Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 μM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.     

Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.     

Pivotal Role of Mitochondrial Calcium Uptake in Neural Cell Apoptosis and Necrosis

Abstract : Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca²⁺]C) and intramitochondrial ([Ca²⁺]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine ; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal ; HNE) insult. STS and HNE each induced an early increase of [Ca²⁺]C followed by delayed increase of [Ca²⁺]M. Overexpression of Bcl-2 blocked the elevation of [Ca²⁺]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca²⁺]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca²⁺]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.     

ROS-Ca(2+) is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca(2+) on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca(2+) concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca(2+) was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ(m)). Then the cytoplasmic Ca(2+) concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

A plant mitochondrial phospholipid hydroperoxide glutathione peroxidase: its precise localization and higher enzymatic activity

A novel cDNA of phospholipid hydroperoxide glutathione peroxidase (PHGPx), which encodes a functional protein capable of complementing the yeast PHGHX-deletion mutant, was recently discovered in radish (Raphanus sativus) and designated as RsPHGPx [Yang X-D, Li W-J, Liu J-Y (2005) Biochim Biophys Acta 1728:199–205]. Sequence alignment suggested that RsPHGPx contains a targeting peptide required for transport to mitochondria, but the experimental evidence for the exact intracellular distribution of RsPHGPx remains to be elucidated. To uncover the cellular localization of plant PHGPx, we first investigated RsPHGPx’s intracellular distribution. Western blot analysis of subcellular fractions using the RsPHGPx antiserum clearly indicated the distribution of RsPHGPx in the radish mitochondrial fraction. Furthermore, a construct expressing the RsPHGPx precursor tagged with green fluorescent protein was introduced into tobacco and yeast cells, and the fusion protein was transported into both mitochondria, indicating that RsPHGPx was indeed localized in mitochondria. To explore the biochemical functions of this enzyme, we tested the enzymatic activity of the recombinant RsPHGPx protein. It displayed GSH-dependent peroxidase activity and exhibited the largest affinity to and the highest catalytic efficiency on phosphatidylcholine hydroperoxide, suggesting that phospholipid hydroperoxide is probably the optimum substrate for RsPHGPx. Furthermore, RsPHGPx showed a much higher V _max value, by two orders of magnitude, than those of all other known plant PHGPxs. Taken together, these results showed evidence for the first time of mitochondrial localization and higher activity of PHGPx in plants and provided a framework for continued studies on the physiological functions of RsPHGPx.     

On the state of calcium ions in isolated rat liver mitochondria IV. Prevention of phosphate-induced mitochondrial destruction by ruthenium red-insensitive calcium release

Ruthenium red prevented the spontaneous calcium release and the accompanying mitochondrial destruction occurring in calcium-loaded mitochondria in the presence of phosphate. Under these conditions delta pH and membrane potential delta psi were preserved and the ruthenium red-induced calcium efflux was low and at a constant rate. On prolonged incubation with calcium prior to addition of ruthenium red increasingly more mitochondrial calcium developed into a pool rapidly dischargeable by ruthenium red. This development was accompanied by stimulation of respiration which was, however, not abolished by ruthenium red as could have been expected if it had been caused by calcium cycling. Calcium therefore altered mitochondria by a different mechanism than by cycling across the inner membrane.     

Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondria

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated. using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.     

On the state of calcium ions in isolated rat liver mitochondria. III. Diversity of ruthenium red action on different calcium pools

Calcium efflux from isolated mitochondria on ruthenium red addition was shown to be biphasic. The rate of efflux from a slowly releasable pool was independent of preincubation. It could be saturated and in extrapolation revealed a maximal rate of 3.6 nmol/(min X mg protein). The efflux from a second, rapidly dischargeable pool was related to calcium added up to 300 nmol/mg protein when a final rate of 15 nmol/(min X mg protein) was reached. The magnitude of the latter pool depended on the time of preincubation in the presence of calcium and correlated with mitochondrial swelling. After ruthenium red addition, a further increase of this pool and spontaneous, destructive calcium release was prevented. Three conclusions are drawn from these results: On preincubation with calcium, part of the mitochondrial calcium develops into a rapidly dischargeable pool. This pool is responsible for mitochondrial alterations resulting in a spontaneous, destructive release of total calcium. Ruthenium red inhibits calcium release by discharging mitochondria from this destructive calcium pool. To avoid artefacts, mitochondrial parameters should be carefully controlled when ruthenium red-insensitive calcium efflux is studied.     

Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Failure of the expression of phospholipid hydroperoxide glutathione peroxidase in the spermatozoa of human infertile males

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.     

Ruthenium red-mediated suppression of Bcl-2 loss and Ca(2+) release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species

The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca(2+) was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca(2+) from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca(2+) levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.     

1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid induces caspase-mediated apoptosis and reactive oxygen species-mediated necrosis in cultured cortical neurons.

Sustained alteration in [Ca(2+)]i triggers neuronal death. We examined morphological and signaling events of Ca(2+)-deficiency-induced neuronal death. Cortical cell cultures exposed to 20 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 microg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4-12 h. An active fragment of caspase-3, a downstream mediator of cytochrome c, was observed within 8 h and cleaved PHF-1-positive tau. Administration of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective inhibitor of caspase-3, selectively prevented the apoptosis component of BAPTA-AM neurotoxicity. In contrast, BAPTA-AM-induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species. Combined treatment with caspase inhibitors and antioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that neurons deficient in [Ca(2+)]i undergo caspase-3-mediated apoptosis and reactive oxygen species (ROS)-mediated necrosis.     

The role of hydroperoxides as calcium release agents in rat brain mitochondria

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.     

Interrelation of active oxygen species,membrane damage and altered calcium functions

Incubation of freshly isolated rat liver mitochondria in the presence of oxygen free radical generating hypoxanthine —xanthine oxidase system led to swelling of mitochondria as measured by the change in optical density, which was reversed by the addition of superoxide dismutase. O₂ ^– in the presence of CaCl₂ enhanced the peroxidative decomposition of mitochondrial membrane lipids along with swelling of the organelle. Free radical generation led to enhancement of monoamine oxidase activity while glutathione peroxidase and cytochrome c oxidase were inhibited. Tertbutyl hydroperoxide (t-BHP) caused mitochondrial swelling through oxidative stress. Incorporation of ruthenium red, which is a Ca²⁺ transport blocker, during assay abolished peroxidative membrane damage and swelling. Dithiothreitol (DTT) accorded protection against t-BHP induced mitochondrial swelling. The above in vitro data suggest a possible interrelationship of active oxygen species, membrane damage and calcium dynamics.     

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.     

Dynamics of intracellular calcium and free radical production during ischemia in pyramidal neurons.

Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction. We investigated the contribution of mitochondrial dysfunction to free radical (FR) overproduction in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. Ischemia-induced FR generation was decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe dihydroRhod-2, revealed that FR production during and after the anoxic episode correlates with the accumulation of mitochondrial calcium. However, the mitochondrial calcium uptake inhibitor Ru360 did not prevent FR generation during ischemia and attenuated it to some degree during reoxygenation. On the other hand, the mitochondrial permeability transition blocker cyclosporinA (CsA) completely arrested both ischemic FR generation and mitochondrial calcium overload, and prevented deterioration of neuronal intrinsic membrane properties. CsA had no effect on the accumulation of intracellular calcium during ischemia-reperfusion. Nicotinamide, a blocker of NAD+ hydrolysis, reproduced the CsA effects on FR generation, mitochondrial calcium accumulation and cytoplasmic calcium increases. These observations suggest that a major determinant of ischemic FR generation in pyramidal neurons is the uncoupling of the mitochondrial respiratory chain, which may be associated with the mitochondrial permeability transition.     

Cadmium induces caspase-independent apoptosis in liver Hep3B cells: role for calcium in signaling oxidative stress-related impairment of mitochondria and relocation of endonuclease G and apoptosis-inducing factor

Cadmium-induced cellular toxicity has been related to necrosis and/or caspase-dependent apoptosis. In the present study, we show that, on cadmium exposure, the human hepatocarcinoma Hep3B cells undergo caspase-independent apoptosis associated with nuclear translocation of endonuclease G and apoptosis-inducing factor, two mitochondrial apoptogenic proteins. Release of these proteins is likely related to calcium-induced alteration of mitochondrial homeostasis. Indeed, it was first preceded by a rapid and sustained increase in cytoplasmic calcium and then by a coincident loss in mitochondrial membrane potential and production of reactive oxygen species. Bapta-AM (acetoxymethyl ester of 5, 5′-dimethyl-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), a calcium chelator, blocked all these events and prevented cadmium-induced apoptosis. Production of reactive oxygen species was inhibited by ruthenium red and rotenone, two mitochondrial inhibitors, and by diphenyleneiodonium, a flavoprotein inhibitor, which also prevented both loss in mitochondrial membrane potential and apoptosis. In addition, Bapta-AM and diphenyleneiodonium were found to almost totally block decreased expression of the mitochondrial anti-apoptotic nuclear factor-κB-regulated bcl-x_L protein in cadmium-treated cells. Taken together, our results show that cadmium induces Hep3B cells apoptosis mainly by calcium- and oxidative stress-related impairment of mitochondria, which probably favors release of apoptosis-inducing factor and endonuclease G.