Dušan Šuput

Achieving the Balance between ROS and Antioxidants: When to Use the Synthetic Antioxidants

Free radical damage is linked to formation of many degenerative diseases, including cancer, cardiovascular disease, cataracts, and aging. Excessive reactive oxygen species (ROS) formation can induce oxidative stress, leading to cell damage that can culminate in cell death. Therefore, cells have antioxidant networks to scavenge excessively produced ROS. The balance between the production and scavenging of ROS leads to homeostasis in general; however, the balance is somehow shifted towards the formation of free radicals, which results in accumulated cell damage in time. Antioxidants can attenuate the damaging effects of ROS in vitro and delay many events that contribute to cellular aging. The use of multivitamin/mineral supplements (MVMs) has grown rapidly over the past decades. Some recent studies demonstrated no effect of antioxidant therapy; sometimes the intake of antioxidants even increased mortality. Oxidative stress is damaging and beneficial for the organism, as some ROS are signaling molecules in cellular signaling pathways. Lowering the levels of oxidative stress by antioxidant supplements is not beneficial in such cases. The balance between ROS and antioxidants is optimal, as both extremes, oxidative and antioxidative stress, are damaging. Therefore, there is a need for accurate determination of individuals oxidative stress levels before prescribing the supplement antioxidants.

Nephrotoxic effects of chronic administration of microcystins -LR and -YR

Acute intoxication with MC-LR induces cytoskeletal alterations, apoptosis and necrosis of hepatocytes resulting in intrahepatic hemorrhage. Preliminary results have shown that chronic treatment of rats with intraperitoneal injections of sublethal doses of microcystins MC-LR and MC-YR could induce not only liver, but also kidney injuries. We aimed to investigate whether the induction of the cytoskeletal changes, apoptosis and necrosis could be the mechanisms involved in the injury of kidney cells in the chronic model of microcystin intoxication. Experimental rats were receiving intraperitoneal injections of MC-LR (10 microg/kg) or MC-YR (10 microg/kg) every second day for 8 months, while control rats were receiving only the vehicle. The histopathological investigation revealed collapsed glomeruli with thickened basement membranes and dilated tubuli filled with eosinophilic casts. Rhodamine-phalloidin labeling showed cytoplasmic aggregation and accumulation of fibrilar actin filaments within the epithelial tubular cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed increased number of TUNEL-positive cells in the kidney cortex and medulla. The pathological changes induced by MC-LR appeared more severe than those induced by MC-YR. The results support the view that at the cellular level, the mechanisms that underly the chronic nephrotoxicity are similar to the mechanisms of the acute hepatotoxicity of microcystins.

Microcystin-LR causes the collapse of actin filaments in primary human hepatocytes

Microcystin-LR (MCLR) is a potent inhibitor of protein phosphatases 1 and 2A and causes alterations in cytoskeletal filaments and morphological changes that underlie apoptosis in rat hepatocytes. It has also been reported that it caused several cases of human deaths and illness. As no study on the effect of microcystins on human hepatocytes was done, yet, the aim of the study is to evaluate the toxicity of MCLR on primary human hepatocytes. The hepatocytes were incubated in 12.5-50 nM MCLR for 3, 6 and 9 h, fixed and stained with fluorescent probes for actin filaments and nuclei. Spectral laser-scanning confocal microscopy revealed that in the MCLR-treated primary human hepatocytes the actin mesh collapsed into the center of the cell, similarly as it has been described for rat hepatocytes. Cells were blebbing, fragmenting, and separated from each other. The nuclei in the affected cells condensed. In conclusion, this study confirms that MCLR is toxic to primary human hepatocytes, and it may be responsible for the liver failure cases observed after acute cyanobacterial poisoning.

In vivo effects of cnidarian toxins and venoms

Cnidarians (Coelenterates), a very old and diverse animal phylum, possess a wide variety of biologically active substances that can be considered as toxins. Anthozoan toxins can be classified into two chemically very different groups, namely polypeptide toxins isolated from sea anemones and diterpenes isolated from octocorals. Cubozoan and scyphozoan protein toxins have been the most elusive cnidarian toxins to investigate - despite a tremendous effort in the past few decades, very few of these large, relatively unstable protein toxins were isolated, but recently this has been achieved for cubozoan venoms. Hydrozoans mainly contain large proteins with physiological mechanisms of action similar to the sea anemone and jellyfish pore-forming toxins. This article will focus on the in vivo physiological effects of cnidarian toxins and venoms; their actions at the cellular level will only be considered to understand their actions at the organ and whole animal levels. An understanding of mechanisms underlying the in vivo toxic effects will facilitate the development of more effective treatments of cnidarian envenomations.

Inhibition of acetylcholinesterase by an alkylpyridinium polymer from the marine sponge, Reniera sarai

Large polymeric 3-alkylpyridinium salts have been isolated from the marine sponge Reniera sarai. They are composed of N-butyl(3-butylpyridinium) repeating subunits, polymerized head-to-tail, and exist as a mixture of two main polymers with molecular weights without counterion of about 5520 and 18900. The monomer analogue of the inhibitor, N-butyl-3-butylpyridinium iodide has been synthesized. This molecule shows mixed reversible inhibition of acetylcholinesterase. The polymers also act as acetylcholinesterase inhibitors and show an unusual inhibition pattern. We tentatively describe it as quick initial reversible binding, followed by slow binding or irreversible inhibition of the enzyme. This kinetics suggests that there are several affinity binding sites on the acetylcholinesterase molecule where the polymer can bind. The first binding favors binding to other sites which leads to an apparently irreversibly linked enzyme-inhibitor complex.

Adaptive response, evidence of cross-resistance and its potential clinical use.

Organisms and their cells are constantly exposed to environmental fluctuations. Among them are stressors, which can induce macromolecular damage that exceeds a set threshold, independent of the underlying cause. Stress responses are mechanisms used by organisms to adapt to and overcome stress stimuli. Different stressors or different intensities of stress trigger different cellular responses, namely induce cell repair mechanisms, induce cell responses that result in temporary adaptation to some stressors, induce autophagy or trigger cell death. Studies have reported life-prolonging effects of a wide variety of so-called stressors, such as oxidants, heat shock, some phytochemicals, ischemia, exercise and dietary energy restriction, hypergravity, etc. These stress responses, which result in enhanced defense and repair and even cross-resistance against multiple stressors, may have clinical use and will be discussed, while the emphasis will be on the effects/cross-effects of oxidants.

Effects of equinatoxin II from Actinia equina (L.) on isolated rat heart: The role of direct cardiotoxic effects in equinatoxin II lethality

Equinatoxin II is a lethal basic protein isolated from the sea anemone Actinia equina (L.) with LD50 in mice 35 microg/kg. The putative cause of death is cardiorespiratory arrest, but the mechanism of cardiotoxicity is poorly understood. It is not clear whether the toxin injected intravenously into an experimental animal reaches the heart in a concentration sufficient to cause direct effects on the heart. Therefore experiments were performed on rats and on isolated rat hearts in order to investigate the possible direct cardiotoxic effects of the toxin. For this reason the hearts were perfused with different concentrations of the toxin and with the effluent from the lungs collected during perfusion of the lungs with equinatoxin II. The results revealed the clear dose-dependent, direct cardiotoxic effects of the toxin and of the effluent from the lungs on Langendorffs heart preparations. The threshold concentration of equinatoxin II causing a drop in the perfusion rate, decreased left ventricular pressure, arrhythmia and increased LDH release, was found to be around 0.1 to 1 nM. With 10 nM equinatoxin II the left ventricular pressure dropped to 14+/-11% of the control, and the coronary flow to 9+/-3%. These effects were followed by arrhythmia and cardiac arrest. The concentration of equinatoxin recovered from the lungs after the perfusion with 100 nM equinatoxin II ranged between 0.8 and 5 nM. The results indicate that direct cardiotoxic effects of equinatoxin II play an important role in the lethal effects of the toxin.

Inhibition of acetylcholinesterase by a pseudozoanthoxanthin-like compound isolated from the zoanthid Parazoanthus axinellae (O. Schmidt).

An ethanolic extract from a zoanthid crust coral Parazoanthus axinellae was lethal to mice and crabs and exhibited anticholinesterase activity. The isolation of several AChE inhibitors with the aid of RP-HPLC is described. The most abundant of the inhibitors present in the P. axinellae extract was identified as pseudozoanthoxanthin or an almost identical compound which belongs to the chemically well-characterized series of tetrazacyclopentazulene natural pigments from the genera Parazoanthus, Epizoanthus, Zoanthus and Palythoa. The inhibitor has a mol. wt of 242 and acts as a competitive inhibitor with a Ki of 4 microM. The inhibitor exhibited a strong blue fluorescence. In vivo action of crude extract and the isolated inhibitor showed a typical picture of systemic acetylcholinesterase inhibition. Atropinization of experimental animals prior to injection of the inhibitor almost entirely neutralized its activity.

Ca2+ and Na+ contribute to the swelling of differentiated neuroblastoma cells induced by equinatoxin-II

Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.

DMSO modulates the pathway of apoptosis triggering

We demonstrate here that distribution of caspase-9 influences the pathway of apoptosis triggering, since caspase-9 is activated efficiently only when it is distributed solely in the cytosol. Caspase-9 moves to the nuclei in a response to cell stress during isolation of primary hepatocytes; this is called preapoptotic cell stress response. The dimethyl sulfoxide (DMSO) treatment cannot prevent the migration of caspase-9 into the nuclei when it is added to primary hepatocytes immediately after isolation; however, it can trigger redistribution of caspase-9 from the nuclei into the cytosol when added 1 day post-isolation. This redistribution is temporary, since caspase-9 returns to the nuclei within 48 hours of DMSO treatment. Thereafter, some caspase-9 is retained in the nuclei of DMSO-treated hepatocytes for longer than in the nuclei of untreated hepatocytes. By measuring caspase activities, we demonstrate that the addition of DMSO to cell culture medium can temporarily normalize the susceptibility of hepatocytes for apoptosis triggering through the intrinsic pathway. DMSO contributes also to the prolonged pathway inactivation, i.e., by extending preapoptotic cell stress response. We propose that DMSO extends the survival of primary hepatocytes by modulating preapoptotic cell stress response, which could be exploited for extending the lifespan of other primary cell cultures.