Improved alkaline comet assay protocol for adherent HaCaT keratinocytes to study UVA-induced DNA damage

Wischermann, K.; Boukamp, Petra; Schmezer, Peter

doi:10.1016/j.mrgentox.2007.02.009

Cited by 13 publications

(10 citation statements)

References 25 publications

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“…The alkaline comet assay was performed either directly after irradiation or after a recovery of 2 or 4 h in complete culture medium, according to Wischermann et al (2007). The neutral comet assay was performed directly after irradiation according to the protocols described by Olive and Banath (Olive et al, 1993).…”

Section: Alkaline and Neutral Comet Assaymentioning

confidence: 99%

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

et al. 2008

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The role of UVA-radiation-the major fraction in sunlight-in human skin carcinogenesis is still elusive. We here report that different UVA exposure regime (4 Â 5 J/cm 2 per week or 1 Â 20 J/cm 2 per week) caused tumorigenic conversion (tumors in nude mice) of the HaCaT skin keratinocytes. While tumorigenicity was not associated with general telomere shortening, we found new chromosomal changes characteristic for each recultivated tumor. Since this suggested a nontelomere-dependent relationship between UVA irradiation and chromosomal aberrations, we investigated for alternate mechanisms of UVA-dependent genomic instability. Using the alkaline and neutral comet assay as well as c-H2AX foci formation on irradiated HaCaT cells (20-60 J/cm 2 ), we show a dosedependent and long lasting induction of DNA single and double (ds) strand breaks. Extending this to normal human skin keratinocytes, we demonstrate a comparable damage response and, additionally, a significant induction and maintenance of micronuclei (MN) with more acentric fragments (indicative of ds breaks) than entire chromosomes particularly 5 days post irradiation. Thus, physiologically relevant UVA doses cause long-lasting DNA strand breaks, a prerequisite for chromosomal aberration that most likely contribute to tumorigenic conversion of the HaCaT cells. Since normal keratinocytes responded similarly, UVA may likewise contribute to the complex karyotype characteristic for human skin carcinomas.

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Section: Alkaline and Neutral Comet Assaymentioning

confidence: 99%

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

et al. 2008

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“…The ability for UVA radiation to generate SSBs and/or alkali‐labile lesions as minor classes of DNA damage has been demonstrated in Chinese ovary cells and human monocytes using the alkaline elution technique, agarose gel electrophoresis or the alkaline comet assay . Similar types of DNA lesions have been shown to be produced in adherent human keratinocytes (HaCaT) that have been exposed to UVA radiation using an improved version of the alkaline comet . As an illustration of the low efficiency for UVA to cleave cellular DNA, it was found that an exposure to a dose of 100 kJ m −2 of UVA radiation was able to increase the tail moment value from 1.91 up to 2.66 in relative units.…”

Section: Uva Radiation‐mediated Oxidation Reactions To Cellular Dnamentioning

confidence: 99%

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation^,

Cadet

Douki

Ravanat

2014

Photochem & Photobiology

279

223

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This review article focuses on a critical survey of the main available information on the UVB and UVA oxidative reactions to cellular DNA as the result of direct interactions of UV photons, photosensitized pathways and biochemical responses including inflammation and bystander effects. UVA radiation appears to be much more efficient than UVB in inducing oxidatively generated damage to the bases and 2-deoxyribose moieties of DNA in isolated cells and skin. The UVA-induced generation of 8-oxo-7,8-dihydroguanine is mostly rationalized in terms of selective guanine oxidation by singlet oxygen generated through type II photosensitization mechanism. In addition, hydroxyl radical whose formation may be accounted for by metal-catalyzed Haber-Weiss reactions subsequent to the initial generation of superoxide anion radical contributes in a minor way to the DNA degradation. This leads to the formation of both oxidized purine and pyrimidine bases together with DNA single-strand breaks at the exclusion, however, of direct double-strand breaks. No evidence has been provided so far for the implication of delayed oxidative degradation pathways of cellular DNA. In that respect putative characteristic UVA-induced DNA damage could include single and more complex lesions arising from one-electron oxidation of the guanine base together with aldehyde adducts to amino-substituted nucleobases.

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“…In the past, researchers have used a wide variety of solutions for UV illumination, ranging from home solarium lamps to state-of-the-art solar simulators. By far the most common source of UV light is a lamp with one or more fluorescent tubes [10][11][12][13][14], ranging from simple hand-held lamps [14] or home solariums [10] to UV-crosslinkers [12] and therapeutic illuminators designed for medical use [13]. The second most common type of UV light sources for medical research are solar simulators [15][16][17], which usually employ a high-power Xenon arc lamp with an optical output spectrum very similar to that of natural sunlight.…”

Section: Introductionmentioning

confidence: 99%

“…Even fluorescent tubes, which are still among the more efficient light sources, emit approximately 90% of their energy in the form of heat [18]. Researchers combat the problem of excessive heat dissipation with ventilation [10], ice blocks [11], cooling blocks connected to a thermostated bath [16], etc.…”

Section: Introductionmentioning

confidence: 99%

Compact UV LED Lamp with Low Heat Emissions for Biological Research Applications

et al. 2019

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Much biomedical research focuses on the effects of UV light on human cells. UV light sources are a prerequisite for such research. This paper presents the design and achieved performance of a UVA (Ultraviolet A: 320–400 nm) and a UVB (Ultraviolet B: 290–320 nm) LED-based lamp suitable for use in bioassays, as well as inside an incubator. Numerical simulations were used to optimise the number, layout and output power of LEDs to achieve good irradiance homogeneity while maintaining low costs. Design was optimised for the efficient transfer of generated heat away from the irradiated samples through the heatsink at the back of the lamps. The average irradiance of the target surface by the UVA lamp was 70.1 W/m2 with a maximum deviation of 4.9%, and the average irradiance by the UVB lamp was 3.1 W/m2 with a maximum deviation of 4.8%. With the UVA and UVB lamps, the temperature of samples undergoing irradiation in the incubator rises from 37 to 42 °C within 40 and 67 min, respectively. This by far exceeds the required UV irradiation time in most cases. Tests on Jurkat and HEK-293 cell cultures confirmed the suitability of our lamps for biomedical research.

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Improved alkaline comet assay protocol for adherent HaCaT keratinocytes to study UVA-induced DNA damage

Cited by 13 publications

References 25 publications

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation^,

Compact UV LED Lamp with Low Heat Emissions for Biological Research Applications

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Improved alkaline comet assay protocol for adherent HaCaT keratinocytes to study UVA-induced DNA damage

Cited by 13 publications

References 25 publications

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation,

Compact UV LED Lamp with Low Heat Emissions for Biological Research Applications

Contact Info

Product

Resources

About

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation^,