31 MDD patients and 21 healthy controls were recruited in the present study. Proton magnetic resonance spectroscopy was used to assess the concentration levels of N-acetylaspartate, choline (Cho), and creatine (Cr) in brain regions of interest, including prefrontal white matter (PWM), anterior cingulate cortex (ACC), thalamus, lentiform nucleus, and cerebellum. The mRNA expression levels of ATP7A were measured using polymerase chain reaction (SYBR Green method). The correlations between mRNA expression levels of ATP7A and/or ceruloplasmin levels and neuronal biochemical metabolite ratio in the brain regions of interest were evaluated.ResultsThe decline in the mRNA expression levels of ATP7A and the increase in ceruloplasmin levels exhibited a significant correlation in MDD patients. In addition, negative correlations were noted between the decline in mRNA expression levels of ATP7A and the increased Cho/Cr ratios of the left PWM, right PWM, and right ACC in MDD patients. A positive correlation between elevated ceruloplasmin levels and increased Cho/Cr ratio of the left PWM was noted in MDD patients.ConclusionsThe findings suggested that the decline in the mRNA expression levels of ATP7A and the elevated ceruloplasmin levels induced oxidation that led to the disturbance of neuronal metabolism in the brain, which played important roles in the pathophysiology of MDD. The decline in the mRNA expression levels of ATP7A and the elevated ceruloplasmin levels affected neuronal membrane metabolic impairment in the left PWM, right PWM, and right ACC of MDD patients.     

ABC transporters at the blood–brain barrier

Introduction: The blood–brain barrier (BBB) possesses an outstanding ability to protect the brain against xenobiotics and potentially poisonous metabolites. Owing to this, ATP binding cassette (ABC) export proteins have garnered significant interest in the research community. These transport proteins are predominantly localized to the luminal membrane of brain microvessels, where they recognize a wide range of different substrates and transport them back into the blood circulation.

Areas covered: This review summarizes recent findings on these transport proteins, including their expression in the endothelial cell membrane and their substrate recognition. Signaling cascades underlying the expression and function of these proteins will be discussed as well as their role in diseases such as Alzheimer’s disease, epilepsy, amyotrophic lateral sclerosis and brain tumors.

Expert opinion: ABC transporters represent an integral part of the human transportome and are of particular interest at the blood–brain barrier they as they significantly contribute to brain homeostasis. In addition, they appear to be involved in myriad CNS diseases. Therefore studying their mechanisms of action as well as their signaling cascades and responses to internal and external stimuli will help us understand the pathogenesis of these diseases.     

Metabolic effects of the HIV protease inhibitor – saquinavir in differentiating human preadipocytes

BackgroundThe iatrogenic, HIV-related lipodystrophy is associated with development of the significant metabolic and cardiovascular complications. The underlying mechanisms of antiretroviral (ARV) drugs are not completely explored.MethodsThe aim of the study was to characterize effects of the protease inhibitor (PI) – saquinavir (SQV) on metabolic functions, and gene expression during differentiation in cells (Chub-S7) culture.ResultsSQV in concentrations observed during antiretroviral therapy (ART) significantly decreased mitochondrial membrane potential (MMP), oxygen consumption and ATP generation. The effects were greater in already differentiated cells. This was accompanied by characteristic changes in the expression of the genes involved in endoplasmic reticulum (ER) stress, and differentiation (lipid droplet formation) process such as: WNT10a, C/EBPα, AFT4, CIDEC, ADIPOQ, LPIN1.ConclusionsThe results indicate that SQV affects not only metabolic (mitochondrial) activity of adipocytes, but affects the expression of genes related to differentiation and to a lesser extent to cell apoptosis.     

Cobra (Naja Naja Atra) Membrane Toxin Isoforms: Structure and Function

Abstract

Two membrane toxins, termed MT-I and MT-II, were purified to HPLC homogeneity from the venom of Naja naja atra. The NH₂-terminal sequences of the two isoforms were determined. When compared with the known sequences of membrane toxins, we concluded they are CTX-I and CTX-III (from Naja naja atra), respectively. Membrane toxins are basic peptides typified by a chain of 60 amino acids long. Their pi is about 10 and Mr is 6,000-7,000. About half of the amino acids are hydrophobic.

There is lytic synergism between membrane toxins and phospholipase A₂. Membrane toxins, which are different from neurotoxins, are capable of depolarizing muscle cells. The toxins are able to kill cancer cells in vitro. Electrocardiograph of cat to which membrane toxin was applied shows magnificent changes. A positive correlation exists between hydrophobicities and activities of the toxins to inhibit protein kinase C (PKC) activity. All the effects are the result of action of the toxins on cell membranes. In addition, structure-activity relationships are investigated with the available comparative data for membrane toxins centering on LD₅₀, erythrocyte lysis, and muscle cell depolarization.     

Toxicity of thallium on isolated rat liver mitochondria: The role of oxidative stress and MPT pore opening

Thallium(I) is a highly toxic heavy metal; however, up to now, its mechanisms are poorly understood. The authors' previous studies showed that this compound could induce reactive oxygen species (ROS) formation, reduced glutathione (GSH) oxidation, membrane lipid peroxidation, and mitochondrial membrane potential (MMP) collapse in isolated rat hepatocyte. Because the liver is the storage site of thallium, it seems that the liver mitochondria are one of the important targets for hepatotoxicity. In this investigation, the effects of thallium on mitochondria were studied to investigate its mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with different concentrations of thallium (25–200 µM). Thallium(I)‐treated mitochondria showed a marked elevation in oxidative stress parameters accompanied by MMP collapse when compared with the control group. These results showed that different concentrations of thallium (25–200 µM) induced a significant (P < 0.05) increase in mitochondrial ROS formation, ATP depletion, GSH oxidation, mitochondrial outer membrane rupture, mitochondrial swelling, MMP collapse, and cytochrome c release. In general, these data strongly supported that the thallium(I)‐induced liver toxicity is a result of the disruptive effect of this metal on the mitochondrial respiratory complexes (I, II, and IV), which are the obvious causes of metal‐induced ROS formation and ATP depletion. The latter two events, in turn, trigger cell death signaling via opening of mitochondrial permeability transition pore and cytochrome c expulsion. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 232–241, 2015.     

吉西他滨介导胰腺癌细胞三磷酸腺苷结合转运体G2质膜移位诱发耐药的研究

目的探讨三磷酸腺苷结合转运体G2(ABCG2)与胰腺癌采用吉西他滨化疗抵抗的相互关系。方法观察胰腺癌手术患者的癌和癌旁组织免疫组化及胰腺癌细胞系中ABCG2的表达,流式细胞术测定胰腺癌细胞系在吉西他滨刺激0、3、12、24、48、72 h后的ABCG2膜表达结果,选择高表达水平的PANC-1细胞株进行相关机制试验。观察吉西他滨作用0、15、30、45、120、180 min后P-AKT表达水平,及用PI3K/AKT抑制剂LY294002或AKT si RNA阻断AKT表达后ABCG2表达水平和生存率变化。免疫荧光共聚焦技术观察在对照、吉西他滨、LY294002和吉西他滨+LY294002 4种实验条件下细胞膜膜表面的ABCG2蛋白表达变化。结果 ABCG2蛋白表达量在胰腺癌组织中较癌旁组织明显增高,ABCG2+细胞在不同病理分化程度的胰腺癌细胞中均有表达。胰腺癌细胞系在吉西他滨刺激后总ABCG2水平无明显变化,而ABCG2+细胞数有明显增加。PANC-1在吉西他滨刺激后细胞总ABCG2蛋白表达无明显变化;p-AKT蛋白表达水平呈现增加趋势;ABCG2+细胞数明显增加,并呈时间相关性。LY294002或LY294002+吉西他滨处理细胞后ABCG2总蛋白表达无明显变化,si RNA-AKT2基因敲除或LY294002处理后,肿瘤细胞对吉西他滨的敏感性明显增加。免疫荧光共聚焦表明吉西他滨调节胰腺癌PI3K/AKT信号分子,介导ABCG2质膜移位。结论吉西他滨活化PI3K/AKT信号分子,促进ABCG2质膜移位,抑制胰腺癌化疗敏感性。     

Kaurane diterpenes as mitochondrial alterations preventive agents under experimental oxidative stress conditions

Context: Foliol, linearol, and sidol are the most common diterpenes found in Sideritis L. spp. (Lamiaceae) with a wide range of demonstrated properties including anti-inflammatory, antioxidant, and anti-apoptotic effects.

Objective: For the first time, the present work was studied for the potential protective role of these kaurane-type diterpenes on mitochondrial oxidative stress induced by H₂O₂ in the human astrocytoma U373-MG cell line and in the rat adrenal pheochromocytoma PC12 cell line.

Materials and methods: Mitochondrial protection was assayed at 5 and 10?µM concentrations for 24?h (for kaurane diterpenes) and H₂O₂ as oxidative stress inducer (0.1?mM for PC12 cells and 1?mM for U373-MG, for 30?min). ATP concentration was determined by high-performance liquid chromatography (HPLC), and changes in mitochondrial membrane potential, caspase-3 activity as well as in cytosolic and mitochondrial calcium levels were assessed by fluorometric techniques, by using specific fluorescent probes.

Results: Pretreatments for 24?h with linearol and sidol, prior to H₂O₂ exposure, acted as mitochondrial alterations preventive agents by increasing membrane potential (over 40–60% in PC12 cells and over 10–20% in U373-MG), restoring both cytosolic and mitochondrial calcium homeostasis (linearol at 10?µM caused a 3.5-fold decrease in cytosolic calcium concentration in PC12 cells), decreasing caspase-3 activity (over 1.25–1.5-fold for linearol and sidol) and avoiding ATP depletion (linearol increased over 20% ATP level in both cell types).

Conclusion: Our results suggest that linearol and sidol could provide protective activity by targeting mitochondria in response to the deleterious changes induced by H₂O₂.     

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na⁺-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na⁺-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na⁺-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na⁺ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na⁺-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A₂ (cPLA₂). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA₂ specific inhibitor AACOCF₃. ATP depletion-induced alterations in Na⁺-dependent phosphate uptake and cell viability were prevented by AACOCF₃. Inhibition of Na⁺-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA₂ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.     

Underlying mechanisms of cytotoxicity in HepG2 hepatocarcinoma cells exposed to arsenic,cadmium and mercury individually and in combination

BackgroundToxicity data regarding combinational exposure of humans to arsenic, cadmium and mercury is scarce. Although hepatotoxicity has been reported, limited information is available on their mechanistic underpinnings. The cytotoxic mechanisms of these metals were determined in HepG2 hepatocarcinoma cell lines after individual and combinational exposure.MethodsHepG2 cells were exposed to heavy metals (sodium arsenite, cadmium chloride, and mercury chloride) individually or in combination for 24 h, after which cell density, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), reduced glutathione (GSH), adenosine triphosphate (ATP) and caspase-3/7 activity was assessed.Results and discussionCadmium (IC₅₀ = 0.43 mg/L) and the combination (0.45 mg/L, arsenic reference) were most cytotoxic, followed by arsenic (6.71 mg/L) and mercury (28.23 mg/L). Depolarisation of the ΔΨm and reductions in ROS, GSH and ATP levels occurred. Arsenic, cadmium and the combination increased caspase-3/7 activity, while mercury reduced it.ConclusionThe combination produced a greater, albeit mechanistically similar, cytotoxicity compared to individual metals. Cytotoxicity was dependent on altered mitochondrial integrity, redox-status, and bioenergetics. Although the combination's cytotoxicity was associated with caspase-3/7 activity, this was not true for mercury. Heavy metal interactions should be assessed to elucidate molecular underpinnings of cytotoxicity.     

Toxicity of new synthetic amphetamine drug mephedrone On Rat Heart mitochondria: a warning for its abuse

Abstract

1.?Mephedrone, a new and popular amphetamine drug, is widely abused and is still legal in some parts around the world. Little data on mechanisms involved in mephedrone induced cardiotoxicity are available.

2.?Therefore, we decided to explain the mechanisms of mephedrone cardiotoxicity by using mitochondria isolated from rat heart. The isolated heart mitochondria were incubated with different concentrations of mephedrone (5, 10 and 20?µM).

3.?Results showed that mephedrone induced mitochondrial dysfunction via an increase in mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) collapse, mitochondrial swelling and damage in the mitochondrial outer membrane (MOM) which is associated with the cytochrome c release. Our results showed that decrease of ATP levels is an indicator of disturbance in oxidative phosphorylation. Also, mephedrone increased the caspase-3 activity.

4.?According to the results, we suggest that mephedrone induced cardiotoxicity is the result of a disruptive effect on the mitochondrial respiratory chain and induction of ROS-mediated apoptosis signaling in heart cardiomyocytes.     

Melatonin-induced augmentation of collagen deposition in cultures of fibroblasts and myofibroblasts is blocked by luzindole – a melatonin membrane receptors inhibitor

BackgroundMelatonin has been proven to have a regulatory influence on collagen accumulation in different types of wound. It was found to inhibit collagen accumulation in the superficial wound model but increase it in the myocardial infarction scar. The aim of the study is to determine the mechanism of melatonin action in the two wound types in rats.MethodsCells were isolated from both the superficial wound (subcutaneously inserted polypropylene net) and myocardial infarction scar (induced by ligation of the left coronary artery) and were identified by electron microscopy.ResultsLong-shaped cells forming whirl-like structures in culture (mainly identified as fibroblasts) were isolated from the superficial wound model, while myofibroblasts growing in a formless manner were acquired from the infarcted heart scar. Melatonin (10^–7 M) increased collagen accumulation in both fibroblast and myofibroblast cultures. Luzindole (10^–6 M), the blocker of both MT₁ and MT₂ melatonin membrane receptors, inhibited the effect of melatonin on the two types of cells.ConclusionRegardless of various healing potentials demonstrated by the tested cells (different cell composition, growth and organization), their response to melatonin was similar. Moreover, in the two investigated cultures, augmentation of the collagen content by melatonin was reversed by luzindole, which indicates the possibility of melatonin membrane receptor involvement in that process. The present results suggest that the increased melatonin-stimulated deposition of collagen observed in the infarcted heart of rats could be dependent on activation of the melatonin membrane receptors on scar myofibroblasts.     

Trends in alternative tobacco use among light, moderate, and heavy smokers in adolescence, 1999-2009

ObjectiveTo examine trends in alternative tobacco product (ATP) use (smokeless tobacco, cigars, and bidis/cloves) among a national sample of adolescent cigarette smokers (light, moderate, and heavy) during 1999–2009.MethodA secondary analysis of data from the 1999–2009 National Youth Tobacco Survey was performed to investigate the tobacco behaviors of 6th through 12th graders enrolled in public and private schools in the United States. Long-term trends in ATP use were analyzed using logistic regression – controlling for sex, grade, and race/ethnicity – and simultaneously assessing linear and higher order time effects and their interaction with cigarette smoking status.ResultsDuring 1999–2009, increases in smokeless tobacco use and decreases in bidis/cloves use were observed across all smoking groups. For cigars, declines were observed for heavy and moderate smokers, but levels returned to baseline levels in 2009. Cigar use among light smokers was less variable. Rates of any ATP were highest among heavy smokers and lowest among light smokers.ConclusionTrends in cigarette and SLT use increased dramatically in the past decade, and this increase is evident across all cigarette smoker types. Implications for tobacco surveillance, prevention and cessation programs, and tobacco control policies are discussed.     

CEREBROVASCULAR AND METABOLIC EFFECTS OF BRADYKININ, ATP AND HYPERCAPNIA IN THE GOAT

1. Cerebral blood flow (CBF) and cerebral O₂ metabolism (CMRO₂) changes were measured in these studies during treatment with three reputed cerebral vasodilators: bradykinin, adenosine triphosphate (ATP) and increased arterial Pco₂. 2. Intracerebrovascular bradykinin infusions increased CMRO₂ and CBF to a similar degree. Intramaxillary ATP infusions increased CBF to an extent greater than that required for increases seen in CMRO₂. Hypercapnia increased CBF without a significant change in CMRO₂. 3. Results suggest that increases in cerebral metabolism may mediate most, some and none of the cerebrovascular changes produced by bradykinin, ATP or hypercapnia, respectively.