safranine-t and Carcinoma--Ehrlich-Tumor

Mitochondria are considered as the main source of reactive oxygen species (ROS) in the cell. For this reason, they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids, and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O(2). Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, convert superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H(2)O. In this chapter, we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depends on the metabolic state of this organelle.

Topics: Animals; Benzimidazoles; Brain; Calcium; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Fractionation; Cell Line, Tumor; Electron Transport; Fibroblasts; HeLa Cells; Humans; Hydrogen Peroxide; Membrane Potential, Mitochondrial; Mice; Microscopy, Confocal; Mitochondria; Oxygen Consumption; Phenazines; Reactive Oxygen Species; Superoxides

The effect of the local anesthetic bupivacaine on transmembrane potential of mitochondria within Ehrlich ascites tumor cells has been investigated by the safranine method. The following summarizes the results. 1) When Ehrlich ascites tumor cells were added into a medium containing safranine, a differential spectrum with a decrease at 520 nm appeared. The addition of bupivacaine did not reverse the response but resulted in a spectrum similar to that observed with deenergized mitochondria, with a maximum at 550 nm and a minimum at 495 nm. 2) The addition of glucose to bupivacaine- or rotenone-treated cells also produced a shift in safranine spectrum similar to that observed upon energization of mitochondrial membranes by ATP, thus suggesting that glycolytically generated ATP was responsible for this spectral change. 3) The ability of bupivacaine to decrease the membrane potential in mitochondria within Ehrlich ascites tumor cells was due to two different effects: inhibition of the energy-conserving site 1 of the respiratory chain and uncoupling by a true protonophoretic mechanisma.

Topics: Adenosine Triphosphate; Anesthetics, Local; Animals; Bupivacaine; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carcinoma, Ehrlich Tumor; Energy Metabolism; Glucose; Intracellular Membranes; Male; Membrane Potentials; Mice; Mitochondria; Oxidative Phosphorylation; Phenazines; Rotenone; Solubility; Tumor Cells, Cultured; Uncoupling Agents

Topics: Animals; Carcinoma, Ehrlich Tumor; Dextrans; Dicyclohexylcarbodiimide; Indicators and Reagents; Intracellular Membranes; Membrane Potentials; Mice; Mitochondria; Phenazines; Spectrophotometry; Time Factors