Biomedical subjects

H de Groot

Publications and source records attributed to H de Groot.

At least 37 records · Page 2Linked to original sources

PubMed2000

Protection by glycine against hypoxic injury of rat hepatocytes: inhibition of ion fluxes through nonspecific leaks.

BACKGROUND/AIMS: Glycine has long been shown to exert strong protective effects against hypoxic injury of hepatocytes. Recently, it was suggested that glycine exerts this protection via inhibition of ligand-gated chloride channels, thereby secondarily inhibiting sodium influx. The purpose of this study was to examine this suggestion. METHODS: Cultured rat hepatocytes were incubated under normoxic and hypoxic conditions. Loss of viability was determined by release of lactate dehydrogenase. Cytosolic ion concentrations were measured using digital fluorescence microscopy. RESULTS: Glycine prevented the hypoxic increase in cytosolic sodium and strongly protected against hypoxic injury. The amino acid was not only protective in Krebs-Henseleit buffer but also in a chloride-free modification thereof and offered additional protection in a sodium-free medium (which already yielded substantial protection in its own right). Glycine also prevented the hypoxic release of the anionic fluorescent dye Newport Green and appeared to prevent the hypoxic entrance of the "nonphysiological" cations cobalt and nickel. CONCLUSION: The results strongly argue against inhibition of ligand-gated chloride channels as being responsible for the potent protective effect of glycine against hypoxic injury of hepatocytes. Instead, they suggest that glycine prevents the formation of nonspecific leaks for small ions including sodium, thereby providing protection.

Adenosine Triphosphate↗

PubMed2000

In vitro effects of hydrogen peroxide on the cochlear neurosensory epithelium of the guinea pig.

Reactive oxygen species (ROS) have been postulated to be involved in drug ototoxicity and noise-induced hearing loss. Hydrogen peroxide (H(2)O(2))-induced cell damage in the inner ear was investigated using the neurosensory epithelium of a guinea pig cochlea. Hair cells and supporting cells of the epithelium incubated in Hanks' balanced salt solution were viable up to 6 h. After 2 h of treatment with 0.2 mM H(2)O(2) about 85% of the outer hair cells lost their viability. In contrast inner hair cells slowly began to die after 2 h of H(2)O(2) treatment. The Deiters cells and Hensen cells did not show any signs of damage in the presence of H(2)O(2). Nifedipine, a calcium channel blocker, Quin-2 AM, an intracellular calcium chelator, and 2,2'-dipyridyl, a membrane-permeable iron chelator, all provided partial protection against H(2)O(2)-induced outer hair cell death. The combination of both chelators showed an additional protective effect. The antioxidants N-acetylcysteine and glutathione-monoethyl ester completely protected against H(2)O(2) damage. These results suggest that calcium, iron, and thiol homeostasis play a crucial role in hair cell death caused by H(2)O(2).

2,2'-Dipyridyl↗

PubMed2000

Carbon dioxide but not bicarbonate inhibits N-nitrosation of secondary amines. Evidence for amine carbamates as protecting entities.

Hydrogen carbonate (bicarbonate, HCO(3)(-)) has been proposed to accelerate the decomposition of N(2)O(3) because N-nitrosation of morpholine via a nitric oxide/oxygen mixture ((*)NO/O(2)) was inhibited by the addition of HCO(3)(-) at pH 8.9 [Caulfield, J. L., Singh, S. P., Wishnok, J. S., Deen, W. M., and Tannenbaum, S. R. (1996) J. Biol. Chem. 271, 25859-25863]. In the study presented here, it is shown that carbon dioxide (CO(2)) is responsible for this kind of protective effect because of formation of amine carbamates, whereas an inhibitory function of HCO(3)(-) is excluded. N-Nitrosation of morpholine (1-10 mM) at pH 7.4-7.5 by the (*)NO-donor compounds PAPA NONOate and MAMA NONOate (0.5 mM each) was not affected by the presence of large amounts of HCO(3)(-) (up to 100 mM) in aerated aqueous solution. Similar results were obtained by replacing the (*)NO-donor compounds with authentic (*)NO (900 microM). In agreement with data from the study cited above, (*)NO/O(2)-mediated formation of N-nitrosomorpholine (NO-Mor) was indeed inhibited by about 45% in the presence of 50 mM HCO(3)(-) at pH 8.9. However, 500 MHz (13)C NMR analysis with (13)C-enriched bicarbonate revealed that significant amounts of morpholine carbamate are formed from reaction of equilibrated CO(2) with morpholine (1-100 mM) at pH 8.9, but only to a minor extent at pH 7. 5. The protective effect of morpholine carbamate formation is explained by a significantly reduced charge density at nitrogen. This view is supported by the results of density functional theory/natural population analysis, i.e., quantumchemical calculations for morpholine and morpholine carbamate. In agreement with its lower pK(a), another secondary amine, piperazine, had already produced significant amounts of piperazine carbamate at pH 7. 4 as shown by (13)C NMR spectrometry. Consequently, and in contrast to morpholine, N-nitrosation of piperazine (2 mM) by both (*)NO/O(2) (PAPA NONOate, 0.5 mM) and the (*)NO/O(2)(-)(*)-releasing compound SIN-1 (1 mM) was inhibited by about 66% in the presence of 200 mM HCO(3)(-).

Bicarbonates↗

PubMed2000

Dietary linoleic acid divergently affects immune responsiveness of growing layer hens.

The effects of linoleic (LA)- and linolenic acid (LNA)-enriched diets on humoral and in vivo cellular immune responses to keyhole limpet hemocyanin (KLH)-dinitrophenyl (DNP) and Mycobacterium butyricum were studied in growing layer hens. Pullets were fed one of three diets: a control, LA enriched, or LNA enriched. Pullets were assigned to one of three immunization treatments: KLH-DNP, M. butyricum, or PBS. The LA-enriched diet enhanced the antibody response to KLH in pullets immunized with KLH-DNP. On the other hand, the antibody response to M. butyricum in M. butyricum-immunized birds was decreased by feeding an LA-enriched diet. In vitro lymphocyte proliferation in the presence of Concanavalin A was affected by the interaction between diet and immunization. Neither cutaneous hypersensitivity to KLH nor to M. butyricum was affected by the diet. The BW gain before immunization was not affected by the diet, but after immunization, the LA-enriched diet enhanced growth in birds immunized with M. butyricum. Diets had various effects on organ weights. We concluded that dietary linoleic acid enrichment of the diet has an antigen-dependent divergent effect on the antibody response. The dietary LNA effect on the antibody response is less pronounced and is opposite to that of the LA effect.

Animals↗

PubMed2000

Nitric oxide detection and visualization in biological systems. Applications of the FNOCT method.

Fluorescent Nitric Oxide Cheletropic Traps (FNOCTs) were applied to specifically trap nitric oxide (NO) with high sensitivity. The fluorescent o-quinoid pi-electron system of the FNOCTs (lambda(exc) = 460 nm, lambda(em) = 600 nm) reacts rapidly with NO to a fluorescent phenanthrene system (lambda(exc) = 380 nm, lambda(em) = 460 nm). The cyclic nitroxides thus formed react further to non-radical products which exhibit identical fluorescence properties. Using the acid form of the trap (FNOCT-4), NO release by spermine NONOate and by lipopolysaccharide (LPS)-activated alveolar macrophages were studied. A maximum extracellular release of NO of 37.5 nmol h(-1) (10(6) cells)(-1) from the macrophages was determined at 11 h after activation. Furthermore, intracellular NO release by LPS-activated macrophages and by microvascular omentum endothelial cells stimulated by the Ca2+ ionophore A-23187, respectively, was monitored on the single cell level by means of fluorescence microscopy. After loading the cells with the membrane-permeating acetoxymethylester derivative FNOCT-5, which is hydrolyzed to a non-permeating dicarboxylate by intracellular hydrolases, NO formation by the endothelial cells started immediately upon stimulation, whereas start of NO production by the macrophages was delayed with a variation between 4 and 8 h for individual cells. These results demonstrate that the FNOCTs can be used to monitor NO release from single cells, as well as from NO-donating compounds, with high sensitivity and with temporal and spatial resolution.

Animals↗

PubMed1999

[Prevalence of IgE-mediated allergy to natural rubber latex in operation room personnel of Rotterdam].

OBJECTIVE: To determine the prevalence of IgE-mediated latex allergy in workers in an operation room (OR) complex. DESIGN: Descriptive. METHOD: Regular OR staff members of Rotterdam University Hospital were tested in May-July 1998 for latex allergy and cross-reacting allergens. Questionnaires, serologic testing and skin prick tests with different extracts were used. RESULTS: The study group comprised 163 persons (response: 70%), 30 men and 133 women, with a mean age of 38 years (range: 18-60). Twenty-three persons (14.1%) had specific IgE antibodies against latex. Persons with an atopic constitution ran an increased risk of latex sensitization (odds ratio: 4.3; 95% confidence interval: 1.6-11.4). Of these 23 persons, 16 showed symptoms of urticaria, angio-oedema, rhinoconjunctivitis and/or dyspnoea. CONCLUSION: IgE-mediated allergy to natural rubber latex was prevalent in 23/163 (14.1%) of the OR personnel.

Adult↗

PubMed1999

Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide.

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlled processes. Under nonenzymatic conditions, a couple of one-electron oxidants easily oxidize NADH to the NAD(.) radical. This radical reduces molecular oxygen to the superoxide radical (O-(2)) at a near to the diffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H(2)O(2)). Because peroxynitrite can act as a one-electron oxidant, the reaction of NAD(P)H with both authentic peroxynitrite and the nitric oxide ((. )NO) and O-(2) releasing compound 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with an efficiency of approximately 25 and 8% in the absence and presence of bicarbonate/carbon dioxide (HCO(3)(-)/CO(2)), respectively. NADH reacted 5-100 times faster with peroxynitrite than do the known peroxynitrite scavengers glutathione, cysteine, and tryptophan. Furthermore, NADH was found to be highly effective in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO(3)(-)/CO(2). Reaction of NADH with authentic peroxynitrite resulted in the formation of NAD(+) and O-(2) and, thus, of H(2)O(2) with yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave virtually the same results; however, two remarkable exceptions were recognized. First, the efficiency of NADH oxidation increased to 60-90% regardless of the presence of HCO(3)(-)/CO(2), along with an increase of H(2)O(2) formation to about 23 and 35% relative to the amounts of added SIN-1 and NADH. Second, and more interesting, the peroxynitrite scavenger glutathione (GSH) was needed in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH half-maximal in the presence of HCO(3)(-)/CO(2). Similar results were obtained with NADPH. Hence, peroxynitrite or radicals derived from it (such as, e.g. the bicarbonate radical or nitrogen dioxide) indeed oxidize NADH, leading to the formation of NAD(+) and, via O-(2), of H(2)O(2). When peroxynitrite is generated in situ in the presence of HCO(3)(-)/CO(2), i.e. under conditions mimicking the in vivo situation, NAD(P)H effectively competes with other known scavengers of peroxynitrite.

Free Radical Scavengers↗

PubMed1999

Protection against hydrogen peroxide cytotoxicity in rat-1 fibroblasts provided by the oncoprotein Bcl-2: maintenance of calcium homoeostasis is secondary to the effect of Bcl-2 on cellular glutathione.

The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by dl-buthionine-(S, R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism.

Acetylcysteine↗

PubMed1999

Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK.

The intracellular pool of chelatable iron is considered to be a decisive pathogenetic factor for various kinds of cell injury. We therefore set about establishing a method of detecting chelatable iron in isolated hepatocytes based on digital fluorescence microscopy. The fluorescence of hepatocytes loaded with the fluorescent metal indicators, phen green SK (PG SK), phen green FL (PG FL), calcein, or fluorescein desferrioxamine (FL-DFO), was quenched when iron was added to the cells in a membrane-permeable form. It increased when cellular chelatable iron available to the probe was experimentally decreased by an excess of various membrane-permeable transition metal chelators. The quenching by means of the ferrous ammonium sulfate + citrate complex and also the "dequenching" using 2,2'-dipyridyl (2,2'-DPD) were largest for PG. We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calibration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of excess 2,2'-DPD. Using this method, we found 9.8 +/- 2.9 micromol/L (mean +/- SEM; n = 18) chelatable iron in rat hepatocytes, which constituted 1.0% +/- 0.3% of the total iron content of the cells as determined by atomic absorption spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 +/- 1.3 micromol/L; mean +/- SEM; n = 8), which were used for comparison. This method allowed us to record time courses of iron uptake and of iron chelation by different chelators (e.g., deferoxamine, 1,10-phenanthroline) in single, intact cells.

Animals↗

PubMed1999

Allergy to bumblebee venom. III. Immunotherapy follow-up study (safety and efficacy) in patients with occupational bumblebee-venom anaphylaxis.

BACKGROUND: The objective was to investigate whether venom immunotherapy with bumblebee venom (BBV) is safe and effective. METHODS: Eleven patients with severe occupational anaphylaxis caused by stings of bumblebees were studied. Sensitization to bumblebee venom was confirmed by skin tests and RAST. Immunotherapy was started with bumblebee venom extract by the semirush procedure, because these patients showed a primary sensitization to Bombus venom, and a low or absent degree of cross-reactivity with honeybee venom. IgE titer and skin tests with bumblebee venom were performed yearly. Efficacy was evaluated by means of in-hospital sting challenge and/or occupational field stings from bumblebees. RESULTS: All patients reached maintenance dose in 6 weeks without severe side-effects. During the follow-up period (1.5-5 years), three systemic reactions in two patients were seen in 20 bumblebee stings. However, these reactions were milder than the index sting. CONCLUSIONS: Immunotherapy with bumblebee venom is safe and effective, and is comparable with honeybee and yellow-jacket venom immunotherapy.

Adult↗

PubMed1999

Cold-induced apoptosis in cultured hepatocytes and liver endothelial cells: mediation by reactive oxygen species.

When cultured hepatocytes were incubated in cell culture medium at 4 degreesC for up to 30 h and then returned to 37 degreesC, blebbing of the plasma membrane, cell detachment, chromatin condensation and margination, enhanced nuclear stainability with Hoechst 33342, ruffling of the nuclear membrane, and DNA fragmentation occurred. Similar to hepatocytes, cultured liver endothelial cells exhibited blebbing, chromatin condensation and margination, marked nuclear condensation, and increased stainability with Hoechst 33342 when exposed to hypothermia/rewarming. In both cell types, the occurrence and extent of these alterations were dependent on the duration of the cold incubation period. This cold-induced apoptosis was inhibited by hypoxia, by an array of free radical scavengers/antioxidants, and by iron chelators. However, the extent of the protection by the different antioxidants was different in the two cell types: iron chelators provided complete protection in liver endothelial cells but only partial protection in hepatocytes, whereas lipophilic antioxidants such as alpha-tocopherol provided complete protection in both cell types. During cold incubation, and especially during rewarming, lipid peroxidation occurred. These results suggest that the formation of reactive oxygen species (ROS) is a key mediator of cold-induced apoptosis, with ROS formation being completely iron-mediated in liver endothelial cells and partially iron-mediated in hepatocytes.

Animals↗

PubMed1998

Hydrogen peroxide formation by reaction of peroxynitrite with HEPES and related tertiary amines. Implications for a general mechanism.

Organic amine-based buffer compounds such as HEPES (Good's buffers) are commonly applied in experimental systems, including those where the biological effects of peroxynitrite are studied. In such studies 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), a compound that simultaneously releases nitric oxide (.NO) and superoxide (O-2), is often used as a source for peroxynitrite. Whereas in mere phosphate buffer H2O2 formation from 1.5 mM SIN-1 was low ( approximately 15 microM), incubation of SIN-1 with Good's buffer compounds resulted in continuous H2O2 formation. After 2 h of incubation of 1.5 mM SIN-1 with 20 mM HEPES about 190 microM H2O2 were formed. The same amount of H2O2 could be achieved from 1.5 mM SIN-1 by action of superoxide dismutase in the absence of HEPES. The increased H2O2 level, however, could not be related to a superoxide dismutase or to a NO scavenger activity of HEPES. On the other hand, SIN-1-mediated oxidation of both dihydrorhodamine 123 and deoxyribose as well as peroxynitrite-dependent nitration of p-hydroxyphenylacetic acid were strongly inhibited by 20 mM HEPES. Furthermore, the peroxynitrite scavenger tryptophan significantly reduced H2O2 formation from SIN-1-HEPES interactions. These observations suggest that peroxynitrite is the initiator for the enhanced formation of H2O2. Likewise, authentic peroxynitrite (1 mM) also induced the formation of both O-2 and H2O2 upon addition to HEPES (400 mM)-containing solutions in a pH (4.5-7.5)-dependent manner. In accordance with previous reports it was found that at pH >/=5 oxygen is released in the decay of peroxynitrite. As a consequence, peroxynitrite(1 mM)-induced H2O2 formation ( approximately 80 microM at pH 7.5) also occurred under hypoxic conditions. In the presence of bicarbonate/carbon dioxide (20 mM/5%) the production of H2O2 from the reaction of HEPES with peroxynitrite was even further stimulated. Addition of SIN-1 or authentic peroxynitrite to solutions of Good's buffers resulted in the formation of piperazine-derived radical cations as detected by ESR spectroscopy. These findings suggest a mechanism for H2O2 formation in which peroxynitrite (or any strong oxidant derived from it) initially oxidizes the tertiary amine buffer compounds in a one-electron step. Subsequent deprotonation and reaction of the intermediate alpha-amino alkyl radicals with molecular oxygen leads to the formation of O-2, from which H2O2 is produced by dismutation. Hence, HEPES and similar organic buffers should be avoided in studies of oxidative compounds. Furthermore, this mechanism of H2O2 formation must be regarded to be a rather general one for biological systems where sufficiently strong oxidants may interact with various biologically relevant amino-type molecules, such as ATP, creatine, or nucleic acids.

Amines↗

PubMed1998

Cold preservation of isolated rabbit proximal tubules induces radical-mediated cell injury.

BACKGROUND: Reactive oxygen species (ROS) are involved in reperfusion injury after preservation. Recent studies in isolated endothelial cells and hepatocytes suggested the occurrence of ROS-mediated injury during the period of cold incubation. In the present study, formation of ROS and subsequent cell injury were studied in freshly isolated rabbit proximal tubules (PTs). METHODS: PTs were incubated in University of Wisconsin (UW) solution, Euro-Collins solution, or a modified Krebs-Henseleit buffer under aerobic conditions for up to 94 hr at 4 degrees C. ROS formation and cell death were assessed as lipid peroxidation (formation of thiobarbituric acid-reactive substances [TBARS]) and release of lactate dehydrogenase, respectively. The involvement of ROS was further investigated in UW solution using compounds that might interfere with ROS formation. In addition, tubules were studied under anaerobic conditions (gassing with 95% N2/5% CO2). RESULTS: Cold preservation of rabbit PTs in any of the solutions under aerobic conditions caused progressive lipid peroxidation and concomitant cell injury. Addition to UW solution of inhibitors of ROS formation, in particular 2,2'-dipyridyl, or removal of oxygen by gassing with 95% N2/5% CO2, prevented lipid peroxidation and protected rabbit PTs against cold injury. Both the nitric oxide (NO) synthase inhibitor L-NAME and dexamethasone, which blocks the inducible NO synthase, were ineffective. The cytoprotectant glycine affected neither TBARS formation nor lactate dehydrogenase release. CONCLUSIONS: Cold preservation of renal PTs under aerobic conditions caused cell injury even in the specially designed preservation solution UW. Cell injury is caused by iron-dependent, NO synthase-independent ROS formation.

Adenosine↗

PubMed1998

The critical role of Hepes in SIN-1 cytotoxicity, peroxynitrite versus hydrogen peroxide.

The cytotoxicity of the superoxide anion radical- and nitric oxide-releasing compound SIN-1 to L929 cells was studied in Krebs-Henseleit buffer. pH 7.4, in the presence and absence of Hepes. SIN-1 cytotoxicity was significantly higher in the presence of Hepes than in the absence of Hepes. The available amount of peroxynitrite formed from SIN-1, however, was significantly decreased by Hepes as indicated by decreased oxidation of dihydrorhodamine 123. On the other hand, Hepes largely increased the formation of H2O2 from SIN-1. Catalase protected the L929 cells from SIN-1 cytotoxicity in the buffer with Hepes. In the buffer without Hepes catalase did not have any protective effect. In contrast, tyrosine and tryptophan provided significant protection against SIN-1 cytotoxicity independent of the presence of Hepes. These results demonstrate that the immediate toxic agent formed from SIN-1 decisively depends on the presence of Hepes. In its absence cytotoxicity is most likely mediated by peroxynitrite while in the presence of Hepes, cytotoxicity is conveyed by co-operative action of hydrogen peroxide and reactive nitrogen species.

Animals↗

PubMed1998

Auxiliary liver transplantation with arterialization of the portal vein for acute hepatic failure.

Six adult patients suffering from acute hepatic failure and with a high urgent status underwent heterotopic auxiliary liver transplantation. In four of these patients, the portal vein of the liver graft was arterialized in order to leave the native liver and the liver hilum untouched and to be able to place the liver graft wherever space was available in the abdomen. The arterial blood flow via the portal vein was tapered by the width of the anastomosis. Two patients died, one of sepsis on postoperative day 17 (POD), the other after 3 months due to a severe CMV pneumonia. There were no technically related deaths. The native liver showed early regeneration in all cases. In one patient, the auxiliary graft was removed 6 weeks after transplantation. Four weeks later, he had to undergo orthotopic retransplantation due to a recurrent fulminant failure of the recovered native liver. This patient is alive more than 1 year after the operation. We conclude that heterotopic auxiliary liver transplantation with portal vein arterialization is a suitable approach to bridging the recovery of the acute failing native liver.

Acute Disease↗

PubMed1998

Cold-induced release of reactive oxygen species as a decisive mediator of hypothermia injury to cultured liver cells.

The mechanisms of hypothermia-induced cell injury are still unclear. The present study provides experimental evidence for the involvement of reactive oxygen species in hypothermia injury: cultured rat hepatocytes incubated in cold (4 degrees C) Krebs-Henseleit buffer or cell culture medium were injured under normoxic conditions and even more so under hyperoxic conditions, whereas the hepatocytes were protected under hypoxic conditions. During warm (37 degrees C) incubation in cell culture medium, on the other hand, cell injury was minimal under normoxic conditions, only slightly increased under hyperoxic conditions, but substantially increased under hypoxic conditions. The injury occurring during cold normoxic incubation was also largely decreased by the addition of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide, the hydroxyl radical scavenger dimethyl sulfoxide, the flavonoid silibinin, or the transition metal chelator 2,2'-dipyridyl to the medium, or by preincubating the cells with the iron chelator deferoxamine or the lipophilic antioxidant alpha-tocopherol before the hypothermic incubation. In addition, marked lipid peroxidation was observed during cold incubations without inhibitors, but not during warm incubations. Similar results were obtained with cultured rat liver endothelial cells. These results suggest that in hepatocytes and in liver endothelial cells, cold-induced release of reactive oxygen species, most likely of hydroxyl radicals, is the main injurious factor under hypothermic conditions.

2,2'-Dipyridyl↗

PubMed1998

Calcium vs. iron-mediated processes in hydrogen peroxide toxicity to L929 cells: effects of glucose.

H2O2 toxicity was studied in L929 cells in the presence and absence of glucose. The data obtained in the absence of glucose suggest a Ca2+-dependent mechanism of cell injury. No evidence was found for any involvement of iron in the process. In particular, cell injury was unaffected by the intracellular iron chelators 2,2'-dipyridyl and deferoxamine or by the hydroxyl radical scavengers DMSO and DMPO. On the other hand, the intracellular Ca2+ chelator BAPTA/AM provided significant protection. The cytosolic Ca2+ level rapidly and consistently increased after H2O2 addition, prior to visible bleb formation and loss of cell viability. Additionally, GSH not only prevented cell death but also significantly decreased cytosolic calcium accumulation. In the presence of glucose, however, Ca2+ does not seem to play any role in H2O2 toxicity. Cell death is now mainly mediated by iron: the iron chelators and hydroxyl radical scavengers prevented cell injury, the increase in cytosolic Ca2+ was significantly less pronounced, and BAPTA/AM did not exert any protection under these conditions. Hence, the metabolic state of the L929 cells, as given by the availability of glucose, decisively determines the biochemical mechanism of H2O2 cell injury.

Animals↗

PubMed1998

Ca2+-dependent cytotoxicity of H2O2 in L929 cells: the role of H2O2-induced Na+-influx.

We investigated the mechanism by which H2O2 mediates an increase in [Na+]i in L929 cells and the relevance of this Na+ load for H2O2-induced cell injury. [Na+]i increased early after exposure to H2O2 as monitored by fluorescence spectrophotometry of cells loaded with SBFI. The omission of Na+ from the incubation buffer significantly reduced H2O2-cytotoxicity. This protection could not be mimicked by inhibition of either the Na+/H+-antiporter, the Na+/HCO3- -cotransporter, or the Na+/K+/2Cl- -cotransporter by using Hoechst 694 (0.02 mM) or 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS) (0.02 mM) or furosemide (1 mM) and bumetanide (0.5 mM). Only the blocker of the Na+/Ca2+-exchanger bepridil (0.2 mM) significantly reduced H2O2-cytotoxicity but without interfering with the increase in [Na+]i. H2O2 caused a rapid and sustained increase in [Ca2+]i, which was significantly reduced in bepridil pretreated cells and after replacing extracellular Na+ by choline. H2O2 was found to initiate a cellular uptake of unphysiological Ni2+ by using Newport Green diacetate as fluorescent dye. Our data suggest that H2O2 mediates Na+-influx across the plasma membrane rather unspecifically than through specific transporters. The protective effect of bepridil against H2O2-cytotoxicity occurs as a consequence of a reduced cellular Ca2+-uptake. We conclude that H2O2-mediated unspecific accumulation of Na+ seems to favor a Ca2+-influx into the cells, which takes place on the Na+/Ca2+-exchanger operating in reverse mode in exchange for Na+-efflux. Therefore, H2O2-induced cellular Na+ accumulation appears to play a permissive rather than a triggering role in H2O2-mediated cell injury.

Animals↗