Phototoxicity evaluation--Tetrahymena thermophila as an alternative model

Interest in utilizing an alternative to animal method for toxicological evaluation has received considerable attention due to cost effectiveness and the ethical issues involving animal experimentation. Alternative methods for phototoxicity evaluation are significant because of growing concern over increasing health effects due to stratospheric ozone depletion resulting in an increasing penetration of ultraviolet light-B radiation (UVB, 290-320 nm) which contributes to activation of chemical and biological molecules to potential phototoxic agents. The classic rabbit eye-irritancy test referred to as Draize test has been the subject of severe criticism by animal welfare groups. Dermal toxicity test using guinea pigs and mouse tail phototoxicity test is time consuming and requires a large number of laboratory animals. In photohaemolysis assay some of the phototoxic agents (such as riboflavin) react with the membrane proteins of the erythrocyte. However, in vitro test system using protozoa offers a promising alternative means of phototoxicity evaluation. Our previous studies have demonstrated that synergistic action of photochemically reactive agents and sunlight produces lethal effects to Paramecium but the protozoan has not received serious consideration for use as an alternative model for phototoxicity evaluation. In the present communication we have described the potential application of Tetrahymena as an alternative model to study the radiation-induced changes both in the presence or absence of photoreactive chemical agents. This model is likely to provide scope for studying the biological effects of environmental UVB radiation, DNA damage and defence against oxidative stress.     

The photodynamic effect of methylene blue and toluidine blue on <Emphasis Type="Italic">Candida albicans</Emphasis> is dependent on medium conditions

Due to the increased number of immunocompromised patients, the infections associated with the pathogen of the genus Candida and other fungi have increased dramatically. Photodynamic antimicrobial chemotherapy (PACT) has been presented as a potential antimicrobial therapy, in a process that combines light and a photosensitizing drug, which promotes a phototoxic response by the treated cells. In this work, we studied the effects of the different medium conditions during PACT, using either methylene blue (MB) or toluidine blue (TB) on Candida albicans. The inhibition of the growth produced by PACT was decreased for different pH values (6.0, 7.0, and 8.0) in a buffered medium. The phototoxic effects were observed only in the presence of saline (not buffered medium). PACT was modulated by calcium in a different manner using either MB or TB. Also when using MB both verapamil or sodium azide were able to decrease the phototoxic effects on the C. albicans. These results show that PACT is presented as a new and promising antifungal therapy, however, new studies are necessary to understand the mechanism by which this event occurs.     

The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus

Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P>1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1-1000 microM). Dye illumination (total light dose of 3.15 J cm(-1) over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (phi'delta: 0.18-1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.     

Multiple hypersensitivity to mutagens in a cell strain (46BR) derived from a patient with immuno-deficiencies

46BR is a fibroblast cell strain established from an individual with hypogammaglobulinaemia. The cells are unique in showing hypersensitivity to the lethal effects of a wide range of DNA-damaging agents. Thus they are hypersensitive to gamma- and 254-nm UV-irradiation and show a limited capacity to repair potentially lethal gamma-irradiation damage when compared with fibroblasts from normal individuals. A slight hypersensitivity to mitomycin C was also revealed but we were not able to discriminate 46BR from normals with 4-nitroquinoline oxide. The cells were hypersensitive to the alkylating agents, dimethyl sulphate, methyl methanesulphonate, ethyl methanesulphonate, N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea but not N-ethyl-N-nitrosourea. A consideration of the spectra of DNA lesions produced by these alkylating agents together with the sensitivity to ionising radiation and mitomycin C suggests that 46BR cells are defective in a repair step that is common to all agents. We suggest that the cells are defective in DNA polymerisation or ligation. Support for this suggestion comes from the absence of any hypersensitivity to N-ethyl-N-nitrosourea since its major reaction products are not removed by excision pathways that require polymerisation and ligation.     

Role of flavonoids in controlling the phototoxicity of Hypericum perforatum extracts.

Hypericum perforatum extracts are used mainly as oral antidepressants. Depending on source the extracts contain various amounts of phenylpropanes, flavonol derivates, biflavones, proathocyanidines, xanthones, phloroglucinoles, some amino acids, naphtodianthrones (hypericines) and essential oil constituents. The therapeutic use of Hypericum perforatum extracts however is limited by their phototoxic potential. It was the aim of the present study to investigate the phototoxic potential of 3 Hypericum perforatum extracts from different sources as well as some of its main constituents. In order to systematically study the phototoxic potential we established a modified neutral red assay utilizing an immortalized human keratinocyte cell line (HaCaT cells) as substrate and UVA irradiation. This modified neutral red assay was found to be a simple and reliable method for detecting phototoxic effects of reference agents and plant extracts. The validity of this method was demonstrated with known phototoxic compounds like chloropromazine and psoralenes like 5-MOP. Hypericum perforatum extracts demonstrated cytotoxicity and photocytotoxicity in a dose and UVA-dose dependent manner. Hypericine itself also evoked severe phototoxic effects and was thus identified as the main phototoxic constituent. Among the tested flavonoids quercitrin was found to be cytotoxic, while rutin unexpectedly demonstrated phototoxicity whereas quercitrin was effective to control the phototoxic activity of Hypericum perforatum extracts.     

Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl-N′-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN₁ and SN₂ mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O⁶ position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.     

HEMOLYTIC ACTIVITY OF HETEROCAPSA CIRCULARISQUAMA (DINOPHYCEAE) AND ITS POSSIBLE INVOLVEMENT IN SHELLFISH TOXICITY

Heterocapsa circularisquama Horiguchi is lethal to shellfish, particularly bivalves such as pearl oysters ( Pinctada fucata Gould). No detrimental effects of this flagellate on fish have been observed thus far. In this study, we found that H. circularisquama causes mammalian erythrocytes to lyse. Among the erythrocytes tested, rabbit erythrocytes showed the highest susceptibility, whereas erythrocytes from cattle, sheep, and human were relatively insensitive. Heterocapsa triquetra Stein, which is morphologically similar to H. circularisquama but not toxic to bivalves, showed no hemolytic activity toward rabbit erythrocytes. Culture supernatant or ultrasonic-ruptured cells of H. circularisquama showed only weak hemolytic activity. Hemolytic activity was found in the ethanol extract of H. circularisquama cells, suggesting that the hemolytic agents may be more stable in ethanol than in aqueous solution. Both an intact flagellate cell suspension and the ethanol extract caused morphological changes and eventual collapse of unfertilized eggs of Pacific oyster. Furthermore, the ethanol extract was lethal to the microzooplankton rotifer Brachionus plicatilis Müller, which is highly sensitive to H. circularisquama. Our results suggest that a hemolytic toxin produced by H. circularisquama may be one of the causative agents responsible for the shellfish toxicity.     

Inhibition of TNF-alpha produced by Kupffer cells protects against the nonspecific liver toxicity of immunotoxin anti-Tac(Fv)-PE38, LMB-2

LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas: exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-alpha by these cells. Following LMB-2 administration to mice, the levels of TNF-alpha in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-alpha in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-alpha produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.     

Caffeine alone causes DNA damage in Chinese hamster ovary cells

Caffeine has been shown to enhance the lethal effect of DNA-damaging agents in mammalian cells, and the potentiation by caffeine of this effect is generally interpreted as the result of inhibition by caffeine of the repair of damaged DNA. However, the mechanism by which caffeine enhances the lethal effect of DNA-damaging agents has not yet been elucidated. During studies on the effect of caffeine on DNA repair, we found by alkaline elution analysis that caffeine alone produced DNA strand breaks or alkali labile sites in Chinese hamster ovary cells. The amount of DNA breakage or alkali labile sites depended on the concentration of caffeine. We propose that DNA breakage induced by caffeine may be involved in the enhancement of the lethal effect of DNA-damaging agents.     

Purification and partial characterization of a toxic serine protease produced by pathogenic Vibrio alginolyticus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11, having a pI of 4.3 and a molecular weight of approximately 33 kD. The toxin was completely inhibited by FeCl2 but partially inhibited by 3,4-dichloroisocoumarin (3,4-DCI), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CuCl2 and ZnCl2. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 microgram protein/g body weight. The haemolymph withdrawn from the moribund prawns injected with the toxic protease was unable to clot. The coagulogen in the kuruma prawn plasma showed an increased migration rate after incubation with this serine protease, and a plasma colour change from blue to pink was recorded. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium. The 33 kD protease was therefore a toxic protease produced by V. alginolyticus strain Swy.     

Cytotoxic and DNA-damaging effects of 1,2-bis(sulfonyl)hydrazines on human cells of the Mer+ and Mer- phenotype

A series of 1,2-bis(sulfonyl)hydrazines with the capacity to function as alkylating agents have been evaluated for their toxicity towards Mer- HT29 and Mer- BE cells, and for their ability to produce DNA damage expressed as single-strand breaks and DNA interstrand cross-links. Compounds of this class with methylating potential showed a marked difference in their capacity to inhibit the growth of Mer- and Mer+ cells, being considerably more toxic to BE Mer- cells. Dose-dependent DNA single-strand breaks were induced by these agents, with the quantity of breaks produced in Mer- and Mer+ cells being essentially the same. Maintenance of these lesions did not appear to explain the differential in toxicity to BE and HT29 cells. A chloroethylating compound of this class was also more toxic to Mer- BE cells than to Mer+ HT29 cells, but the differential toxicity was considerably less than that of the methylating agents of the series. The chloroethylating agent did not produce measurable single-strand breaks of the DNA of treated cells, but caused more DNA interstrand cross-links in Mer- cells than in Mer+ cells. Thus, DNA interstrand cross-links may be at least in part responsible for the cell kill produced by this agent. The findings suggest that methylating and chloroethylating derivatives of the 1,2-bis(sulfonyl)hydrazine family have different biochemical determinants of their cytodestructive actions.     

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme.

An extracellular lethal toxin produced by Aeromonas salmonicida was purified by fast-protein liquid ion-exchange chromatography. The toxin is composed of glycerophospholipid:cholesterol acyltransferase (GCAT) (molecular mass, 25 kilodaltons) aggregated with lipopolysaccharide (LPS), the GCAT/LPS complex having a molecular mass of about 2,000 kilodaltons, estimated by gel filtration chromatography. The toxin is lethal for Atlantic salmon (Salmo salar L.) at a concentration of 0.045 micrograms of protein per g of body weight. The toxin is a hemolysin (T-lysin, active on fish erythrocytes), leukocytolysin, and cytotoxin. Antiserum to the purified toxin neutralized the lethal toxicity of the crude extracellular toxins, indicating this toxin to be the major lethal factor produced by A. salmonicida. In the crude extracellular products, small amounts of free GCAT were also present. This has been purified, and its activities and properties have been compared with those of the GCAT/LPS complex. The presence of LPS did not influence the GCAT activity of the enzyme with egg yolk or phosphatidylcholine (lecithin) as a substrate, but the specific hemolytic activity and lethal toxicity was about eightfold higher in the complexed form. Furthermore, the free GCAT was more susceptible to proteolytic and heat inactivation than was the GCAT/LPS complex. Recombination of LPS (phenol extracted from extracellular products of A. salmonicida) with free GCAT enhanced the hemolytic activity, lethal toxicity, and heat stability of the latter but did not influence its lecithinase activity. In native polyacrylamide gel electrophoresis, the GCAT/LPS complex and the recombined GCAT-LPS both showed a high-molecular-mass band which did not enter the gel, while the free GCAT produced a single band with low molecular mass. In isoelectric focusing gels, the GCAT/LPS and recombined GCAT-LPS produced a nonfocusing smear with pIs from pI 5.0 to 5.8, while the free GCAT produced a single band with pI 4.3. These data show that free GCAT can combine with LPS to produce a high-molecular-mass complex with enhanced toxicity and heat stability compared with those of free GCAT, similar to the preexisting GCAT/LPS complex, and indicate that the LPS moiety of the toxin plays an active role in toxicity.     

Potent phototoxicity of marine bunker oil to translucent herring embryos after prolonged weathering

Pacific herring embryos (Clupea pallasi) spawned three months following the Cosco Busan bunker oil spill in San Francisco Bay showed high rates of late embryonic mortality in the intertidal zone at oiled sites. Dead embryos developed to the hatching stage (e.g. fully pigmented eyes) before suffering extensive tissue deterioration. In contrast, embryos incubated subtidally at oiled sites showed evidence of sublethal oil exposure (petroleum-induced cardiac toxicity) with very low rates of mortality. These field findings suggested an enhancement of oil toxicity through an interaction between oil and another environmental stressor in the intertidal zone, such as higher levels of sunlight-derived ultraviolet (UV) radiation. We tested this hypothesis by exposing herring embryos to both trace levels of weathered Cosco Busan bunker oil and sunlight, with and without protection from UV radiation. Cosco Busan oil and UV co-exposure were both necessary and sufficient to induce an acutely lethal necrotic syndrome in hatching stage embryos that closely mimicked the condition of dead embryos sampled from oiled sites. Tissue levels of known phototoxic polycyclic aromatic compounds were too low to explain the observed degree of phototoxicity, indicating the presence of other unidentified or unmeasured phototoxic compounds derived from bunker oil. These findings provide a parsimonious explanation for the unexpectedly high losses of intertidal herring spawn following the Cosco Busan spill. The chemical composition and associated toxicity of bunker oils should be more thoroughly evaluated to better understand and anticipate the ecological impacts of vessel-derived spills associated with an expanding global transportation network.     

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis in the gut of mosquito larvae results in further activation of the Bacillus sphaericus toxin.

Gut proteases from the larvae of the mosquito Culex pipiens convert the 43-kilodalton (kDa) toxin from Bacillus sphaericus 2362 to a 40-kDa peptide. The 50% lethal concentration of this peptide for tissue culture-grown cells of Culex quinquefasciatus was 1.0 microgram/ml (as determined by the intracellular ATP assay), 54-fold less than that of the 43-kDa peptide. Gut proteases from Anopheles gambiae and Aedes aegypti, as well as bovine pancreatic trypsin, also converted the 43-kDa protein to a 40-kDa peptide which was indistinguishable from the peptide formed by the proteases from C. pipiens with respect to its toxicity to tissue culture-grown cells of C. quinquefasciatus. Evidence for the in vivo conversion of the 43-kDa protein to the 40-kDa peptide was also obtained from experiments in which larvae of C. pipiens, Anopheles gambiae, and Aedes aegypti were fed crystals from B. sphaericus 2362. By using the exclusion of trypan blue as an indication of cell viability, it was shown that chitobiose, chitotriose, N-acetylmuramic acid, and N-acetylneuraminic acid decreased the toxicity of the 40-kDa peptide (from 100 to 50% mortality at about 10 mM concentrations of these sugars). Muramic acid, N-acetylgalactosamine, and N-acetylglucosamine were less effective, while several sugars had no effect, suggesting that the 40-kDa toxin binds to specific receptors on the cell membrane. The 40-kDa protein was less toxic to tissue culture-grown cells of Anopheles gambiae and Aedes dorsalis, and the same sugars which reduced the toxicity for cells of C. quinquefasciatus were also effective in reduction of toxicity for these cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxide formation

Several types of cultured fibroblasts, including chick embryo, human and mouse, were killed by the addition of sodium ascorbate at final concentrations of 0.05–0.25 mM to cultures at the time of inoculation or to attached cells. Ascorbate did not affect the attachment of cells to the substratum. The effect on chick embryo fibroblasts was visible by fours hours and by six hours almost all cells had swelled and were becoming detached. By 24 hours detached cells had either lysed or become crenated in appearance. Other end-diol reducing agents and also glutathione and cysteine were effective while gulonolactone, a non-reducing analogue of ascorbate, was ineffective. Preincubation of medium containing ascorbate but no cells, conditions which result in degradation of the vitamin, led to loss of toxicity, indicating that a degradation product was not the lethal agent and that a component of the medium was not converted to a lethal substance. The lethal effect of both ascorbate and glutathione was prevented by the addition of catalase to the medium, suggesting that H₂O₂ formed by intracellular reactions and then excreted into the medium was the cytotoxic agent. This conclusion was supported by the findings that 0.05 mM H₂O₂ added to chick embryo fibroblasts was lethal and that the effect of this compound on cellular morphology was almost identical to that of ascorbate.     

Blue light-triggered photochemistry and cytotoxicity of retinal

The chemical- and photo- toxicity of chromophore retinal on cells have long been debated. Although we recently showed that retinal and blue light exposure interrupt cellular signaling, a comprehensive study examining molecular underpinnings of this perturbation and its consequences to cellular fate is lacking. Here, we report molecular evidence for blue light excited-retinal induced oxidative damage of polyunsaturated lipid anchors in membrane-interacting signaling molecules and DNA damage in cells using live-cell imaging and in vitro experimentation. The incurred molecular damage irreversibly disrupted subcellular localization of these molecules, a crucial criterion for their signaling. We further show retinal accumulation in lipid-bilayers of cell membranes could enhance the lifetime of retinal in cells. Comparative response-signatures suggest that retinal triggers reactions upon photoexcitation similar to photodynamic therapy agents and generate reactive oxygen species in cells. Additionally, data also shows that exposing retinal-containing cells to sunlight induces substantial cytotoxicity. Collectively, our results explain a likely in vivo mechanism and reaction conditions under which bio-available retinal in physiological light conditions damages cells.     

A quantitative method to evaluate neutralizer toxicity against Acanthamoeba castellanii.

A standard methodology for quantitatively evaluating neutralizer toxicity against Acanthamoeba castellanii does not exist. The objective of this study was to provide a quantitative method for evaluating neutralizer toxicity against A. castellanii. Two methods were evaluated. A quantitative microtiter method for enumerating A. castellanii was evaluated by a 50% lethal dose endpoint method. The microtiter method was compared with the hemacytometer count method. A method for determining the toxicity of neutralizers for antimicrobial agents to A. castellanii was also evaluated. The toxicity to A. castellanii of Dey-Engley neutralizing broth was compared with Page's saline. The microtiter viable cell counts were lower than predicted by the hemacytometer counts. However, the microtiter method gives more reliable counts of viable cells. Dey-Engley neutralizing medium was not toxic to A. castellanii. The method presented gives consistent, reliable results and is simple compared with previous methods.     

Clonidine protection from soman and echothiophate toxicity in mice

The influence of clonidine on the toxicity produced by two irreversible, organophosphate cholinesterase inhibitors, soman and echothiophate, was studied in mice. At lethal doses, soman produced whole body tremor but no muscle fasciculation; at lethal doses, echothiophate produced muscle fasciculations but no whole body tremor. Pretreatment with clonidine protected against several toxic manifestations of soman, but had little effect on echothiophate toxicity. In addition to its documented effects on acetylcholine metabolism, clonidine was found to be a weak inhibitor of acetylcholinesterase. At certain concentrations, clonidine protected the enzyme from permanent inactivation by soman. These findings indicate that the toxicity of soman and echothiophate reflect primarily central and peripheral actions, respectively, and that clonidine has a much greater protective effect versus the centrally-acting agent. Moreover, direct interactions with acetylcholinesterase may contribute to clonidine protection from cholinesterase inhibitor toxicity.