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PubMed1998

Triclosan: cytotoxicity, mode of action, and induction of apoptosis in human gingival cells in vitro.

The in vitro cytotoxicology of triclosan, the active ingredient in some mouthrinses and dentifrices used in the prevention and treatment of gingivitis and plaque, was studied using the Smulow-Glickman (S-G) human gingival epithelial cell line. The 24 h midpoint cytotoxicity value was 0.05-0.06 mM triclosan as assessed with the neutral red (NR) assay. Triclosan is used in dentifrices in combination with either zinc citrate or sodium fluoride (NaF). The sequence of potencies of these test agents, as assessed with the NR assay, was triclosan>zinc citrate>>NaF; combinations of triclosan + zinc citrate and triclosan + NaF were additive in their toxicities. Damage to the integrity of the plasma membrane, as assessed by the leakage of lactic acid dehydrogenase during a 3-h exposure, was initially evident with 0.1 mM triclosan. When exposed to triclosan for 3 d, a lag in the growth kinetics of the S-G cells was first observed at 0.01 mM triclosan. A reduction in attachment of S-G cells to dentin chips, previously exposed to triclosan for 1 h, was noted at 0.25 mM triclosan and greater. Triclosan-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis of extracted oligonucleosomal fragments.

Anti-Infective Agents, Local↗
PubMed2003

Cytotoxicity of bisphenol A glycidyl methacrylate on cytochrome P450-producing cells.

Of the cytochrome P450 (CYP) family of carcinogen-activating enzymes, CYP3A is the major form found in human livers. The purpose of this study was to investigate the cytotoxic effects of dental resin monomers after being metabolized by CYP3A4 and CYP3A7, using a colony formation assay and a neutral red assay. Specimen wells were plated with transfected cells derived from the Chinese hamster lung at 100 cells well(-1). The experimental group consisted of CYP-producing 3A4-10 and 3A7-40 cells, while the control group consisted of non-CYP-producing CR-119 cells. Bisphenol A (BPA) and bisphenol A glycidyl methacrylate (Bis-GMA) and a positive control (Aflatoxine Bl) were added separately to each well and cultured for 7 days. After cultivation, the number of the colonies was counted and IC50 values were determined. The data were statistically analysed by a Student's t-test. The resultant of IC50 values indicated that the monomers were not metabolically activated by CYP3A4 or CYP3A7 as compared with the control (P < 0.05). We also confirmed that these monomers act neither as activators nor as inhibitors of CYP3A4 and CYP3A7.

Aflatoxins↗
PubMed2000

Cytotoxicity in pig hepatocytes induced by 8-quinolinol, chloramine-T and natamycin.

The potential cytotoxic effects of the compounds 8-quinolinol, chloramine-T and natamycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH), 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) efflux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR20, NR50, and NR80, and MTT20, MTT50 and MTT80 enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. Also for the parameters LDH and GSH, chloramine-T appears to be less cytotoxic than natamycin and 8-quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs, therefore avoiding possible extrapolation problems due to species differences.

Animals↗
PubMed1998

Pro-oxidant role of superoxide dismutase in ultraviolet-A-induced lipid peroxidation in cultured normal human skin fibroblasts.

Exposure of cultured normal human skin fibroblasts to ultraviolet A triggers lipid peroxidation. In sharp contrast with the tert-butylhydroperoxide-induced lipid peroxidation, the ultraviolet-A-induced lipid peroxidation is inhibited by treating cells with diethyldithiocarbamate. Diethyldithiocarbamate decreases superoxide dismutase activity and, to a lesser extent, the total glutathione level. Catalase and glutathione peroxidase, however, are unaffected. The decrease in the superoxide dismutase activity parallels an inhibition of H2O2 formation in both irradiated and unirradiated cells. The protection against lipid peroxidation may thus be associated with superoxide dismutase inhibition. Membrane damage revealed by neutral red uptake is not prevented by diethyldithiocarbamate.

Antioxidants↗
PubMed1994

Effects of organotins on rat brain astrocytes in culture.

The interaction of triethyltin (TET) and trimethyltin (TMT) with rat brain astrocytes in vitro was investigated. Both compounds are highly neurotoxic after in vivo application, cause neurobehavioral changes, and elicit neuronal and glial responses in the CNS. In this study, 5-week-old cultures were exposed to TMT or TET (0.1-2.5 microM) for 24 h. A concentration-dependent cytotoxicity was observed for both agents by vital dye uptake assay using neutral red (NR). The order of potency for half-maximal cytotoxicity (NR-50) was TET (0.7 microM) > TMT (2.5 microM), in agreement with results found after in vivo administration. TET and TMT caused similar morphological changes: large holes extending through the plasma membrane appeared initially in the flattened cell bodies, cytoplasmic extensions were retracted, and long cellular processes formed. Later, the cell bodies rounded up and had only a few extremely long and thin processes. Indirect immunofluorescence staining using anti-vimentin and anti-glial fibrillary acidic protein (GFAP) antibodies revealed that the orderly array of the intermediate filament system was severely disturbed. At lower concentrations, an increased bundling was observed, and at higher concentrations the disassembly of the intracellular framework was seen, and cellular staining appeared rather diffuse. Western blot analysis of cellular extracts was carried out to determine the protein levels of GFAP and vimentin. In this culture system, TET and TMT caused an almost two-fold increase in the levels of GFAP at concentrations around and below NR-50, indicating that astrocytes react to organotins independently of neuronal signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts.

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

Cell Differentiation↗
PubMed1998

Shiga toxin 1 elicits diverse biologic responses in mesangial cells.

BACKGROUND: Shiga toxin 1 (Stx1) is a causative agent in hemolytic uremic syndrome (HUS). Its receptor, the glycosphingolipid globotriaosylceramide (Gb3), is expressed on cultured human endothelial and mesangial cells. Mesangial cell injury in HUS ranges from mild cellular edema to severe mesangiolysis and eventual glomerulosclerosis. We hypothesized that, in addition to endothelial cells, mesangial cells are targets of Stx1. METHODS: Human mesangial cells were exposed to Stx1. Protein synthesis was measured using [35S]-methionine/cysteine. Cell viability was measured as the lysosomal uptake of Neutral Red. Monocyte chemotactic peptide (MCP-1) mRNA and protein were analyzed by Northern blotting and ELISA. RESULTS: Stx1 (0.25 to 2500 ng/ml) resulted in a dose-dependent inhibition of protein synthesis. This effect of Stx1 was potentiated by preincubation of the cells with interleukin-1alpha (IL-1alpha; 2 ng/ml) or tumor necrosis-alpha (TNF-alpha; 500 U/ml). Stx1 had little effect on mesangial cell viability during the first 24 hours of exposure to Stx1. However, prolonged incubation with Stx1 for 48 and 72 hours resulted in a 68% and 80% decrease in cell-viability, respectively. Stx1 elicited a dose and time dependent increase in the levels of MCP-1 mRNA, an effect that was potentiated by preincubation with IL-1alpha. CONCLUSION: These data indicate that mesangial cells are susceptible to the effects of Stx1 in vitro. Stx1 exerts a spectrum of biologic effects on mesangial cells ranging from activation of chemokine genes to a lethal toxic injury. Immunoinflammatory cytokines potentiate the effects of Stx1. Thus, glomerular pathology in HUS may also result from a direct effect of Stx1 on mesangial cells.

Bacterial Toxins↗
PubMed1998

Cultured human glomerular mesangial cells express the C5a receptor.

BACKGROUND: While the association of complement activation and glomerulonephritis has been recognized for decades, the pathogenic mechanisms of complement-mediated glomerular damage are incompletely understood. Expression of the C5a receptor in the kidney suggests that C5a could play a direct role in initiating or promoting glomerulonephritis. METHODS: Expression of the C5a receptor by cultured human glomerular mesangial cells (HGMC) was examined by immunofluorescence, by FACS analysis and by reverse transcriptase-polymerase chain reaction (RT-PCR). Potential mitogenic effects were examined by analysis of neutral red dye uptake after treatment with recombinant C5a (rC5a). The production of cytokines [interleukin-1 (IL-1), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1)] and growth factors [transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF-AB)] by mesangial cells stimulated with rC5a was examined by ELISA of cell culture supernatants. RESULTS: Expression of the C5a receptor by the cultured HGMC was demonstrated by both immunofluorescence and FACS. The presence of mRNA encoding the receptor was confirmed by RT-PCR. Treatment of HGMC in vitro with rC5a resulted in mild cellular proliferation. No IL-1 was detected despite stimulation with up to 100 nM rC5a. Concentrations of IL-8 and TGF-beta did not increase beyond basal levels in control samples at any level of stimulation. Mean MCP-1 concentrations and PDGF-AB concentrations increased by 40% and 70% above control values 48 hours post-stimulation (P = 0.01 and P = 0.003, respectively). CONCLUSIONS: These data indicate that the C5a receptor is expressed on HGMC in vitro, and may play a role in mediating glomerular injury by promoting cellular proliferation and the production of cytokines and growth factors.

Antigens, CD↗
PubMedDate not supplied

Development of a model to demonstrate photosensitizer-mediated viral inactivation in blood.

A model has been developed to demonstrate the use of photodynamic treatment (PDT) to eradicate viral contaminants from donated blood and blood products. Whole blood, spiked with vesicular stomatitis virus (VSV), was treated with the photosensitizer benzoporphyrin derivative-monoacid ring A (BPD-MA). After light activation of BPD-MA, a neutral red dye uptake assay was carried out to determine virus inactivation. Various drug incubation times and light intensities were tested as well as red cell lysis and distribution of VSV in blood. At BPD-MA concentrations between 2 and 4 micrograms per mL in whole blood, up to 10(7) VSV were inactivated. Several photosensitizers were also tested with this model to determine their relative efficacy in viral inactivation.

Blood↗
PubMed1996

Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction.

BACKGROUND & AIMS: Tacrine administration (1-3 mg/kg) may lead to sinusoidal concentrations in the micromolar range and produce liver dysfunction in 50% of recipients. The aim of this study was to determine the cellular effects of tacrine that account for liver dysfunction. METHODS: The effects of tacrine on mitochondrial function were determined in isolated rat liver mitochondria, cultured rat hepatocytes, and isolated human lymphocytes. RESULTS: In vitro, tacrine was taken up by rat liver mitochondria, decreased their membrane potential, and stimulated their respiration. Ex vivo, respiration was increased in rat mitochondria isolated 30 minutes after the administration of 2 mg of tacrine per kilogram. After 7 days of culture, tacrine (2.5 mumol/L) decreased rat hepatocyte adenosine triphosphate levels. Ten micromolar decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium reduction and neutral red uptake without modifying cell glutathione, the morphology of the endoplasmic reticulum, or protein synthesis. Tacrine (1.25 mumol/L) decreased levels of adenosine triphosphate in human lymphocytes. CONCLUSIONS: The weak base tacrine exerts a protonophoric effect in mitochondria that wastes energy and decreases levels of adenosine triphosphate in rat and human cells. These effects are visible after clinically relevant doses of tacrine and might be involved in tacrine-induced liver dysfunction.

Animals↗
PubMed2003

Isolation and culture of adult and fetal rabbit bladder smooth muscle cells and their interaction with biopolymers.

PURPOSE: The aim of this study was to test the feasibility of isolation and culture of adult and fetal rabbit bladder smooth muscle cells (SMCs) and comparison of their interactions with different types of biodegradable biopolymers in cell culture. METHODS: Bladder SMCs isolated from adult and fetus rabbits were identified by immunostaining for smooth muscle alpha-actin and myosin. Growth kinetics of SMCs were estimated using population doubling time (PDT) and thymidine labeling index (TLI). Poly (D, L-lactide-co-glycolide; PLGA) copolymers were synthesized at 85:15 and 75:25 monomer ratios. The porous scaffolds prepared from these polymers were seeded with SMCs. The study compared the effectiveness of adsorbing fibronectin and fetal calf serum (FCS) on these biopolymers. The cells grown on these polymers were quantified using a neutral red uptake assay. RESULTS: Over 90% of the 2 cell populations stained positive for SMC marker proteins. Fetal SMCs were seen to emerge from the tissue after 3 to 4 days, whereas adult SMCs were seen after 5 to 6 days. However, estimated PDT of fetal and adult SMCs was 85.2 and 54.5 hours, respectively, and TLI of adult SMCs was also higher than with fetal SMCs. Proliferation on 75:25 PLGA was better than for 85:15 and for both biopolymers; adsorption of FCS significantly affected cell attachment relative to fibronectin. CONCLUSIONS: Although fetal SMCs were shown to emerge from explants early after seeding onto dishes, doubling time and S-phase fraction of adult bladder SMCs were markedly higher than of fetal derived cells. Adsorption of serum proteins significantly enhances the attachment of both fetal and adult SMCs to biopolymers.

Adsorption↗
PubMed1999

Cytotoxicity of trans-dehydrocrotonin from Croton cajucara on V79 cells and rat hepatocytes.

The cytotoxicity of trans-dehydrocrotonin (DHC), an antiulcerogenic diterpene from Croton cajucara (Euphorbiaceae), was assessed on a V79 fibroblast cell line and on rat hepatocytes. Three independent endpoints for cytotoxicity were evaluated: DNA content, MTT reduction and neutral red uptake (NRU). For the V79 cells IC50 values of 253 and 360 microM were obtained for the NRU and MTT tests. The cytotoxic effect of DHC was time exposure dependent and no ability to recover after treatment was observed. For the rat hepatocytes IC50 values of 8, 300 and 400 microM for the MTT, DNA and NRU assays were obtained. The greater toxicity observed for the MTT test was inhibited when the experiment was performed using non-fresh hepatocytes in an age-dependent fashion. The treatment of V79 cells with the conditioned medium resulting after hepatocyte incubation with DHC showed an enhancement of MTT reduction without any evident toxic effects on fibroblasts. These results suggest that DHC has basal cytotoxic effects as observed on V79 fibroblasts and expresses a selective cytotoxicity after its metabolization by the hepatocytes. The bioactivation of DHC is mediated by cytochrome P450 and could generate metabolites that have no toxicity for V79 fibroblasts.

Animals↗
PubMed2001

[Human cultured lens cells. II. Characterization of established lens cell lines and test of their suitability for cytotoxicity tests].

BACKGROUND: Human lens cells in culture may be valuable tools to discover cataractogenic risk factors. Here we report on the characterization of established human lens cells and their use in cytotoxicity tests. MATERIAL AND METHODS: Adhesion dependence was tested by an autoradiographic method. Soft agar test was performed to secure anchorage-independent growth. Laser scan microscopy was used for measuring the nucleus-cytoplasmic relationship. The cytoskeleton was visualized by fluorescence microscopy. Investigations on cytotoxicity were done by neutral red cytotoxicity assay and 3H-thymidine incorporation. The labeling index was determined by the BrdU-method. RESULTS: Two lens cell lines were transformed into fast growing cells. They are characterized by a partial loss of adhesion dependence, cell growth in soft agar, high cloning efficiency and reduced serum requirement. However, the nucleus-cytoplasmic relationship did not change very much in comparison to non-transformed lens cells. The organization of the cytoskeleton was cell shape dependent. The intermediate filaments were from the vimentin type. The established cell lines proved to be highly sensitive to ethanol and mitomycin C. CONCLUSIONS: The established human lens cell lines are well suited to screen for cytotoxic substances in vitro which might be cataractogenic risk factors in vivo.

Animals↗
PubMed1994

[Vital preservation of cartilage transplants with tissue culture methods].

Cartilage grafting is one of the most common procedures in plastic surgery. Since storage of both autologous and allogenic cartilage is necessary, different preservation methods have been used with more or less success. The use of chemical preservation procedures like formaldehyde, Merthiolate or Cialit lead to a loss of the vitality of the graft. This work presents a study of the cell vitality and the matrix of cartilage grafts stored in different solutions (formaldehyde, saline, RPMI 1640, Ham F-12 and DMEM 4500) during 150 days. The cell viability was assessed using tissue sections (neutral red supravital staining and trypan blue dye exclusion test) and isolated cells (trypan blue dye exclusion test and cell adhesion in monolayer culture). The state of the cartilage matrix was analysed by means of the azan, alcian and toluidine blue staining). Cartilage immersed in formaldehyde solution lost 100% of the vitality after a storage period of 10 days, the one immersed in saline solution after 30 days. Cartilage stored in tissue culture media retained its vitality (> 85%) during the whole storage time. Histological staining methods showed a decrease of the staining intensity after 10 days storage in formaldehyde and after 30 days storage in saline solution. No differences in vitality and the matrix staining were found among all three culture media. Our results suggest that viable cartilage tissue can be successfully stored for a long time using tissue culture methods.

Cartilage↗
PubMed2006

Ecotoxicological assessment of sediment, suspended matter and water samples in the upper Danube River. A pilot study in search for the causes for the decline of fish catches.

GOALS, SCOPE AND BACKGROUND: Fish populations, especially those of the grayling (Thymallus thymallus), have declined over the last two decades in the upper Danube River between Sigmaringen and Ulm, despite intensive and continuous stocking and improvement of water quality since the 1970s. Similar problems have been reported for other rivers, e.g. in Switzerland, Great Britain, the United States and Canada. In order to assess if ecotoxicological effects might be related to the decline in fish catch at the upper Danube River, sediment, suspended matter and waste water samples from sewage treatment plants were collected at selected locations and analyzed in a bioanalytical approach using a battery of bioassays. The results of this pilot study will be used to decide if a comprehensive weight-of-evidence study is needed. METHODS: Freeze-dried sediments and suspended particulate matters were extracted with acetone in a Soxhlet apparatus. Organic pollutants from sewage water were concentrated using XAD-resins. In order to investigate the ecotoxicological burden, the following bioassays were used: (1) neutral red assay with RTL-W1 cells (cytotoxicity), (2) comet assay with RTL-W1 cells (genotoxicity), (3) Arthrobacter globiformis dehydrogenase assay (toxicity to bacteria), (4) yeast estrogen screen assay (endocrine disruption), (5) fish egg assay with the zebrafish (Danio rerio; embryo toxicity) and (6) Ames test with TA98 (mutagenicity). RESULTS AND DISCUSSION: The results of the in vitro tests elucidated a considerable genotoxic, cytotoxic, mutagenic, bacteriotoxic, embryotoxic and estrogenic burden in the upper Danube River, although with a very inhomogeneous distribution of effects. The samples taken from Riedlingen, for example, induced low embryo toxicity, but the second highest 17beta-estradiol equivalent concentration (1.8 ng/L). Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, with clear-cut dose-response relationships for the embryotoxic effects of contaminated sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that--at least for fish eggs--the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The effect observed most frequently in the fish egg assay was a developmental delay. A comparison of our own results with locations along the rivers Rhine and Neckar demonstrated similar or even higher ranges of ecotoxicological burdens in the Danube River. CONCLUSIONS: The complex pattern of ecotoxicological effects caused by environmental samples from the Danube River, when assessed in an in vitro biotest battery using both acute and more specific endpoints, showed that integration of different endpoints is essential for appropriate hazard assessment. Overall, the ecotoxicological hazard potential shown has indeed to be considered as one potential reason for the decline in fish catches at the upper Danube River. However, based on the results of this pilot study, it is not possible to elucidate that chemically induced alterations are responsible for the fish decline. RECOMMENDATIONS AND PERSPECTIVES: In order to confirm the ecological relevance of the in vitro results for the situation in the field and especially for the decline of the grayling and other fishes, further integrated investigations are required. For linking the weight of evidence obtained by in vitro assays and fish population investigations, the application of additional, more specific biomarkers (e.g. vitellogenin induction, EROD and micronucleus assay) has been initiated in fish taken from the field as well as in situ investigations.

Animals↗
PubMed1976

Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis.

Intracellular parasites (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, and some Chlamydiae) may promote their survival within the host by acting from within phagosomes to prevent phagolysosome formation, thus avoiding exposure to the lysosomal hydrolases. The present studies demonstrate that when sulfatides of M. tuberculosis (anionic trehalose glycolipids largely responsible for the neutral red reactivity of virulent strains) are administered to cultured mouse peritoneal macrophages, they accumulate in the secondary lysosomes, which are rendered incompetent for fusion with phagosomes containing suitable target particles such as viable yeasts. This antifusion effect is also exhibited when small amounts of sulfatide are introduced directly into phagosomes by attachment to the target yeasts prior to their ingestion. The sulfatides evidently exert a selective inhibitory influence on membrane fusion, analogous to what occurs typically when macrophage cultures are infected with tubercle bacilli. This effect may be due to ionic interaction between the polyanionic micelles of bacterial sulfatide and organelle membranes, modifying the latter and inducing dysfunction.

Lysosomes↗
PubMed1996

Acridones: a chemically new group of protonophores.

Although the interaction of proton-conducting ionophores (protonophores) with photosynthetic electron transport has been extensively studied during the past decade, the mode of action of protonophores remained uncertain. For a better understanding of the molecular mechanism of the action of protonophores, the introduction of chemically new types of molecules will be required. In this work, we demonstrate that acridones (9-azaanthracene-10-ones) completely fulfill this requirement. At low concentrations of acridones, the thermoluminescence bands at +20 degrees C and +10 degrees C were strongly inhibited, while normal electron transport activity was retained. This indicates that the concentrations of S2 and S3 states involved in the generation of these bands are reduced. At higher concentrations, an increased activity of electron transport was observed, which is attributed to the typical uncoupler effect of protonophores. Indeed, acridones accelerate the decay of the electrochromic absorbance change at 515 nm and also inhibit the generation of the transmembrane proton gradient, measured as an absorbance transient of neutral red. Variable fluorescence induction was quenched even at low concentrations of acridones but was restored by either a long-term illumination or high light intensity. Acridones, similarly to other protonophores, promoted the autooxidation of the high-potential form of cytochrome b559 and partially converted it to lower potential forms. These results suggest that acridones, acting as typical protonophores, uncouple electron transport, accelerate the deactivation of the S2 and S3 states on the donor side, and facilitate the oxidation of cytochrome b559 on the acceptor side of photosystem II.

Journal Article↗
PubMed2000

NADPH oxidase is an O2 sensor in airway chemoreceptors: evidence from K+ current modulation in wild-type and oxidase-deficient mice.

Pulmonary neuroepithelial bodies (NEBs) are presumed airway chemoreceptors that express the putative O(2) sensor protein NADPH oxidase and O(2)-sensitive K(+) channels K(+)(O(2)). Although there is a consensus that redox modulation of K(+)(O(2)) may be a common O(2)-sensing mechanism, the identity of the O(2) sensor and related coupling pathways are still controversial. To test whether NADPH oxidase is the O(2) sensor in NEB cells, we performed patch-clamp experiments on intact NEBs identified by neutral red staining in fresh lung slices from wild-type (WT) and oxidase-deficient (OD) mice. In OD mice, cytochrome b(558) and oxidase function was disrupted in the gp91(phox) subunit coding region by insertion of a neomycin phosphotransferase (neo) gene. Expression in NEB cells of neo mRNA, a marker for nonfunctional gp91(phox), was confirmed by nonisotopic in situ hybridization. In WT cells, hypoxia (pO(2) = 15-20 mmHg; 1 mmHg = 133 Pa) caused a reversible inhibition ( approximately 46%) of both Ca(2+)-independent and Ca(2+)-dependent K(+) currents. In contrast, hypoxia had no effect on K(+) current in OD cells, even though both K(+) current components were expressed. Diphenylene iodonium (1 microM), an inhibitor of the oxidase, reduced K(+) current by approximately 30% in WT cells but had no effect in OD cells. Hydrogen peroxide (H(2)O(2); 0.25 mM), a reactive oxygen species generated by functional NADPH oxidase, augmented K(+) current by >30% in both WT and OD cells; further, in WT cells, H(2)O(2) restored K(+) current amplitude in the presence of diphenylene iodonium. We conclude that NADPH oxidase acts as the O(2) sensor in pulmonary airway chemoreceptors.

4-Aminopyridine↗