SEARCH · PubMed Health

Results for “TETRAZOLIUM COMPOUNDS”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,531 records · Page 85Linked to original sources

PubMed2007

Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds.

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.

Antimalarials↗

PubMed2006

Epigallocatechin gallate inhibits nitric oxide-induced apoptosis in rat PC12 cells.

Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.

Animals↗

PubMedDate not supplied

Brain injury and protective effects of hypothermia using triphenyltetrazolium chloride in neonatal rat.

Colorless 2,3,5-triphenyltetrazolium chloride (TTC) is reduced by enzymes in functioning mitochondria to a red-colored compound, and has been used to differentiate areas of viable tissue from areas of infarction in adult animals. TTC was used to study the central nervous system protective effects of hypothermia on the neonatal rat exposed to hypoxia and ischemia. The effect of hypothermia on survival and weight gain was also determined. Seven-day-old Wistar rats with right carotid artery ligation were exposed to 3 hours of 8% oxygen and maintained at either 37 degrees C (n = 22) or 30 degrees C (n = 18). The survivors were sacrificed 2 days later and brain slices exposed to TTC. These slices were photographed and the percentage of damage to the right brain was estimated gravimetrically from the stained and unstained areas of enlarged images. The mean weight gains were 4.2 +/- 1.2 gm in the 30 degrees C group and -1.0 +/- 2.8 gm in the 37 degrees C group (P < .001). The survival in the 37 degrees C group was 77% and in the 30 degrees C group 100% (P < .025). The mean percentage damage to the right side of the brain in the 37 degrees C group was 45.5% (range: 0-87.5%); there was no detectable damage in any of the 30 degrees C group pups (P < .0001). In our study, TTC proved to be a rapid and simple method for assessing central nervous system injury in the neonatal rat. This study also confirms that moderate hypothermia is protective against hypoxic-ischemic brain injury.

Animals↗

PubMed2002

Induction of breast cancer resistance protein by the camptothecin derivative DX-8951f is associated with minor reduction of antitumour activity.

DX-8951f (exatecan mesylate), a new water-soluble derivative of camptothecin, is currently being evaluated in phase II clinical trials. Resistance may be acquired when treating cancer patients with DX-8951f. Therefore, we selected a subline of the human ovarian cancer cell line A2780 for resistance against DX-8951f to investigate possible mechanisms of resistance. This DX-8951f-resistant subline, designated 2780DX8 (resistance factor=9.3), displayed a typical cross-resistance pattern including compounds, such as topotecan (resistance factor =34), SN-38 (resistance factor =47), mitoxantrone (resistance factor =59) and doxorubicin (resistance factor =2.9), which have previously been associated with the expression of breast cancer resistance protein. 2780DX8 cells did not show changes in the topoisomerase I gene, in topoisomerase I protein levels or catalytic activity. Overexpression of breast cancer resistance protein could be detected, both at the mRNA and protein level, while staining for Pgp, MRP1, or LRP was negative. GF120918, an inhibitor of breast cancer resistance protein, was able to reverse the DX-8951f-induced resistance in 2780DX8 cells. In vivo experiments in well-established 2780DX8 human tumour xenografts demonstrated that the growth inhibition induced by CPT-11 was more affected by breast cancer resistance protein expression than that of DX-8951f. These data indicate for the first time that DX-8951f is able to induce breast cancer resistance protein as a mechanism of resistance. Breast cancer resistance protein, however, results in only minor reduction of antitumour activity of DX-8951f which is an advantage over topotecan and CPT-11/SN-38.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells.

PURPOSE: The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. METHODS: A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. RESULTS: Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. CONCLUSIONS: These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.

Apoptosis↗

PubMed2005

Proteolytic cleavage of extracellular secreted {alpha}-synuclein via matrix metalloproteinases.

Although alpha-synuclein is the main structural component of the insoluble filaments that form Lewy bodies in Parkinson disease (PD), its physiological function and exact role in neuronal death remain poorly understood. In the present study, we examined the possible functional relationship between alpha-synuclein and several forms of matrix metalloproteinases (MMPs) in the human dopaminergic neuroblastoma (SK-N-BE) cell line. When SK-N-BE cells were transiently transfected with alpha-synuclein, it was secreted into the extracellular culture media, concomitantly with a significant decrease in cell viability. Also the addition of nitric oxide-generating compounds to the cells caused the secreted alpha-synuclein to be digested, producing a small fragment whose size was similar to that of the fragment generated during the incubation of alpha-synuclein with various MMPs in vitro. Among several forms of MMPs, alpha-synuclein was cleaved most efficiently by MMP-3, and MALDI-TOF mass spectra analysis showed that alpha-synuclein is cleaved from its C-terminal end with at least four cleavage sites within the non-Abeta component of AD amyloid sequence. Compared with the intact form, the protein aggregation of alpha-synuclein was remarkably facilitated in the presence of the proteolytic fragments, and the fragment-induced aggregates showed more toxic effect on cell viability. Moreover, the levels of MMP-3 were also found to be increased significantly in the rat PD brain model produced by the cerebral injection of 6-hydroxydopamine into the substantia nigra. The present study suggests that the extracellularly secreted alpha-synuclein could be processed via the activation of MMP-3 in a selective manner.

Alzheimer Disease↗

PubMed2006

Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria.

Cadmium (Cd) is a well-known toxic compound for the kidney in vivo and in vitro. It has been demonstrated to induce nephrotoxicity via in part by apoptotic cell death, but the precise mechanism is still unclear. Therefore, we have studied the effects of Cd on HEK 293 cells and investigated the mechanisms of Cd-induced apoptosis. Studies of morphology and oligonucleosomal DNA fragmentation demonstrated that 30-60 microM Cd induced apoptosis as early as 6-9h with strong effects on MTT activity, whereas 120 microM Cd revealed mainly necrosis, and the result of flow cytometry confirmed it. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 expression was observed, subsequently, release of cytochrome c (Cyt c) and activation of caspase-3 were detected, suggesting a caspase-dependent pathway. Meanwhile, mitochondrial AIF was released to cytoplasm and nucleus, suggesting a caspase-independent pathway. Furthermore, when cells were transfected with pcDNA3/Bcl-2 before exposed to CdCl(2), alleviated apoptosis was assessed by part of the apoptotic features in this study. Taken together, our results showed that CdCl(2) caused time- and dose-dependent apoptosis or even necrosis in HEK 293 cells depending on the exposure conditions. The apoptotic events may involve mitochondrial disruption including both caspase-dependent and -independent pathways.

Apoptosis↗

PubMed2005

Disodium Disuccinate Astaxanthin (Cardax) attenuates complement activation and reduces myocardial injury following ischemia/reperfusion.

Carotenoids are a naturally occurring group of compounds that possess antioxidant properties. Most natural carotenoids display poor aqueous solubility and tend to form aggregates in solution. Disodium disuccinate astaxanthin (DDA; Cardax) is a water-dispersible synthetic carotenoid that rapidly and preferentially associates with serum albumin, thereby preventing the formation of supramolecular complexes and facilitating its efficacy after parenteral administration. This study investigated the ability of DDA to reduce inflammation and myocardial injury in a rabbit model of ischemia/reperfusion. DDA (50 mg/kg/day) or saline was administered i.v. for 4 consecutive days before the initiation of the protocol for induction of myocardial ischemia/reperfusion. On the 5th day, rabbits underwent 30 min of coronary artery occlusion, followed by a 3-h reperfusion period. Myocardial infarct size, as a percentage of the area at risk, was calculated for both groups. Infarct size was 52.5 +/- 7.5% in the vehicle-treated (n = 9) and 25.8 +/- 4.7% in the DDA-treated (n = 9) animals (p < 0.01 versus vehicle; mean myocardial salvage = 51%). To evaluate the anti-inflammatory effects of DDA, complement activity was assessed at the end of reperfusion using a red blood cell lysis assay. DDA administration significantly reduced (p < 0.01) the activation of the complement system in the serum. The current results, coupled with the well established antioxidant ability of carotenoids, suggest that the mechanism(s) of action by which DDA reduces the tissue damage associated with reperfusion injury may include both antioxidant and anticomplement components.

Adjuvants, Immunologic↗

PubMed2004

Comparative study of PM2.5 - and PM10 - induced oxidative stress in rat lung epithelial cells.

Accurate estimation of the exposure-response relationship between ambient urban particulate matters (PM) and public health is important for regulatory perspective of ambient urban particulate matters (PM). Ambient PM contains various transition metals and organic compounds. PM10 (aerodynamic diameter less than 10 micro m) is known to induce diverse diseases such as chronic cough, bronchitis, chest illness, etc. However, recent evaluation of PM2.5 (aerodynamic diameter less than 2.5 micro m) against health outcomes has suggested that the fine particles may be more closely associated with adverse respiratory health effects than particles of larger size. This study was performed to evaluate PM2.5-induced oxidative stress in rat lung epithelial cell in order to provide basic data for the risk assessment of PM2.5. PM2.5 showed higher cytotoxicity than PM10. Also, PM 2.5 induced more malondialdehyde (MDA) formation than PM10. In Hoechst 33258 dye staining and DNA fragmentation assay, apopotic changes were clearly detected in PM2.5 treated cells in compared to PM10. Expression of catalase mRNA was increased by PM2.5 rather than PM10. PM2.5 induced higher Mth1 mRNA than PM10. In pBR322 DNA treated with PM2.5, production of single strand breakage of DNA was higher than that of PM10. In Western blot analysis, PM2.5 induced more Nrf-2 protein, associated with diverse transcriptional and anti-oxidative stress enzymes, compared to PM10. Our data suggest that PM2.5 rather than PM10 may be responsible for PM-induced toxicity. Additional efforts are needed to establish the environmental standard of PM2.5.

Air Pollutants↗

PubMed2005

Beta-carbolines induce apoptosis in cultured cerebellar granule neurons via the mitochondrial pathway.

N-butyl-beta-carboline-3-carboxylate (betaCCB) is, together with 2-methyl-norharmanium and 2,9-dimethylnorharmanium ions, an endogenously occurring beta-carboline. Due to their structural similarities with the synthetic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), harman and norharman compounds have been proposed to be involved in the pathogenesis of Parkinson's disease. While also structurally related, betaCCB has received much less interest in that respect although we had previously demonstrated that it induces the apoptotic cell death of cultured cerebellar granule neurons (CGNs). Herein, we have investigated the molecular events leading to CGN apoptosis upon betaCCB treatment. We first demonstrated that betaCCB-induced apoptosis occurs in neurons only, most likely as a consequence of a specific neuronal uptake as shown using binding/uptake experiments. Then we observed that, in betaCCB-treated CGNs, caspases 9, 3 and 8 were successively activated, suggesting an activation of the mitochondrial pathway. Consistently, betaCCB also induced the release from the mitochondrial intermembrane space of two pro-apoptotic factors, i.e. cytochrome c and apotptosis inducing factor (AIF). Interestingly, no mitochondrial membrane depolarisation was associated with this release, suggesting a mitochondrial permeability transition pore-independent mechanism. The absence of any neuroprotective effect provided by two mPTP inhibitors, i.e. cyclosporine A and bongkrekic acid, further supported this hypothesis. Together, these results show that betaCCB is specifically taken up by neuronal cells where it triggers a specific permeabilization of the outer mitochondrial membrane and a subsequent apoptotic cell death.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Prevention of cisplatin-induced kidney epithelial cell apoptosis with a Cu superoxide dismutase-mimetic [copper2II(3,5-ditertiarybutylsalicylate)4(ethanol)4].

Copper(2)(II)(3,5-ditertiarybutylsalicylate)(4)(ethanol)(4), Cu(2)(II)(3,5-DTBS)(4)(Eth)(4), was synthesized and characterized for evaluation as an anti-apoptotic superoxide dismutase (SOD)-mimetic in an in vitro 50 microM cis-diamminedichloroplatinum(II), [Pt(II)(NH(3))(2)(Cl)(2)]-treated kidney proximal tubule epithelial cell (LLC-PK) preparation. Synthesized Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) was characterized by elemental analysis, FTIR spectrophotometry, and X-ray crystallography. The IC(50) for SOD-mimetic reactivity of Cu(2)(II)(3,5-DTBS)(4)(Eth)(4), determined with the xanthine/xanthine oxidase/nitroblue tetrazolium (NBT) system, was found to be 2.69 microM for the binuclear chelate. Pretreatment of LLC-PK cells with 20 microM Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) prevented 50 microM Pt(II)(NH(3))(2)(Cl)(2)-induced and superoxide-mediated apoptosis. This SOD-mimetic significantly suppressed Pt(II)(NH(3))(2)(Cl)(2)-induced translocation of pro-apoptotic Bax from the cytosol to the inner mitochondrial membrane, prevented Pt(II)(NH(3))(2)(Cl)(2)-induced release of cytochrome c from the inner mitochondrial membrane and the appearance of cytochrome c in the cytosol, and prevented conversion of procaspase-3 to active caspase-3. Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) treatment inhibited Pt(II)(NH(3))(2)(Cl)(2)-mediated tubular cell injury by preventing activation of cellular mechanisms that lead to proximal tubule kidney cell death. Based on these observations, Pt(II)(NH(3))(2)(Cl)(2)- induced O(2)(-)-mediated apoptosis can be mechanistically overcome with a small molecular mass SOD-mimetic, Cu(2)(II)(3,5-DTBS)(4)(Eth)(4). Prior treatment of patients who are to undergo treatment with Pt(II)(NH(3))(2)(Cl)(2) for their neoplastic disease with Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) may be beneficial to these patients.

Animals↗

PubMed1997

Photodynamic therapy inhibits transforming growth factor beta activity associated with vascular smooth muscle cell injury.

PURPOSE: The multifunctional cytokine, transforming growth factor beta 1 (TGF-beta), plays an important role in the development of injury-associated intimal hyperplasia (IH). Strategies to suppress local TGF-beta activity may have a clinical potential to prevent restenosis caused by IH. Photodynamic therapy (PDT) involves the local generation of cytotoxic free radicals by light activation of photosensitizer dyes and has been shown to inhibit experimental IH. This study investigated whether PDT-generated free radicals can affect TGF-beta activity in a biologic system using vascular smooth muscle cells (SMCs). METHODS: The release and activation of TGF-beta by injured SMCs in culture was compared between mechanical injury and PDT. Mechanical injury was induced with a rubber policeman, and PDT was performed with the photosensitizer chloroaluminum sulfonated phthalocyanine (5 micrograms/ml) and 675 nm laser light at subtherapeutic 10 J/cm2 and the in vivo therapeutic dose of 100 J/cm2. Cell viability was assessed by the tetrazolium salt conversion assay, and active and total (active + latent) TGF-beta was determined by enzyme-linked immunosorbent assay in the conditioned media of SMCs 24 hours after treatment. Functional TGF-beta activity was assessed by inhibition of endothelial cell mitogenesis. RESULTS: Both forms of injury severely reduced (p < 0.0005) SMC viability to less than 15%. In untreated SMC conditioned media, only 14.5% of the total TGF-beta was active (27.7 +/- 8.7 pg per 1 x 10(5) cells). However, after mechanical injury and PDT with 10 J/cm2, there was a significant increase (p < 0.02) in active TGF-beta (60.1 +/- 10.1 pg and 48.6 +/- 21.0 pg, respectively), despite a total reduction of approximately 50%. In contrast to this result, PDT with 100 J/cm2 did not result in increased levels of active TGF-beta (8.1 +/- 3.5 pg), despite having similar levels of total TGF-beta. Consequently, the conditioned media of SMCs that had 100 J/cm2 PDT did not inhibit endothelial cell mitogenesis as compared with the conditioned media of SMCs with mechanical injury and 10 J/cm2 PDT (p < 0.0002). CONCLUSIONS: This report describes two novel findings: (1) injury to SMCs in vitro induces the conversion of biologically latent TGF-beta to active TGF-beta; and (2) the therapeutic PDT dose interferes with this injury activation process. This study substantiates the concept of local cytokine inhibition by PDT in a biologic system and provides new insights into the mechanisms of PDT-mediated inhibition of experimental IH.

Aluminum↗

PubMed2006

Glucocorticoids exacerbate hypoxia-induced expression of the pro-apoptotic gene Bnip3 in the developing cortex.

Neonatal administration of the synthetic glucocorticoid, dexamethasone (DEX) retards brain growth, alters adult behaviors and induces cell death in the rat brain, thereby implicating glucocorticoids as developmentally neuroendangering compounds. Glucocorticoids also increase expression of pro-apoptotic Bcl-2 family members and exacerbate expression of hypoxic responsive genes. Bnip3 is a pro-apoptotic Bcl-2 family member that is upregulated in response to hypoxia. In these studies, we investigated the interactions of glucocorticoid receptor and hypoxia in the regulation of Bnip3 mRNA in cortical neurons. Using quantitative real time reverse transcription-polymerase chain reaction, we found that DEX treatment of postnatal days 4-6 rat pups caused a significant increase in Bnip3 mRNA expression compared with vehicle controls. A significant increase in Bnip3 mRNA was also measured in primary cortical neurons 72 h after treatment with RU28362, a glucocorticoid receptor selective agonist. In primary cortical neurons, hypoxia increased Bnip3 mRNA expression and this was exacerbated with RU28362 treatment. To elucidate the mechanism of glucocorticoid- and hypoxia-mediated regulation of Bnip3 transcription, a Bnip3 promoter-luciferase reporter construct was utilized in primary cortical neurons. Upregulation of the Bnip3 promoter was mediated by a single glucocorticoid response element and a hypoxic response element. Bnip3 overexpression in primary cortical neurons significantly increased cell death, which is dependent on the Bnip3 transmembrane domain. However, despite the increased expression of Bnip3 following glucocorticoid and hypoxia treatment, corresponding decreases in cell survival were minimal. These studies identify a novel pathway in the developing cortex through which glucocorticoids may enhance a metabolic insult, such as hypoxia.

Analysis of Variance↗

PubMed2005

Cytotoxic and genotoxic effects of tert-butyl hydroperoxide on Chinese hamster B14 cells.

The organic hydroperoxide, tert-butyl hydroperoxide (t-BHP), is a useful model compound to study mechanisms of oxidative cell injury. In the present work, we examined the features of the interactions of this oxidant with Chinese hamster B14 cells. The aim of our study was to reveal a possible role of structural modifications in membranes and loss of DNA integrity in t-BHP-induced cell injury and death. The tert-butyl hydroperoxide treatment (100-1000 microM, 37 degrees C for 1h) did not decrease cell viability (as measured by cell-specific functional activity with an MTT test), but completely prevented cell growth. We observed intracellular reduced glutathione (GSH) oxidation and total glutathione (GSH+GSSG) depletion, a slight increase in the level of lipid-peroxidation products, an enhancement of membrane fluidity, intracellular potassium leakage and a significant decrease of membrane potential. At oxidant concentrations of 100-1500 microM, a significant damage to DNA integrity was observed as revealed by the Comet assay. The inhibition of cell proliferation (cell-growth arrest) may be explained by genotoxicity of t-BHP, by disturbance of the cellular redox-equilibrium (GSH oxidation) and by structural membrane modifications, which result in ion-non-selective pore formation. The disturbance in passive membrane permeability and the DNA damage may be the most dramatic cell impairments induced by t-BHP treatment. The presence of another oxidant, hypochlorous acid (HOCl), completely prevented t-BHP-induced DNA strand breaks, perhaps due to extracellular oxidation of t-BHP by HOCl.

Analysis of Variance↗

PubMed1997

Effect of CP101,606, a novel NR2B subunit antagonist of the N-methyl-D-aspartate receptor, on the volume of ischemic brain damage off cytotoxic brain edema after middle cerebral artery occlusion in the feline brain.

BACKGROUND AND PURPOSE: The purpose of this study was to test the hypothesis that the neuroprotective compound CP101,606 will ameliorate the increase in lactate, retard the development of cytotoxic edema, and decrease the infarct volume after ischemic stroke. METHODS: Seventeen adult cats were allocated to control (n = 7) and CP101,606-treated groups (n = 10). Transorbital middle cerebral artery occlusion was performed under anesthesia. Extracellular fluid lactate by microdialysis as well as infarct volume measurement by triphenyltetrazolium chloride (TTC)-stained section, with and without neuroprotective agents, was used to determine the value of these potential "surrogate markers" of ischemic damage. RESULTS: The control group showed an increased dialysate lactate (15.5% increase) at 30 minutes and a peak (332.0% increase) in dialysate lactate at 1 hour after middle cerebral artery occlusion compared with the drug-treated group. Significant differences between control and drug-treated groups were seen in the rate of fall of the apparent diffusion coefficient at both 1 and 5 hours. A close correlation was seen between the 1- and 5-hour apparent diffusion coefficient maps and the TTC-stained sections. There was a significantly smaller lesion in the CP101,606-treated group (62.9% reduction in infarct size compared with the control group; P < .001). CONCLUSIONS: CP101,606 ranks very highly among the current neuroprotection candidates for clinical trials, and its excellent safety record in both animals and phase II studies in conscious, moderate head injury patients suggests that it will be highly effective in human occlusive stroke.

Animals↗

PubMed1996

Oxidative stress in acidic conditions increases the production of inositol phosphates in chick retinal cells in culture.

The effect of oxidative stress on the production of [3H]inositol phosphates (InsP) by retinal cells in culture was analyzed. The process of oxidation was induced by incubating the cells with ascorbic acid and ferrous sulphate, and increased extent of oxidation was obtained by varying the pH from neutral to moderate acidosis (pH 6.5). The oxidative process significantly reduced cell viability (about 15%) by decreasing the capacity of mitochondria dehydrogenases to reduce tetrazolium salts, but had no effect on the leakage of lactate dehydrogenase. The production of [3H]InsP, in the absence of receptor activation, was increased dose dependently by oxidative stress. Maximal increases to 189 +/- 7%, 197 +/- 13%, and 329 +/- 22% were observed, respectively, for inositol monophosphates (InsP1), inositol bisphosphates (InsP2), and inositol trisphosphates (InsP3), at 2.5 nmol thiobarbituric acid reactive substances (TBARS)/mg protein. The response to cholinergic receptor activation was slightly decreased in cells oxidized in acidic conditions. Antagonists of glutamate receptors failed to inhibit the enhancement in InsP that occurred upon cellular oxidation, suggesting that the effect was not mediated by activation of glutamate receptors. Cellular oxidation increased by about two fold the uptake of 45Ca2+ in the absence of agonist stimulation. However, stimulation of phospholipase C by Ca2+ did not mediate the increase in [3H]InsP upon cell oxidation in acidic conditions, because the addition of 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1-H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C-dependent processes, did not affect the production of [3H]InsP in oxidized cells. Nevertheless, U-73122 significantly inhibited carbachol- and K(+)-stimulated accumulation of [3H]InsP. Furthermore, the enhancement of [3H]InsP induced by ascorbate/Fe2+ was still observed in the absence of external Ca2+. This increase in the production of InsP did not substantially induce the release of Ca2+ from internal stores. The results suggest that both Ca(2+)-dependent and Ca(2+)-independent pathways are involved in oxidative stress-mediated InsP increment, and that the enzymes of the InsP metabolism may be affected by oxidation.

Animals↗

PubMed2001

Differential effects of non-genotoxic carcinogens and proliferating agents on cell growth, survival and apoptosis in hepatic cells in vitro.

Many nongenotoxic carcinogen's (ngc) produce hyperplastic lesions from which neoplastic foci may arise. Modulation of the rate of apoptosis by some ngc's within these lesions may be critical to their mechanism of tumour promotion but some may be cytotoxic. To establish if these compounds are apoptotic or necrotic in vitro, three ngc's (12-0-tetradecanoyl phorbol-13-acetate (TPA); nickel, and di(2-ethylhexyl-phthalate (DEHP), two noncarcinogenic hepatoproliferating agents (1,4-dichlorobenzene (DCB; HGF) and an in vitro genotoxic reference compound (7-hydroxy-2-acetylaminofluorene (70H2AAF) were used to induce mitogenic or growth responses in two liver cell-lines HepG2 and JTC-15. MTT and 3H-thymidine incorporation assays were used to measure cell growth and DNA replicative activity respectively. Rates of apoptosis were assayed using FITC-annexin V with propidium iodide staining and flow cytometry. Responses in HepG2 cells were HGF (proliferation at > or = 3 ng/ml), TPA (cell growth at > or = 8 ng/ml), DEHP (proliferation at > or = 0.05 microg/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.001 microg/ml and 100 ng/ml respectively. An equivocal result was obtained for DCB. Responses in JTC-15 cells were HGF (proliferation, 3 ng/ml), TPA (DNA replication, 10 ng/ml), and DEHP (cell mass, 2.5 microl/ml). NiCl2 and 70H-2AAF were cytotoxic above 0.01 microg/ml and 110 mg/ml respectively. Equivocal results were obtained for DCB. In flow cytometry assays apoptotic and necrotic populations were not clearly separable. Approximate rates of apoptosis in HepG2 were: control 8.7%; DEHP, 10.19%. NiCl2, 12.67%; 70H2AAF, 16.56%; TPA, 19.72%; HGF, 23.73%; DCB, 24.59%; positive apoptotic control (taxol) 26.94%. These data show apoptosis was increased in chemically activated populations of HepG2. The ngc, DEHP, unexpectedtly produced proliferation in HepG2 and almost totally suppressed apoptosis in vitro in HepG2 relative to the non-carcinogenic hepatoproliferators. The rate of apoptosis induced by the ngc TPA was not considered to be sufficiently different to the rates of apoptosis induced by the noncarcinogenic hepatoproliferators. The results emphasize the importance of considering necrotic reactions from effects on apoptosis in detecting non-genotoxic carcinogens.

Apoptosis↗

PubMed1990

Histochemical localization of formaldehyde dehydrogenase in the rat.

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or ten-micrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD+, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowman's and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose (M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560-567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.

Administration, Inhalation↗