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Biomedical subjects

S Orrenius

Publications and source records attributed to S Orrenius.

At least 163 records · Page 9Linked to original sources
PubMed1993

Effects of glutathione and chelating agents on copper-mediated DNA oxidation: pro-oxidant and antioxidant properties of glutathione.

The exposure of DNA to H2O2 in the presence of Cu(II) and a reducing agent is known to result in the induction of a variety of oxidative lesions, including DNA strand breaks and base modifications. Since the reducing agent glutathione occurs in cell nuclei at relatively high concentrations, and copper exists in nuclei associated with chromatin, the present study was undertaken to evaluate the ability of GSH to promote copper-mediated free radical damage to DNA. When compared with ascorbate, GSH was found to be inefficient in the promotion of damage to DNA. Parallel ESR spin trapping measurements indicated that GSH inhibits free radical formation by copper ions in the presence of H2O2, ascorbate, and DNA. The protective effect of GSH is attributed to its stabilization of copper in the +1 oxidation state, thereby compromising its ability to participate in free radical generating reactions. Consequently, it is suggested that the GSH in cell nuclei serves to prevent, rather than promote, copper-dependent damage to DNA. In contrast, in the presence of 1,10-phenanthroline, GSH stimulated free radical formation and DNA damage. This is attributed to the failure of GSH to remove copper(I) from 1,10-phenanthroline. Therefore, under these conditions, GSH serves primarily to redox cycle the reactive 1,10-phenanthroline-copper complex.

Ascorbic Acid↗
PubMed1993

Ca2+ and endonuclease activation in radiation-induced lymphoid cell death.

Exposure of lymphoid cells to X-irradiation caused an increase in cytosolic Ca2+ ([Ca2+]i) in rat thymocytes but not in rat splenocytes. The Ca2+ elevation in rat thymocytes was transitory and by 2 h postirradiation, when the chromatin degradation began to be appreciable, the [Ca2+]i had returned to control level. Inhibitors of RNA and protein syntheses prevented the radiation-induced [Ca2+]i rise in thymocytes. Pretreatment of both cell types with Ca2+ chelators also prevented DNA fragmentation, chromatin condensation, and cell death caused by X-irradiation. These data suggest that Ca2+ plays an important role in the perpetuation of apoptosis in both thymocytes and splenocytes although a Ca2+ elevation, which may serve as a signal in thymocytes, does not appear to be required to initiate radiation-induced DNA fragmentation in splenocytes.

Animals↗
PubMed1993

Modifications of Ca2+ signaling by inorganic mercury in PC12 cells.

The effects of different levels of inorganic mercury (Hg2+) on depolarization- or agonist-stimulated Ca2+ signals were studied in PC12 cells. Exposure to 50-300 nM Hg2+ did not alter the resting cytosolic free Ca2+ concentration ([Ca2+]i), but enhanced the Ca2+ response to KCL-induced depolarization. Patch-clamp experiments revealed that these Hg2+ concentrations increased the voltage-dependent Ca2+ current through L-type channels. Also, Hg2+ treatment amplified the intracellular [Ca2+]i transients elicited by extracellular ATP. In contrast, the Ca2+ increase stimulated by bradykinin was unaffected. At slightly higher concentrations (1 to 2 microM), Hg2+ caused a sustained rise of the resting [Ca2+]i. This increase did not occur in Ca(2+)-free medium and was prevented by pretreatment with NiCl2 or with the L-type Ca2+ channel blockers, verapamil and nifedipine. Hg2+ did not mobilize Ca2+ from intracellular stores sensitive to thapsigargin, 2,5-di-(tert-butyl)-benzohydroquinone, or caffeine. At 2 microM, Hg2+ inhibited the [Ca2+]i transients elicited by bradykinin, ATP, or KCl-induced depolarization. The loss of the intracellular Ca2+ response to bradykinin was independent from the Ca2+ overload elicit by Hg2+; instead, it was associated with inhibition of polyphosphoinositide generation. Exposure to the lower Hg2+ concentrations (0.3-0.5 microM) greatly potentiated NGF-induced PC12 cell differentiation. Conversely, treatment with 2 microM Hg2+ caused cell death. Our results show that inorganic mercury has selective and different effects on Ca2+ signaling in PC12 cells depending on the concentration, within a narrow range.

Adenosine Triphosphate↗
PubMed1993

Chelation of intracellular zinc triggers apoptosis in mature thymocytes.

BACKGROUND: Thymocyte apoptosis has been shown to be regulated by intracellular levels of cations. Elevation of [Ca2+]i can induce cell death by apoptosis, whereas, Zn2+ prevents it. EXPERIMENTAL DESIGN: A membrane permeable metal ion chelator, N,N,N'N',-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), was used to examine the role of intracellular zinc in thymocyte (rat and human) apoptosis. Characteristics of apoptosis that were assessed included: chromatin degradation into oligosomal-sized fragments and nuclear condensation. The necessity for protein synthesis in TPEN-induced apoptosis was ruled out using the inhibitors, cycloheximide and emetine. Metal ion specificity for TPEN was established by competition with exogenous cations. FACS analysis was employed to determine the phenotype of the TPEN-sensitive thymocyte populations. RESULTS: TPEN induced DNA fragmentation within 4 hours of exposure before the onset of cell death (6 hours). Addition of equimolar exogenous Zn2+ or Cu2+, but not Mn2+ or Fe2+, at the initiation of culture prevented TPEN-induced apoptosis. A membrane impermeable chelator, diethylenetriaminepentaacetic acid, did not induce thymocyte apoptosis indicating that chelation of intracellular Zn2+ was required to trigger DNA fragmentation. The identity of the critical intracellular Zn(2+)-binding site(s) is currently unknown, but it appears that increased thymocyte [Ca2+]i may displace Zn2+ from these intracellular sites. TPEN treatment resulted in the depletion of thymocytes having a mature phenotype with respect to CD3, CD4, and CD8. Moreover, lymph node cells were more sensitive to TPEN than thymocytes. CONCLUSIONS: These experiments show that Zn2+ chelation has disparate effects on immature and mature T cells, and suggest that zinc availability controls the cell death or selection pathway during thymopoiesis.

Animals↗
PubMed1992

Cyclosporin A protects hepatocytes against prooxidant-induced cell killing. A study on the role of mitochondrial Ca2+ cycling in cytotoxicity.

Cyclosporin A (CsA) is a potent inhibitor of the prooxidant-induced release of Ca2+ from isolated mitochondria. In this investigation, pretreatment of hepatocytes with CsA before exposure to the prooxidants tert-butyl hydroperoxide (tBH), cumene hydroperoxide or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) prevented the loss of cell viability. HPLC analysis of adenine and pyridine nucleotide concentrations in hepatocytes treated with 3,5-Me2-NAPQI showed a rapid depletion of ATP prior to the loss of cell viability versus the maintenance of near control levels of ATP in hepatocytes treated with CsA before 3,5-Me2-NAPQI. In 3,5-Me2-NAPQI-exposed hepatocytes there was also a rapid loss of cellular NAD+ which could be accounted for initially by a transient increase in NADP+. Measurement of the intracellular Ca2+ pools showed an early depletion of the mitochondrial Ca2+ pool in hepatocytes exposed to 3,5-Me2-NAPQI, tBH or cumene hydroperoxide; this loss was prevented by CsA. In conclusion, these results show that CsA protected hepatocytes from prooxidant injury by preventing mitochondrial Ca2+ cycling and subsequent mitochondrial dysfunction. This suggests that in prooxidant injury, excessive Ca2+ cycling is an early and important event leading to mitochondrial damage and subsequently to cell death.

Animals↗
PubMed1992

m-iodobenzylguanidine increases the mitochondrial Ca2+ pool in isolated hepatocytes.

The incubation of isolated hepatocytes with the inhibitor of protein mono ADP-ribosylation, m-iodobenzylguanidine (MIBG), resulted in an increase in the size of the mitochondrial Ca2+ pool, without alteration of the non-mitochondrial Ca2+ store(s). This increase was abolished when the cytosolic free Ca2+ concentration ([Ca2+]i) was buffered by prior loading of the cells with fluo 3. Elevating [Ca2+]i by releasing the endoplasmic reticular Ca2+ store with 2,5-di-(tert-butyl)-1,4-hydroquinone resulted in a synergistic increase in the magnitude of the mitochondrial Ca2+ pool. A role for protein ADP-ribosylation in the intracellular regulation of mitochondrial Ca2+ homeostasis is suggested.

3-Iodobenzylguanidine↗
PubMed1992

Butylated hydroxytoluene prevents cumene hydroperoxide-induced Ca2+ release from liver mitochondria by inhibiting pyridine nucleotide hydrolysis.

The mechanism by which the free radical scavenger butylated hydroxytoluene (BHT) prevents cumene hydroperoxide-induced Ca2+ release from rat liver mitochondria was studied. In Ca(2+)-loaded mitochondria cumene hydroperoxide induced a rapid oxidation and subsequent hydrolysis of the pyridine nucleotides. In the presence of BHT, pyridine nucleotide oxidation by cumene hydroperoxide occurred but was reversible as hydrolysis was prevented by BHT. However, the addition of BHT directly to rat liver submitochondrial particles did not inhibit NAD+ hydrolysis or the formation of ADP-ribose from NAD+. Thus, whilst BHT prevented NAD+ hydrolysis in isolated mitochondria, this appeared not to be due to a direct effect of BHT on the NADase. It is concluded that the mechanism of action of BHT on cumene hydroperoxide-induced Ca2+ release from mitochondria involves the inhibition of pyridine nucleotide hydrolysis by an indirect mechanism rather than the radical scavenging properties of BHT.

Adenosine Diphosphate Ribose↗
PubMed1992

Evidence for two pathways of receptor-mediated Ca2+ entry in hepatocytes.

Receptor-mediated Ca2+ entry was studied in fura-2-loaded isolated hepatocytes. Emptying of internal Ca2+ stores by treatment with either the Ca(2+)-mobilizing hormone vasopressin or the inhibitors of the microsomal Ca2+ pump, 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ) or thapsigargin, stimulated Ca2+ entry, as indicated by a rise in the cytosolic free Ca2+ concentration after Ca2+ was added to cells suspended in nominally Ca(2+)-free medium. The enhancement of Ca2+ entry was proportional to the degree of depletion of the intracellular Ca2+ pool and occurred also after removal of vasopressin from its receptor. In contrast, the stimulation of Mn2+ entry by vasopressin required the continuous presence of the agonist, since it was prevented by the addition of vasopressin receptor antagonist. This effect was observed under conditions where refilling of the agonist-sensitive pool was prevented by using nominally Ca(2+)-free medium. Unlike vasopressin, tBuBHQ or thapsigargin did not stimulate Mn2+ entry. These results suggest the existence of two pathways for receptor-mediated Ca2+ entry in hepatocytes, a 'capacitative' pathway that is sensitive to the Ca2+ content in the Ins(1,4,5)P3-sensitive Ca2+ pool and does not allow Mn2+ entry, and a second pathway that depends on receptor occupation, seems to require a second messenger for activation, and permits influx of Mn2+.

Animals↗
PubMed1992

Demonstration of nuclear compartmentalization of glutathione in hepatocytes.

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury.

Animals↗
PubMed1992

Tumor necrosis factor alpha induces apoptosis in mammary adenocarcinoma cells by an increase in intranuclear free Ca2+ concentration and DNA fragmentation.

The incubation of human mammary adenocarcinoma cells (BT-20) with tumor necrosis factor alpha in the absence or presence of cycloheximide resulted in progressive DNA fragmentation. This was preceded by a sustained increase in intracellular free Ca2+ concentration and was not detected in cells pretreated with intracellular Ca2+ chelators, calmodulin antagonists, or activators of protein kinase C. Image analysis of fura-2-loaded BT-20 cells treated with tumor necrosis factor alpha revealed that, in many cells, the initial increase in Ca2+ level occurred in a cellular region that corresponded to the localization of the nucleus. Our findings suggest that tumor necrosis factor alpha can promote an increase in intranuclear free Ca2+ which, in turn, may stimulate Ca(2+)-dependent endonuclease activity, resulting in DNA fragmentation and apoptosis.

Adenocarcinoma↗
PubMed1992

N-acetyl-p-benzoquinone imine induces Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis.

The mechanism of N-acetyl-p-benzoquinone imine (NAPQI)-induced release of Ca2+ from rat liver mitochondria was investigated. The addition of NAPQI or 3,5-Me2-NAPQI (a dimethylated analogue of NAPQI with only oxidizing properties) to mitochondria resulted in the rapid and extensive oxidation of NADH and NADPH. High-performance liquid chromatographic analysis of mitochondrial pyridine nucleotides revealed that the formation of NAD+ and NADP+ was followed by a time-dependent net loss of total pyridine nucleotides as a result of their hydrolysis, with the formation of nicotinamide. Preincubation of the mitochondria with cyclosporin A completely prevented the quinone imine-stimulated release of sequestered Ca2+ from mitochondria. Cyclosporin A did not affect the ability of NAPQI or 3,5-Me2-NAPQI to oxidize NAD(P)H but prevented the quinone imine-induced hydrolysis of the pyridine nucleotides. Although there was no detectable change in total protein-bound ADP-ribose content during quinone imine-induced Ca2+ release from mitochondria, meta-iodobenzylguanidine, a competitive inhibitor of protein mono(ADP-ribosylation), prevented Ca2+ release by NAPQI and 3,5-Me2-NAPQI; meta-iodobenzylguanidine did not inhibit the quinone imine-induced NAD(P)H oxidation and only partially blocked hydrolysis of the oxidized pyridine nucleotides. It is concluded that NAPQI causes the oxidation of mitochondrial NADH and NADPH, and stimulates Ca2+ release as a result of the further hydrolysis of the oxidized pyridine nucleotides and protein mono(ADP-ribosylation).

3-Iodobenzylguanidine↗
PubMed1992

Calcium ions and oxidative cell injury.

Exposure of mammalian cells to oxidative stress induced by oxidation-reduction-active quinones and other prooxidants results in depletion of intracellular glutathione, followed by modification of protein thiols and loss of cell viability. Protein thiol modification during oxidative stress is normally associated with impairment of various cell functions, including inhibition of agonist-stimulated phosphoinositide metabolism, disruption of intracellular Ca2+ homeostasis, and perturbation of normal cytoskeletal organization. The latter effect appears to be responsible for formation of the numerous plasma membrane blebs typically seen in cells exposed to cytotoxic concentrations of prooxidants. Following disruption of thiol homeostasis in prooxidant-treated cells, there is impairment of Ca2+ transport and subsequent perturbation of intracellular Ca2+ homeostasis, resulting in a sustained increase in cytosolic Ca2+ concentration. This increase in Ca2+ can cause activation of various Ca(2+)-dependent degradative enzymes (e.g., phospholipases, proteases, endonucleases), which may contribute to cell death. In contrast to the cytotoxic effects of excessive oxidative damage, low levels of oxidative stress can lead to activation of enzymes involved in cell signaling. In particular, the activity of protein kinase C is markedly increased by oxidation-reduction-cycling quinones through a thiol/disulfide exchange mechanism, which may represent a mechanism by which prooxidants can modulate cell growth and differentiation.

Animals↗
PubMed1992

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.

Adenosine Triphosphate↗
PubMed1992

Ca(2+)-dependent mechanisms of cytotoxicity and programmed cell death.

There is increasing evidence that the calcium ion plays a critical role in both toxic cell killing and programmed cell death. Thus, in a variety of experimental systems a perturbation of intracellular Ca2+ homeostasis due to increased Ca2+ influx and/or inhibition of Ca2+ extrusion has been found to be an early event in the development of cell injury. It is clear that sustained increases in intracellular Ca2+ can activate cytotoxic mechanisms which result in perturbations of cellular structure and function. For example, the stimulation of Ca(2+)-dependent proteases can result in a disruption of cytoskeletal organization and the formation of surface protrusions (blebs) and Ca(2+)-mediated phospholipase activation can result in an impairment of mitochondrial function with collapse of membrane potential and cessation of ATP synthesis. The activation of a Ca2+, Mg(2+)-dependent nuclear endonuclease is associated with chromatin cleavage and appears to play a crucial role in programmed cell death (apoptosis) in the immune system and other tissues. There is also recent evidence that this process may be responsible for the immunotoxicity of dioxins and organotin compounds and involved in the killing of adenocarcinoma cells by tumor necrosis factor alpha. Although calcium ions appear to be required for endonuclease activity during apoptosis, this process is also influenced by other factors, e.g. protein kinase C activity, intracellular polyamine and Zn2+ levels, chromatin structure, etc. Thus, the regulation of endonuclease activity under both physiological and toxicological conditions appears to be complex and to involve multiple factors.

Animals↗
PubMed1992

Thiol modification and cell signalling in chemical toxicity.

Exposure of cells to thiol oxidizing agents can result in the modification of key proteins involved in cell signalling. Such changes have been shown to affect agonist-stimulated phosphoinositide metabolism, activation of protein kinases and intracellular Ca2+ signals, which result in abnormalities in cell metabolisms and growth. Here, we show that moderate levels of oxidants potentiate growth signals and either enhance cell proliferation or facilitate cell differentiation, whereas inhibition of growth signals by higher oxidant concentrations can block cell proliferation and activate programmed cell death (PCD). Finally, a general alteration of multiple signalling pathways associated with increased catabolic reactions results in cell death by necrosis. Our data suggest that oxidant interaction with cell signalling systems may exert opposite effects, depending on the dose, and that oxidative reactions may either mimic growth factor stimulation and stimulate cell proliferation or inhibit growth signals and activate PCD, in the same cell systems.

Animals↗
PubMed1992

Cytotoxic and genotoxic effects of styrene-7,8-oxide in neuroadrenergic Pc 12 cells.

Exposure of Pc 12 cells to styrene-7,8-oxide (SO) (0.5-1 mM) caused a rapid increase in cytosolic Ca2+, depletion of intracellular glutathione and ATP, DNA damage and loss of cell viability. Lower SO concentrations (less than or equal to 100 microM), did not cause loss of cell viability or affect cell growth rate. However, at 30 and 100 microM, SO stimulated the formation of alkali-sensitive, DNA single-strand breaks (SSB). DNA SSB were fully repaired when cells exposed to 30 microM SO were subsequently incubated for 3 h in fresh medium, whereas DNA repair was only partial after exposure to 100 microM SO. When cells exposed to 30 or 100 microM SO were incubated with the inhibitors of repair synthesis 1-beta-D-arabinofuranosyl-cytosine (AraC) and hydroxyurea (HU), SSB accumulated, indicating the involvement of the excision-repair system in the removal of DNA lesions. A SO adduct with guanine at the N7 position was detected in the DNA extracted from treated cells. SO did not induce the formation of double-strand breaks, interstrand cross-links, or DNA-protein cross-links. Although cells exposed to 30 or 100 microM SO underwent normal cell division, latent DNA damage was retained for up to 14 subsequent replicative cycles. In addition, SO-treated cells partially lost their normal ability to differentiate in response to nerve growth factor (NGF) stimulation. NGF failed to induce differentiation in cells that had replicated for 20 generations after exposure to 100 microM SO. Spontaneous differentiation stimulated by high-density culture was also inhibited in SO-treated cells. These results indicate that non-lethal concentrations of SO can cause modifications that compromise the ability of Pc 12 cells to respond to NGF and differentiate.

Adrenal Gland Neoplasms↗