cytochrome-c-t and cupric-chloride

One mechanism of the anticancer action of anthracyclines is believed to be oxidative DNA damage. Previously, we reported that doxorubicin induced oxidative DNA damage in the presence of Cu(II). However, the mechanism of pirarubicin-induced oxidative DNA damage has not been well clarified.. DNA damage by pirarubicin in the presence of Cu(II) was analyzed using pBR322 plasmid DNA. O. Pirarubicin induced DNA damage in the presence of Cu(II). Scavenger experiments suggest that reactive species are generated from H. These findings suggest that pirarubicin plus Cu(II) induces oxidative DNA damage in a similar manner to doxorubicin, and Cu(II)-mediated oxidative DNA damage may serve as a common mechanism for antitumor effects of anthracyclines.

Topics: Antibiotics, Antineoplastic; Cations, Divalent; Copper; Cytochromes c; DNA Damage; DNA, Circular; Doxorubicin; Drug Synergism; Electrophoresis, Agar Gel; Humans; Molecular Structure; Oxidation-Reduction; Phenanthrolines; Plasmids; Reactive Oxygen Species; Superoxides

This study is to examine if Cu(2+) can act directly on mitochondria or indirectly by producing reactive oxygen species (ROS), isolated broiler hepatic mitochondria were exposed to different concentrations of Cu(2+) (10, 30, 50 μM). Respiratory chain complex activities, ROS generation, respiratory control ratio (RCR) and mitochondrial membrane potential were investigated. Dose-dependent inhibition of respiratory chain complexes and induction of ROS were observed, which coincided with decreasing RCR both with glutamate + malate or succinate. Further investigation indicated that the membrane potential determined by rhodamine 123 release decreased after CuCl(2) exposure at 30 and 50 μM. In addition, the effects of the antioxidants NAC (200 μM) and GSH (200 μM) were studied at 50 μM Cu(2+). The results indicate that Cu can induce mitochondrial dysfunction in excessive dose and the effect of Cu(2+) exposure on respiratory chain is not site-specific, and antioxidants can protect the mitochondrial function by reducing the formation of free radicals.

Topics: Acetylglucosamine; Animals; Chickens; Copper; Cytochromes c; Electron Transport; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex IV; Female; Glutathione; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Membrane Potentials; Mitochondria, Liver; Oxygen Consumption; Reactive Oxygen Species

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N-acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using (32)P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H(2)O(2) with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Autoradiography; Benzoquinones; Caspase 3; Cell Line, Tumor; Copper; Cytochromes c; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Free Radical Scavengers; Humans; NAD; Neuroblastoma; Phenanthrolines; Phosphorus Isotopes; Salsoline Alkaloids; Tetrahydroisoquinolines; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine 3-Monooxygenase

Astrocytes play a critical neurotrophic and neuroprotective role in the brain, and improper function of these cells may contribute to the onset of neurodegenerative diseases. Because astrocytes are known to be enriched with Cu chaperone proteins, it is important to understand the factors that may lead to cytotoxic effects of Cu on astrocytes. In this report, we demonstrated a dramatic potentiating effect of neocuproine (NCP), a membrane permeable metal chelator, on Cu, but not Fe or Pb, in inducing apoptosis of cultured astrocytes. It was estimated that individually, CuCl2 and NCP only weakly exhibited cytotoxic effects on astrocytes, with EC50 of 180 and 600 microM, respectively. However, NCP at a nontoxic concentration of 10 microM markedly reduced EC50 of Cu to 0.35 microM (physiological concentration) and Cu (10 microM) reduced EC50 of NCP down to 0.06 microM. The mechanisms underlying these dramatic potentiation effects are elucidated. NCP increased the intracellular concentration of Cu in astrocytes and a nonpermeable Cu chelator, bathocuproine disulfonate was able to abolish all of the apoptotic signaling. Cell death was determined to be via apoptosis due to increased reactive oxygen species production, mitochondrial dysfunction, depletion of glutathione and adenosine triphosphate, cytochrome c release, c-Jun N-terminal kinase, and caspase-3 activation, and poly-ADP-ribose polymerase degradation. This finding, coupled with our previous reports, suggests that metal chelators (NCP, dithiocarbamate and disulfiram) should be cautiously used as they may potentiate a cytotoxic effect of endogenous Cu on astrocytes. Their clinical implications in the etiology of neurodegenerative diseases deserve further investigation.

Topics: Animals; Animals, Newborn; Apoptosis; Astrocytes; Caspase 3; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Chelating Agents; Copper; Cytochromes c; DNA Breaks; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Glutathione; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 8; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phenanthrolines; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Reactive Oxygen Species; Signal Transduction; Time Factors

The quantitative influence of copper on the interaction between cytochrome c and sulfite, which is a derivate of sulfur dioxide in the human body, has been studied by fluorescence spectrometry and ultraviolet absorption spectrometry in vitro. The results indicate that copper may intensely combine with protein component and ferroporphyrin component in cytochrome c at the concentration of 0.1 mM, and the respective association constants (K(A)) are 3.77 x 10(4) L mol(-1) and 9.38 x 10(3) L mol(-1). Sulfite has little interaction with the protein component in cytochrome c (K(A) = 0.094 L mol(-1)), at either low concentrations or relatively high concentrations (<0.15 M). However, it can react with the ferroporphyrin component in cytochrome c (K(A) = 4.297 L mol(-1)). After copper is added to the sulfite-cytochrome c binary systems, the reaction between sulfite and the protein component in cytochrome c is obviously strengthened at a low concentration (K(A) = 7.289 L mol(-1)), while the addition of copper merely has a little effect on the interaction between sulfite and the ferroporphyrin component in cytochrome c.

Topics: Air Pollutants; Copper; Cytochromes c; Drug Interactions; Hydrogen-Ion Concentration; Metalloporphyrins; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Sulfites; Temperature