The Protozoan, Paramecium primaurelia, as a Nonsentient Model to Test Laser Light Irradiation:

The Effects of an 808nm Infrared Laser Diode on Cellular Respiration

Andrea Amaroli, Silvia Ravera, Steven Parker, Isabella Panfoli, Alberico Benedicenti and Stefano Benedicenti

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. Unfortunately, conflicting literature has led to the labelling of PBM as a complementary or alternative medicine approach. However, past and ongoing clinical and research studies by reputable investigators have re-established the merits of PBM as a genuine medical therapy, and the technique has, in the last decade, seen an exponential increase in the numbers of clinical instruments available, and their applications. This resurgence has led to a clear need for appropriate experimental models to test the burgeoning laser technology being developed for medical applications. In this context, an ethical model that employs the protozoan, Paramecium primaurelia, is proposed. We studied the possibility of using the measure of oxygen consumption to test PBM by irradiation with an infrared or near-infrared laser. The results show that an 808nm infrared laser diode (1W; 64J/cm2) affects cellular respiration in P. primaurelia, inducing, in the irradiated cells, a significantly (p < 0.05) increased oxygen consumption of about 40%. Our findings indicate that Paramecium can be an excellent tool in biological assays involving infrared and near-infrared PBM, as it combines the advantages of in vivo results with the practicality of in vitro testing. This test represents a fast, inexpensive and straightforward assay, which offers an alternative to both traditional in vivo testing and more expensive mammalian cellular cultures.
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The Role of Biomembranes in Chromium (III)-induced Toxicity In Vitro

Emil Rudolf and Miroslav Cervinka

The role of biomembranes in the chronic toxicity of environmentally occurring chromium acetate hydroxide was investigated by using primary human fibroblasts. Transport of chromium acetate hydroxide across the plasma membrane of the cell, and the effects of chromium (III) ions on the plasma membrane as well as other intracellular membranes, were determined during six weeks of continuous exposure by using atomic absorption spectrometry, observation of cell morphology, membrane integrity assays (for lactate dehydrogenase leakage and lysosomal membrane disruption), and mitochondrial assays (for mitochondrial dehydrogenase activity and mitochondrial transmembrane potential analysis). The type of cell death induced by long-term exposure was determined in terms of phosphatidylserine externalisation, caspase-3 activation, and chromatin fragmentation. Chromium acetate hydroxide, at a concentration of 100μmol/l, accumulated in exposed cells, inflicting plasma membrane damage and suppressing mitochondrial function. Antioxidant co-enzyme Q, at a concentration of 10μmol/l, partially prevented plasma membrane damage and mitochondrial dysfunction. Exposure to chromium acetate hydroxide produced apoptosis, necrosis and an intermediate type of cell death in primary human fibroblasts. These results show that the plasma membrane and mitochondrial membrane are important targets for chronic chromium acetate hydroxide toxicity, and that this in vitro system holds promise for studying the toxicity resulting from long-term exposure to metal ions.
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S-Adenosylmethionine Exerts a Protective Effect Against Thioacetamide-induced Injury in Primary Cultures of Rat Hepatocytes

Halka Lotková, Zuzana Cvervinková, Otto Kucvera, Tomáš Roušar and Pavla Krviváková

S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently-used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide- induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation
periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.
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Evaluation of Mitochondrial Function in Isolated Rat Hepatocytes and Mitochondria during Oxidative Stress

Zuzana Cvervinková, Halka Lotková, Pavla Krvivaková, Tomásv Rousvar, Otto Kucvera, Lukas Tichý, Miroslav Cvervinka, and Zdenevk Drahota

The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert- butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.
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Can Drug Safety be Predicted and Animal Experiments Reduced by Using Isolated Mitochondrial Fractions?

Susana P. Pereira, Gonçalo C. Pereira, António J. Moreno and Paulo J. Oliveira

Mitochondrial toxicity has resulted in the withdrawal of several drugs from the market. One particular example is nefazodone, an anti-depressant withdrawn in the USA due to hepatoxicity caused by drug-induced mitochondrial dysfunction. Drug development and safety testing can involve the use of large numbers of laboratory animals, which, without a decisive pre-screening for mitochondrial toxicity, are often unable to pre-empt higher mortality rates in some patient groups. The use of isolated mitochondria as a screening tool for drug safety can decrease the number of laboratory animals used in pre-clinical studies, thus improving animal welfare and healthcare outcomes and costs. Novel techniques involving highthroughput methods can be used to investigate whether a molecule is a mitochondrial toxicant. Moreover, these screens are mechanistically-based, since the effects of the drug on oxidative phosphorylation, calcium homeostasis and mitochondrial genetics can be assessed. This review is intended to demonstrate that isolated mitochondrial fractions are suitable for predicting drug and general chemical safety in toxicological screenings, thus contributing to the refinement and reduction of animal use in laboratory research.
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