Evaluation of Sunscreen Protection in Human Melanocytes Exposed to UVA or UVB Irradiation Using the Alkaline Comet Assay¶

Abstract

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low‐melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 μL volumes of sunscreens. DNA single‐strand breaks induced directly by UVA at 4°C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37°C were quantified using the comet assay. Tail moments (TM) (tail length ×%tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a χ2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty‐one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.

Assessment of occupational exposure to welding fumes by inductively coupled plasma‐mass spectroscopy and by the alkaline Comet assay

“…A nonlinear regression analysis was performed on the OTM distribution frequencies by using a v 2 function with TableCurve 2D software (version 5.0; Jandel Scientific Software, San Rafael, CA). The calculated degrees of freedom (n) for this function were a quantitative measure of the DNA damage for a sample [Bauer et al, 1998;Jean et al, 2001]. The n was termed OTM v 2 and was used as the sole parameter for assessing levels Environmental and Molecular Mutagenesis.…”

Different Susceptibility of Cells of Porcine Skin and Internal Organs to Ultraviolet A‐Induced Breaking of Nuclear DNA¶

“…The difference between our threshold values and the results of Arimoto‐Kobayashi (53) may originate from differences in the wavelengths of the UV‐A radiation: 337 nm in our case and the less energetic 360 nm in the experiments described in the reference (53). Such a conclusion is supported by the fact that Jean et al (45) using the comet assay, similar to that used by Arimoto‐Kobayashi (53), detected DNA breaks in the human melanocytes at 20 mJ/cm 2 for 312 nm and 400 mJ/cm 2 for 365 nm.…”

“…The temperature of the tissue sample did not exceed +14°C. In such conditions both the degradation processes and the repair of the UV‐A‐induced breaks in the nuclear DNA were significantly slowed down (43–45).…”

“…According to the literature (43–45), the highest number of the UV‐A‐induced DNA breaks can be detected immediately after the irradiation. Therefore, within 1‐3 min after completing the irradiation the material was cut off and frozen in liquid nitrogen (‐197°C) and then stored in a deep freezer (‐80°C) until it was used for terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) assays.…”

UV‐Mediated DNA Strand Breaks in Corneal Epithelial Cells Assessed Using the Comet Assay Procedure¶

“…The Comet assay is a highly sensitive and a validated biomonitoring tool for DNA damage, and in the alkaline version, measures single‐strand and double‐strand breaks (16–18). This technique has been used most often with peripheral lymphocytes, but it has also been applied to other cell types, including lung, liver and retina (19–24). To date, however, there has been no report on its application in corneal epithelial cells.…”