Diisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard

1998 ◽  
Vol 17 (11) ◽  
pp. 606-612 ◽  
Author(s):  
Christopher D Lindsay ◽  
Joy L Hambrook
Keyword(s):  
Phase Contrast ◽  
Gentian Violet ◽  
A549 Cells ◽  
Neutral Red ◽  
A549 Cell Line ◽  
Cytotoxic Effects ◽  
Sulphur Mustard ◽  
Culture Viability ◽  
Contrast Microscopy ◽  
Optimal Effect

The A549 cell line was used to assess the ability of diisopropylglutathione (DIPE) to protect against a 100 mM challenge dose of sulphur mustard (HD) using gentian violet (GV), thiazolyl blue (MTT) and neutral red (NR) assays as indicators of cell culture viability. As part of a continuing study of the efficacy of protective nucleophiles as candidate treatments for HD poisoning, several different combinations of protectant and HD were used to determine the optimal means of protecting A549 cells from the effects of HD. It was found that DIPE (4 mM) could protect cells against the effects of HD though for optimal effect, DIPE had to be present at the time of HD challenge. Cultures protected with DIPE were up to 2.9- fold more viable than HD exposed cells 48 h after HD challenge when using the GV, MTT and NR assays to assess viability. Observations by phase contrast microscopy of GV stained cultures confirmed these findings. Pretreating A549 cultures with DIPE for 1 h followed by its removal prior to HD challenge did maintain cell viability, though at a relatively low level (only up to 1.4- fold more viable than HD only exposed cells). DIPE was also able to protect HD exposed A549 cultures when added to cell cultures at intervals of up to 12 to 15 min after the initial HD exposure, though viability tended to decrease over this period, so that at 1 h, addition of DIPE did not maintain the viability of the cultures. This is the first such report of the anti-HD protectant properties of DIPE in A549 cells. It is concluded that the protection observed against HD is probably largely due to extracellular inactivation of HD by DIPE.

Monoisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard

1997 ◽  
Vol 16 (11) ◽  
pp. 636-644 ◽  
Author(s):  
Christopher D Lindsay ◽  
Joy L Hambrook ◽  
Alison F Lailey
Keyword(s):  
Cell Viability ◽  
Cell Cultures ◽  
A549 Cell ◽  
High Performance ◽  
Gentian Violet ◽  
A549 Cells ◽  
Rapid Onset ◽  
Neutral Red ◽  
Biochemical Basis ◽  
Sulphur Mustard

1 The A549 cell line was used to assess the toxicity of sulphur mustard (HD), using gentian violet (GV) and neutral red (NR) dyes as indicators of cell viability. It was found that exposure to concentrations in excess of 40 ?M HD resulted in a rapid onset of toxicity. 2 The ability of monoisopropylglutathione ester (MIPE) to protect A549 cells against the effects of a 100 ?M challenge dose ofHD was determined using the NR and GV assays. It was found that MIPE (8 mM) could protect cells against the effects ofHD though MIPE had to be present at the time of HD challenge. Cultures protected with MIPE were two times more viable than HD exposed cells 48 h after HD challenge when using the GV and NR assays to assess viability. Observations by phase contrast microscopy of NR and GV stained cultures confirmed these findings. Addition of MIPE after previously exposing the A549 cultures to HD (for up to 5 min) maintained cell viability at 72% compared to 37% for unprotected cultures, after which time viability fell significantly so that at 10 min there was no difference in viability between the MIPE treated and untreated cultures. 3 Pretreating A549 cultures with MIPE for 1 h followed by its removal prior to HD challenge did not maintain cell viability. Treatment of cultures with HD for 1 h followed by addition of MIPE did not maintain the viability of the cultures, thus the window within which it was possible for MIPE to rescue cell cultures from the effects of HD was of short duration. 4 High performance liquid chromatography was used to determine the biochemical basis of the actions of MIPE. It was found that whilst intracellular levels of cysteine were increased up to 40-fold following treatment of A549 cell cultures with MIPE, levels of reduced glutathione did not rise. The lack of protection seen in cultures pretreated with MIPE for 1 h prior to HD exposure suggests that raising intracellular cysteine levels was not an effective strategy for protecting cells from the effects of HD. The protection observed is probably due to extra cellular inactivation of HD by MIPE.

Protection of A549 cells against the toxic effects of sulphur mustard by hexamethylenetetramine

1997 ◽  
Vol 16 (2) ◽  
pp. 106-114 ◽  
Author(s):  
Christopher D Lindsay ◽  
Joy L Hambrook
Keyword(s):  
Cell Cultures ◽  
A549 Cell ◽  
Gentian Violet ◽  
A549 Cells ◽  
Rapid Onset ◽  
Neutral Red ◽  
Toxic Effects ◽  
In Vivo Models ◽  
Sulphur Mustard

The A549 cell line was used as a model of the deep lung to study the toxicity and mechanism of action of sulphur mustard (HD), using the neutral red (NR) dye retention and gentian violet (GV) assays as indices of cell viability. It was found that exposure to concentrations in excess of 40 μM HD resulted in a rapid onset of toxicity. Exposure to 1000 μM HD reduced viability in A549 cell cultures to 61% after 2 h (control cultures=100%), whereas exposure to 40 μM HD did not result in deleterious effects until 26 h at which point viability fell to only 84% (NR assay). Agarose gel electrophoresis of cell cultures exposed to 40 and 1000 μM HD and harvested at 4.5, 19 and 43 h after exposure to HD, indicated that cell death was due to necrosis, despite the observation that at the higher concentration of HD cells displayed many of the features common to cells undergoing apoptotic death. The ability of hexamethylenetetramine (HMT) to protect A549 cells against the effects of an LC50 challenge dose of HD was assessed using the GV and NR assays. It was found that HMT (15 mM) could protect cells against the effects of HD though HMT had to be present at the time of HD challenge. Cultures treated with HD only were 49% viable at 48 h after HD challenge, compared to 101% for protected cultures (NR assay) and 58% and 91% for unprotected and protected cultures respectively using the GV assay. Morphological observations of GV and NR stained cultures confirmed these findings. HMT concen trations of 2.5 to 25 mM were used. Maximal protection against the toxic effects of HD (LC50) was found at 10 to 25 mM HMT. Over this concentration range, HMT did not exert any toxic effects on A549 cells. Pretreatment of A549 cultures with HMT followed by its removal prior to HD challenge had no protective effect. Similarly, treating cultures with HD followed by addition of HMT did not increase the viability of the cultures, even if the HMT was added immediately after HD exposure. HMT was found to protect against the toxic effects ofHD, though it must be present at the time ofHD challenge. A549 cells were found to be a valuable experimental model for studying the toxicology of HD and other lung damaging agents, and for screening other compounds for potential therapeutic efficacy as a prelude to studies with non- transformed cell culture systems and in vivo models.

THE STRUCTURE AND BEHAVIOR OF THE NUCLEI IN SPORES AND GROWING HYPHAE OF MUCORALES: I. MUCOR HIEMALIS AND MUCOR FRAGILIS

1957 ◽  
Vol 3 (5) ◽  
pp. 771-789 ◽  
Author(s):  
C. F. Robinow
Keyword(s):  
Phase Contrast ◽  
Central Body ◽  
Gentian Violet ◽  
Metaphase Plate ◽  
Low Density ◽  
Mucor Hiemalis ◽  
The Past ◽  
New Findings ◽  
Contrast Microscopy ◽  
And Behavior

The behavior of the nuclei in resting and germinating spores and in growing hyphae of Mucor hiemalis has been followed during life with phase contrast microscopy. The nuclei consist of a dense central body, the nucleolus, surrounded by a shell of variable shape composed of optically uniform material of low density. The nuclei have been seen to divide by constriction. One half of the nucleolus and one half-shell of the low density material pass to each daughter nucleus.All phases of nuclear division have been recognized in fixed and stained preparations. The nucleolus is readily stained by iron hematoxylin or gentian violet but is Feulgen-negative. The shell of low density has no marked affinity for hematoxylin and other basic or acid stains although it consists largely of granules and filaments which are Feulgen-positive. In these elements the chromosomes of the nuclei must somehow be contained but the size, shape, and behavior of individual chromosomes cannot yet be described. Division of the mass of chromatinic elements is direct and involves neither spindle nor metaphase plate. It is tentatively proposed that the chromosomes in the resting nucleus are already divided and segregated to opposite sides and that the constriction of the nucleus is merely the consummation of a kind of endomitosis initiated during the terminal stages of the previous division. Supporting observations have been made on Mucor fragilis, several other Mucorales, and two species of Saprolegnia. Uncertainties and controversies of the past can be explained in the light of the new findings.

Morphological and Histochemical Studies of the Chromatoid Body in the Grass-snake, Natrix natrix

1961 ◽  
Vol s3-102 (57) ◽  
pp. 51-58
Author(s):  
BHUPINDER N. SUD
Keyword(s):  
Phase Contrast ◽  
Primary Spermatocyte ◽  
Neutral Red ◽  
Chromatoid Body ◽  
Natrix Natrix ◽  
Late Spermatid ◽  
Final Maturation ◽  
Contrast Microscopy ◽  
Histochemical Tests ◽  
Grass Snake

The chromatoid body in the spermatogenesis of the grass-snake, Natrix natrix, has been studied by the use of phase-contrast microscopy, vital dyes, and histochemical tests. It first appears during the growth of the primary spermatocyte and is also seen in the secondary spermatocyte and late spermatid, but is absent at metaphases of both the maturation divisions, in the early spermatid, and during the final stages of spermateleosis. It does not make any visible contribution to the final make-up of the spermatozoon. In living cells it gives a very low phase change, and is not stained by neutral red or Janus green. The histochemical study reveals that it consists mainly of RNA and of proteins with abundant acidic and basic groups. It is tentatively suggested that its function is to provide basic proteins for the final maturation of the chromatin in the nucleus of late spermatid.

Detachment of biofilm bacteria due to variations in nutrient supply

1998 ◽  
Vol 37 (4-5) ◽  
pp. 211-214 ◽  
Author(s):  
Linda K. Sawyer ◽  
Slawomir W. Hermanowicz
Keyword(s):  
Phase Contrast ◽  
Digital Image Analysis ◽  
Specific Growth ◽  
Nutrient Supply ◽  
Surface Shear Stress ◽  
Surface Shear ◽  
Nutrient Limitations ◽  
Contrast Microscopy ◽  
Biofilm Bacteria ◽  
Observable Parameter

Growth and detachment rates of an environmental isolate of Aeromonas hydrophila attached to a surface were determined under varying nutrient supply conditions in a complex medium. Growth and detachment of cells were observed in real time using phase contrast microscopy in glass parallel plate flow chambers. Surface shear stress was controlled in all experiments at 3 N m−2. Images were taken every 15 min. Digital image analysis was used to determine specific growth and detachment rates. An observable parameter proportional to the nutrient depletion at the surface due to transfer limitations was used to indicate nutrient limitations. Specific detachment rates increased as the depletion parameter increased, indicating that nutrient limitations cause this bacterium to detach at greater rates.

In vitro Cytotoxicity and Genotoxicity Analysis of Ten Tannery Chemicals Using SOS/umu Tests and High-content In vitro Micronucleus Tests

2018 ◽  
Vol 21 (4) ◽  
pp. 262-270 ◽  
Author(s):  
Zehao Huang ◽  
Na Li ◽  
Kaifeng Rao ◽  
Cuiting Liu ◽  
Zijian Wang ◽  
...  
Keyword(s):  
Micronucleus Test ◽  
A549 Cells ◽  
Quantitative Structure Activity Relationship ◽  
In Vitro Cytotoxicity ◽  
Cytotoxic Effects ◽  
Qsar Analysis ◽  
Cell Assays ◽  
Micronucleus Tests ◽  
Damaging Effects

Background: More than 2,000 chemicals have been used in the tannery industry. Although some tannery chemicals have been reported to have harmful effects on both human health and the environment, only a few have been subjected to genotoxicity and cytotoxicity evaluations. Objective: This study focused on cytotoxicity and genotoxicity of ten tannery chemicals widely used in China. Materials and Methods: DNA-damaging effects were measured using the SOS/umu test with Salmonella typhimurium TA1535/pSK1002. Chromosome-damaging and cytotoxic effects were determined with the high-content in vitro Micronucleus test (MN test) using the human-derived cell lines MGC-803 and A549. Conclusion: The cytotoxicity of the ten tannery chemicals differed somewhat between the two cell assays, with A549 cells being more sensitive than MGC-803 cells. None of the chemicals induced DNA damage before metabolism, but one was found to have DNA-damaging effects on metabolism. Four of the chemicals, DY64, SB1, DB71 and RR120, were found to have chromosome-damaging effects. A Quantitative Structure-Activity Relationship (QSAR) analysis indicated that one structural feature favouring chemical genotoxicity, Hacceptor-path3-Hacceptor, may contribute to the chromosome-damaging effects of the four MN-test-positive chemicals.

scholarly journals THU0406 IDENTIFICATION OF INTRACELLULAR VACUOLES IN SYNOVIAL FLUID WITH CALCIUM PYROPHOSPHATE AND MONOSODIUM URATE CRYSTALS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 440.1-441
Author(s):  
M. L. Peral ◽  
I. Calabuig ◽  
A. Martín-Carratalá ◽  
M. Andrés ◽  
E. Pascual
Keyword(s):  
Clinical Practice ◽  
Synovial Fluid ◽  
Phase Contrast ◽  
Calcium Pyrophosphate ◽  
Monosodium Urate ◽  
Fluid Analysis ◽  
Cytoplasmic Vacuoles ◽  
Contrast Microscopy ◽  
Ordinary Light ◽  
Msu Crystals

Background:Synovial fluid analysis using polarized microscopy is the gold standard for the diagnosis of crystal-related arthritis. In our experience, we have noted that, when calcium pyrophosphate (CPP) crystals are observed, they sometimes appear within intracellular vacuoles. However, this phenomenon is not seen in those samples containing monosodium urate (MSU) crystals. This finding has been scantly reported in the literature, but may be useful in clinical practice to ensure accurate crystal identification.Objectives:Our study aims to assess whether the presence of vacuoles contributes to identifying the type of crystal, and also to gauge the frequency of their presentation.Methods:We conducted an observational study in a rheumatology unit between February and June of 2019. Synovial fluids containing CPP or MSU crystals, obtained in daily clinical practice, were consecutively included for analysis. Two observers simultaneously analyzed the presence of vacuoles by ordinary light and phase contrast microscopy in less than 24 hours after their extraction, using a microscope equipped with two viewing stations. The primary study variable was to determine whether CPP and MSU crystals are seen inside intracellular vacuoles, and to calculate the frequency of this finding for each type of crystal, estimating their 95% confidence interval (95% CI) and comparing rates using Fisher’s exact test.Results:Twenty-one samples were obtained. Data is given in the Table. MSU crystals were present in 7 (33.3%) and CPP crystals in 14 (66.6%). Interestingly, none of the MSU samples showed crystal-containing vacuoles (95% CI 0-35.4%). On the contrary, cytoplasmic vacuoles containing crystals were present in all of the CPP samples (95% CI 78.5-100%). The findings were confirmed by phase-contrast microscopy. Differences were statistically significant (p<0.001).Table.SAMPLES ACCORDING TO TYPE OF MICROCRYSTAL(n=21)SAMPLES WITH VACUOLS(UNDER ORDINARY LIGHT)SAMPLES WITH VACUOLS(UNDER PHASE CONTRAST)CPP (14; 66.6%)14 (100%)(95%CI 78.5-100%)14 (100%)(95%CI 78.5-100%)MSU (7; 33.3%)0 (0%)(95%CI 0-35.4%)0 (0%)(95%CI 0-35.4%)Conclusion:The presence of vacuoles may be a useful and easy way to differentiate MSU and CPP crystals when performing synovial fluid microscopy in clinical practice, since it appears to be a distinctive feature in CPP crystal fluids.References:[1]Kohn NN, Hughes RE, McCarty DJ Jr, Faires JS. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: the «pseudogout syndrome». II. Identification of crystals. Ann InternMed. 1962 May;56:738-45.[2]Pascual E, Sivera F, Andrés M. Synovial Fluid Analysis for Crystals. CurrOpRheumatol 2011;23:161-169.[3]McCarty DJ, Koopman WJ. Arthritis and allied conditions: A textbook of rheumatology, volumen 1. Lea &amp;Febiger. 1993.[4]Pascual E, Sivera F. Synovial fluid crystal Analysis. En Gout and other crystal arthropathies. Terkeltaub R ed. Elsevier; 2012: p.20-34.[5]Hwang HS, Yang CM, Park SJ, Kim HA. Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process. Int J Mol Sci. 2015 Dec 8;16(12):29265-77.Image 1. Microscopy with ordinary light. Cells with cytoplasmic vacuoles are observed, as well as abundant intra and extracellular CPP crystals.Image 2. Microscopy with phase contrast technique. Cells with intracellular vacuoles are observed inside which have microcrystals with parallelepiped morphology, compatible with CPP.Disclosure of Interests: :None declared

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