Evaluation of Cytotoxicity Assays of Human Epidermal Keratinocytes Exposed In Vitro to Sulfur Mustard

2003 ◽  
Author(s):  
Susan Kelly ◽  
William Smith ◽  
Clark Gross ◽  
Juanita Guzman
Keyword(s):  
Sulfur Mustard ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Cytotoxicity Assays

Comparison of Keratinocytes and Lymphocytes on Sulfur Mustard-Dependent Changes in Cell Yield and Viability

1998 ◽  
Vol 17 (4) ◽  
pp. 413-430
Author(s):  
Janet Moser ◽  
Henry L. Meier
Keyword(s):  
Cell Count ◽  
Propidium Iodide ◽  
Sulfur Mustard ◽  
Viable Cell ◽  
Peripheral Blood Lymphocytes ◽  
Time Dependent ◽  
Cell Yield ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes

In comparing the effects of sulfur mustard (SM)-induced cell death in adult cultured normal human epidermal keratinocytes (NHEK) and peripheral blood lymphocytes (PBL), we observed an SM concentration-and time-dependent loss of NHEK and PBL viability as measured by propidium iodide exclusion. By this parameter alone, PBL appeared more susceptible to SM-induced cytotoxi-city than NHEK, as previously reported. However, therewas a concentration-and time-dependent loss of NHEK yield that was not observed in PBL. Keratinocyte yield was approximately 46% and 8% of control cells at 12 and 24 hours, respectively, following exposure to 0.3 mM SM. The decrease in NHEK yield was associated with an increase in trypan blue-stained fragments observed on slides. Fragments were not observed on slides of SM-exposed PBL cultures. These results suggest that SM causes fragmentation of cultured NHEK and subsequently results in a lower cell yield. The fragmented dead NHEK are not included in the total cell count when percent viability is determined by propidium iodide exclusion. Therefore, cytotoxicity of SM in NHEK is greater than that reflected by dye exclusion alone. Actual viable cell count, which considers both propidium iodide exclusion and cellyield, indicates NHEK are more susceptible than PBL to SM-induced cytotoxicity, contrary to previously published reports. Cell yield must be taken into account when using in vitro models to study not only viability, but also any quantitative biochemical changes induced by SM.

scholarly journals The Role of Interleukin-6 (IL-6) in Human Sulfur Mustard (HD) Toxicology

2001 ◽  
Vol 20 (5) ◽  
pp. 281-296 ◽  
Author(s):  
Carmen M. Arroyo ◽  
Clarence A. Broomfield ◽  
Brennie E. Hackley
Keyword(s):  
Interleukin 6 ◽  
Time Course ◽  
Structural Changes ◽  
Sulfur Mustard ◽  
Skin Irritation ◽  
Epidermal Keratinocytes ◽  
Early Event ◽  
Mrna Levels ◽  
Human Epidermal Keratinocytes

The authors applied in vitro models of controlled damage to human epidermal keratinocytes (HEKs), human skin fibroblasts (HSFs), and human breast skin tissue (HBST) to examine the mechanism responsible for sulfur mustard (HD)-induced interleukin-6 (IL-6) alterations. Treatment with 100 μM HD for 24 hours resulted in a significant increased amount of IL-6 being secreted by HEKs (HD-exposed to control ratio [E/C] = 4.15 ± 0.07) and by HSFs (E/C = 7.66 ± 0.04). Furthermore, the HD-induced secretion of IL-6 in HEKs was neutralized with monoclonal human IL-6 antibodies. The secretion of IL-6 in HBST supernatant exposed to HD produced conflicting results. Although an increase of IL-6 was observed in control superfusion media from HBST, IL-6 levels were observed to decrease as the concentration of HD increased. Time course of IL-6 mRNA levels were performed using a competitive polymerase chain reaction (PCR) and human IL-6 mRNA assay detection kit in control and HD (100 μM)-treated HEKs cells. IL-6 mRNA transcripts in HD-exposed HEKs were first observed within 2 hours, dropped at 5 to 6 hours, and increased by ® 2.2-fold and 8.5-fold at 24 to 48 hours after HD exposure, respectively, as detected by the Xplore mRNA Quantification System. Surface-enhanced laser desorption ionization (SELDI) mass spectrometry was also applied to study the secretion pattern of IL-6 on lysate preparations of HBST. A peak in the area of 23,194 to 23,226 Da was detected using antibody coupled to the chip. This peak was assigned to correspond to the mass of the IL-6 glycoprotein. Recombinant human IL-6 (rhIL-6) exposed to HD lacked the second disulfide bridge and was partially unfolded, as determined by nuclear magnetic resonance-nuclear Overhauser enhancement and exchange spectroscopy (NMR-NOESY). The disappearance of the resonance peak at 3.54 ppm and the appearance of a new chemical shift at 1.85 ppm suggested that a change in structure had occurred in the presence of HD. From the data, the possibility cannot be excluded that IL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein that indirectly initiates the cascade of events such as skin irritation and blister formation observed in the pathophysiology of HD injury.

Alterations of ultrastructure and elemental composition in cultured human epidermal keratinocytes after treatment with nitrofurazone and silver sulfadiazine

1987 ◽  
Vol 45 ◽  
pp. 676-677
Author(s):  
A. R. Crooker ◽  
M. C. Myers ◽  
T. L. Beard ◽  
E. S. Graham
Keyword(s):  
Electron Microscopy ◽  
Elemental Composition ◽  
Pyrolytic Carbon ◽  
Human Keratinocytes ◽  
Silver Sulfadiazine ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Culture Systems ◽  
Cytotoxicity Endpoints

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.

scholarly journals Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2153
Author(s):  
Raffaella Marina Lecci ◽  
Isabella D’Antuono ◽  
Angela Cardinali ◽  
Antonella Garbetta ◽  
Vito Linsalata ◽  
...  
Keyword(s):  
Antioxidant Activities ◽  
Biological Activities ◽  
Human Keratinocytes ◽  
Ldl Oxidation ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Epidermal Keratinocytes ◽  
Olive Cultivar ◽  
Human Epidermal Keratinocytes ◽  
Apoptotic Effect

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.

scholarly journals 5-Aminolevulinic Acid-Based Photodynamic Therapy Pretreatment Mitigates Ultraviolet A-Induced Oxidative Photodamage

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Hua ◽  
Jiawei Cheng ◽  
Wenbo Bu ◽  
Juan Liu ◽  
Weiwei Ma ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Epidermal Keratinocytes ◽  
Control Group ◽  
Hacat Cells ◽  
Ultraviolet A ◽  
Hairless Mice ◽  
Human Epidermal Keratinocytes ◽  
Experimental Group

Aim. To determine whether 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is effective in combating ultraviolet A- (UVA-) induced oxidative photodamage of hairless mice skin in vivo and human epidermal keratinocytes in vitro. Methods. In in vitro experiments, the human keratinocyte cell line (HaCaT cells) was divided into two groups: the experimental group was treated with ALA-PDT and the control group was left untreated. Then, the experimental group and the control group of cells were exposed to 10 J/m2 of UVA radiation. ROS, O2− species, and MMP were determined by fluorescence microscopy; p53, OGG1, and XPC were determined by Western blot analysis; apoptosis was determined by flow cytometry; and 8-oxo-dG was determined by immunofluorescence. Moreover, HaCaT cells were also treated with ALA-PDT. Then, SOD1 and SOD2 were examined by Western blot analysis. In in vivo experiments, the dorsal skin of hairless mice was treated with ALA-PDT or saline-PDT, and then, they were exposed to 20 J/m2 UVA light. The compound 8-oxo-dG was detected by immunofluorescence. Conclusion. In human epidermal keratinocytes and hairless mice skin, UVA-induced oxidative damage can be prevented effectively with ALA-PDT pretreatment.

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