Optimization of the comet assay for the sensitive detection of PUVA-induced DNA interstrand cross-links
Abstract
Psoralen plus ultraviolet A (PUVA), commonly used for the treatment of hyperproliferative skin disorders, has been found to be associated with an increased risk of squamous cell cancer. Interstrand cross-link (ICL) formation by PUVA treatment is considered the major factor contributing to the carcinogenesis. However, it remains unclear how PUVA causes, or promotes cancers, in humans. As an initial step in understanding the mechanisms of mutagenesis and carcinogenesis of PUVA photochemotherapy, we have optimized and subsequently utilized a modified alkaline comet assay involving a post-lysis gamma-irradiation at 9 Gy to sensitively measure the formation and repair of PUVA-induced ICLs in the immortalized human keratinocyte cell line HaCaT. A clear dose-dependent response of HaCaT cells to PUVA exposure was observed with a combination of a fixed UVA dose at 0.05 J/cm(2) and a dose of 8-methoxypsoralen ranging from 10 to 100 microM. Results also indicated that the ICL repair was concentration dependent. We have also demonstrated that PUVA-induced monoadduct formation, at an estimated ratio of 3:1 to ICLs in the present experimental conditions, does not interfere with the detection of the ICLs in the modified alkaline comet assay. Furthermore, comparison of the amount of ICL formation between the single-dose UVA treatment and a split-dose protocol was performed. The split-dose protocol was believed to generate more ICLs than the single-dose treatment, thus more effective in PUVA photochemotherapy. Our results demonstrate that comparable amounts of ICLs were formed in HaCaT cells for each dose of UVA, using either the split-dose or single-dose protocols.
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A modified alkaline comet assay with a postlysis gamma-irradiation step of 9 Gy measures DNA interstrand cross-links induced in HaCaT cells by 8-MOP plus UVA.
“Recently, a modified version of the alkaline comet assay that includes a postlysis c-irradiation step with a dose of 9 Gy has been designed for measuring DNA ICLs induced in HaCat cells upon treatment with 8-MOP plus UVA radiation (164).”
From Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup> (2012) · cites this paper as [164]
The repair rate of interstrand cross-links is slow, requiring at least 24 h, and decreases with increasing 8-MOP concentration.
“The repair rate of ICLs that is rather slow, requiring at least 24 h to be completed, was found to decrease with an increase in the concentration of 8-MOP (164).”
From Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup> (2012) · cites this paper as [164]
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Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions<sup>†</sup>
Cadet, J., Mouret, S., Ravanat, J., & Douki, T. (2012) · Photochemistry and Photobiology · doi:10.1111/j.1751-1097.2012.01200.x · Cited by 300 · see citing works
- Authors
- Wu, J. H., Wilson, J. B., Wolfreys, A. M., Scott, A., & Jones, N. J.
- Affiliations
- University of Liverpool (GB)
- Unilever (United Kingdom) (GB)
- Corresponding author
- Jones, N. J.
- Venue
- Mutagenesis
- Publisher
- Oxford University Press (OUP)
- Published
- 2008-11-25
- Volume
- 24
- Issue
- 2
- Pages
- 173-181
- ISSN
- 0267-8357 (ISSN-L)
- 1464-3804
- OpenAlex
- W2167782007
- Cited by
- 22
- References
- 51
- Citations by year
- 2026: 2 · 2025: 1 · 2018: 1 · 2016: 3 · 2015: 2 · 2014: 2 · 2013: 2 · 2012: 4
- FWCI
- 0.56
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Mutagenesis (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Health Sciences › Medicine › Dermatology › Skin Protection and Aging
- Keywords
- PUVA, psoralen, interstrand cross-links, DNA damage, DNA repair, alkaline comet assay, gamma irradiation, 8-methoxypsoralen, monoadducts, HaCaT keratinocytes, squamous cell carcinoma, photochemotherapy
- MeSH terms
- Cell Line, Cross-Linking Reagents, DNA, DNA Repair, Dose-Response Relationship, Radiation, Gamma Rays, Humans, Methoxsalen, Furocoumarins, PUVA Therapy, Keratinocytes, DNA Adducts, Comet Assay
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