cytochrome-c-t and Fever

Research over the past 50 years has consistently documented that cell death is an integral part of both normal development and the etiology of birth defects; however, the significance of this cell death has been, until recently, unclear. Research published during the past 15 years has now shown that programmed cell death (PCD) and teratogen-induced cell death are genetically controlled processes (apoptosis) that play important roles in both normal and abnormal development. Therefore, the purpose of this review is to highlight what is known about PCD and teratogen-induced cell death and their relationships to the mechanisms of apoptosis and abnormal development.

Topics: Animals; Apoptosis; Blotting, Western; Caspases; Cell Death; Chick Embryo; Cyclophosphamide; Cytochromes c; Extremities; Fever; Limb Deformities, Congenital; Mice; Teratogens; Tumor Suppressor Protein p53

Psychostimulant methamphetamine (METH) is toxic to striatal dopaminergic and serotonergic nerve terminals in adult, but not in the adolescent, brain. Betulinic acid (BA) and its derivatives are promising anti-HIV agents with some toxic properties. Many METH users, particularly young men, are HIV-positive; therefore, they might be treated with BA or its derivative for HIV infection. It is not known whether BA, or any of its derivatives, are neurotoxic in combination with METH in the adolescent brain. The present study investigated the effects of BA and binge METH in the striatum of late adolescent rats. BA or METH alone did not decrease the levels of dopaminergic or serotonergic markers in the striatum whereas BA and METH together decreased these markers in a BA dose-dependent manner. BA+METH also caused decreases in the levels of mitochondrial complex I in the same manner; BA alone only slightly decreased the levels of this enzyme in striatal synaptosomes. BA or METH alone increased cytochrome c. METH alone decreased parkin, increased complex II and striatal BA levels. These results suggest that METH in combination with BA can be neurotoxic to striatal dopaminergic and serotonergic nerve terminals in the late adolescent brain via mitochondrial dysfunction and parkin deficit. We report a synergistic neurotoxicity of betulinic acid (BA) and methamphetamine (METH) to monoaminergic terminals in the striatum of male late adolescent rats. BA contribution to the neurotoxicity is decreasing mitochondrial complex I whereas METH contribution is decreasing parkin and increasing brain concentration of BA. We propose that clinical use of BA in young male METH users can be neurotoxic.

Topics: Animals; Anti-HIV Agents; Betulinic Acid; Blotting, Western; Brain Chemistry; Caspase 3; Chymotrypsin; Cytochromes c; Dopamine Agents; Dopaminergic Neurons; Dose-Response Relationship, Drug; Electron Transport Complex I; Electron Transport Complex II; Fever; Male; Methamphetamine; Mitochondria; Neostriatum; Nerve Endings; Pentacyclic Triterpenes; Rats; Rats, Sprague-Dawley; Serotonergic Neurons; Synaptosomes; Triterpenes; Ubiquitin-Protein Ligases

Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+ -adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress-induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress-induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.

Topics: Animals; Animals, Newborn; Apoptosis; Calcium; Calcium-Transporting ATPases; Caspase 3; Caspases; Cell Respiration; Cells, Cultured; Cytochromes c; Disease Models, Animal; Fever; Heart Diseases; Heat Stress Disorders; Intracellular Membranes; Male; Membrane Potentials; Microscopy, Electron, Transmission; Mitochondria; Myocytes, Cardiac; Necrosis; Oxidative Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2'-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44 degrees C for 60 min, minimal apoptosis was observed. When combined with nontoxic AAPH (50mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37 degrees C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca2+]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca2+]i and an intracellular Ca2+ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependenton increase in LPO and [Ca2+]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.

Topics: Amidines; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cytochromes c; DNA Fragmentation; Egtazic Acid; fas Receptor; Female; Fever; Flow Cytometry; Free Radicals; HeLa Cells; Humans; Ions; Lipid Peroxidation; Microscopy, Fluorescence; Oxidants; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Temperature; Time Factors; Uterine Cervical Neoplasms

Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown.. Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9.. Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens.. HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.

Topics: Animals; Apoptosis; Blastocyst; Blotting, Western; Caspase 6; Caspase 7; Caspases; Cyclophosphamide; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Fever; Mice; Models, Biological; Oligopeptides; Recombinant Proteins; Staurosporine; Teratogens

Previously, we showed that teratogens such as hyperthermia activate the mitochondrial apoptotic pathway in day nine mouse embryos. Activation of this pathway involves an initial release of cytochrome c from intermembranous spaces of the mitochondria into the cytoplasm. Cytoplasmic cytochrome c then activates a caspase cascade resulting in the orderly demise of the cell. In addition, we showed that teratogens activate the mitochondrial pathway in cells of the neuroepithelium, but not the heart.. To further investigate the role of the mitochondrion in teratogen-induced apoptosis, we used transmission electron microscopy (TEM) to compare mitochondrial morphology in cells of the neuroepithelium and heart of control and hyperthermia-treated embryos. Because we know that the apoptotic pathway is activated some time during the first 5 hr after teratogen exposure is initiated, we assessed mitochondrial morphology at 1, 2.5, and 5 hr after day nine mouse embryos were exposed to hyperthermia (43 degrees C, 15 min).. In neuroepithelial cells of the prosencephalon, abnormally-shaped mitochondria were observed at the 1 hr time point and thereafter, whereas loss of cristae and shrunken mitochondria were noted at the 5 hr time point. In contrast, no obvious changes in mitochondria of heart cells were observed at any of the time points monitored.. These results indicate that teratogen-induced cell death in neuroepithelial cells is temporally correlated with alterations in mitochondrial morphology, whereas the absence of cell death in the heart is correlated with a corresponding lack of change in mitochondrial morphology. Birth Defects Research (Part A), 2003.

Topics: Animals; Apoptosis; Cell Death; Cytochromes c; Cytoplasm; Embryo, Mammalian; Epithelial Cells; Female; Fever; Hot Temperature; Kinetics; Mice; Microscopy, Electron; Mitochondria; Temperature; Teratogens; Time Factors