Albert A. Schothorst

The UV action spectra for the clone-forming ability of cultured human melanocytes and keratinocytes.

Abstract Melanocytes (skin type 2) and keratinocytes were irradiated with UV light of 254, 297, 302, 312 and 365 nm and the survival was measured. Clone‐forming ability was chosen as the parameter for cell survival. Melanocytes were found to be less sensitive to UV light than keratinocytes (a difference of a factor 1.22‐1.92 for the UV‐C and UV‐R wavelengths (254, 297, 301 and 312 nm) and a factor 6.71 for the UV‐A wavelength (365 nm). Because melanin does not appear to protect against the induction of pyrimidine dimers the difference between melanocytes and keratinocytes in the UV‐C and UV‐B region could not be explained by the presence of melanin in the melanocytes. The relatively small difference can be explained by the longer cell cycle of melanocytes, which provides more time for the melanocytes to repair UV damage. In the UV‐A region the difference between melanocytes and keratinocytes was much larger, suggesting that besides the longer cell cycle some additional factors must be involved in protection against UV‐A light.

Physiological Doses of Ultraviolet Irradiation Induce DNA Strand Breaks in Cultured Human Melanocytes, as Detected by Means of an Immunochemical Assay

Abstract— An immunochemical assay, i.e. sandwich enzyme‐linked immunosorbent assay, has been modified to detect UV‐induced damage in cellular DNA of monolayer‐grown human melanocytes. The method is based on the binding of a monoclonal antibody to single‐stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar‐simulated light or γ‐rays. Following physiological doses of UVA, UVB or solar‐simulated light, a dose‐related DNA unwinding comprising a considerable number of single‐strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/1010 Da/kJ/m2 were detected and after UVB irradiation 1.9 ssb/1010 Da/kJ/m2 were seen. One minimal erythema dose of solar‐simulated light induced 2.25 ssb/1010 Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.

PHOTOCHEMICAL DAMAGE TO DNA TREATED WITH CHLORPROMAZINE AND NEAR UV RADIATION UNDER AEROBIC AND ANAEROBIC CONDITIONS

Double stranded salmon sperm DNA in a chlorpromazine (CPZ) solution is damaged when irradiated with near UV light. The damage of irradiated DNA can be estimated by measuring the increase in extinction at 260 nm following incubation at 60°C of the DNA with formaldehyde. Moreover, DNA irradiated in the presence of CPZ or kept in the dark separate quite differently in an aqueous polymer two‐phase system. DNA irradiated in the presence of CPZ seemed to be susceptible to digestion by endonuclease S1, while the endonuclease of Neurospora crassa could not digest this DNA. Irradiation under aerobic conditions seemed to be less disastrous for DNA than under anaerobic conditions.

UV INDUCTION OF CYCLOBUTANE THYMINE DIMERS IN THE DNA OF CULTURED MELANOCYTES FROM FORESKIN, COMMON MELANOCYTIC NEVI and DYSPLASTIC NEVI

Abstract— We compared the induction of cyclobutane thymine dimers after exposure to 302 nm UV in foreskin‐derived melanocytes and melanocytes from nevocellular nevi, as well as in melanocytes cultured from dysplastic nevi, precursor lesions of melanoma, derived from four, three and four individuals, respectively. Cyclobutane thymine dimers were quantified in situ by means of an immunofluorescence assay with a specific monoclonal antibody. A method was developed to compare separately performed experiments in a standardized manner. For melanocytes from each source, we demonstrated a linear relationship between UV dose and immunofluorescence. In nevocellular and dysplastic nevi, two subpopulations could be detected, distinguished by their nuclear size. Large nucleated nevocellular nevus cells were most susceptible to the induction of thymine dimers (49% higher induction compared to induction in foreskin melanocytes), while in normal‐sized nuclei of these nevus cells the same induction of thymine dimers was found as in nuclei from foreskin melanocytes. In contrast, large nucleated dysplastic nevus melanocytes did not differ from the foreskin melanocytes, while normal‐sized nuclei of dysplastic nevus cells showed a lower induction (32% lower induction than in foreskin melanocytes).