Steven E. Freeman

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.

PYRIMIDINE DIMER FORMATION IN HUMAN SKIN

Cyclobutyl pyrimidine dimers are major photoproducts formed upon irradiation of DNA with ultraviolet light. We have developed a method for detecting as few as one pyrimidine dimer per million bases in about 50 ng of non‐radioactive DNA, and have used this method to quantitate dimer yields in human skin DNA exposed in situ to UV. We found that UVA radiation (320–400 nm) produces detectable levels of dimers in the DNA of human skin. We also measured UVB‐induced dimer yields in skin of individuals of differing sun sensitivity and found higher yields in individuals with higher UVB minimal erythema doses and greater sun sensitivity. These approaches should provide important information on damage induced in human skin upon exposure to natural or artificial sources of ultraviolet radiation.

DO PYRIMIDINE DIMER YIELDS CORRELATE WITH ERYTHEMA INDUCTION IN HUMAN SKIN IRRADIATED in situ WITH ULTRAVIOLET LIGHT (275–365 nm)?

Ultraviolet radiation produces erythema in human skin, and damages the DNA of living cells in skin. Previous work showed that broad‐band UV‐B (290–320 nm) radiation produced higher levels of cyclobutyl pyrimidine dimers in DNA of individuals with high UV‐B sensitivity (low minimal erythema dose) than in subjects of low UV‐B sensitivity [Freeman et al. (1986) J. Invest. Dermatol., 86, 34–36]. We examined the relationship between erythema induction and dimer yields in DNA of human skin irradiated in situ with narrow band radiation spanning the wavelength range 275–365 nm. We find that, in general, higher dimer yields are produced per incident photon in volunteers with higher susceptibility to erythema induced by radiation of the same wavelength.

Does exposure of human skin in situ to 385 or 405 nm UV induce pyrimidine dimers in DNA

Abstract— A previous report [Freeman et al.(1986) Photochem. Photobiol.43S, 93S] indicated that irradiation of human skin in situ with 385 or 405 nm radiation produced detectable levels of pyrimidine dimers in DNA. Since these wavelengths are absorbed poorly by DNA, these results suggested that DNA damage was sensitized by other absorbing molecules present in skin. Examination of two experimental aspects of the previous work indicates that (1) the static gel electrophoresis method for DNA dispersion used in lesion determination gave accurate values of the levels of induced dimers, and (2) the DNA damage apparently induced by 385 nm was actually induced by shorter wavelength UV present in the 20 nm bandpass beam of the monochromator. The current results indicate that monochromatic 385 and 405 nm radiation are ineffective in dimer production in human skin in situ.

Quantitation of ultraviolet radiation-induced cyclobutyl pyrimidine dimers in DNA by video and photographic densitometry☆

We have compared video and photographic methods for calculating the number of ultraviolet radiation (uv)-induced pyrimidine dimers in DNA from the bacteriophage T7 exposed to uv (0 to 800 J/m2) from an FS40 sunlamp. DNA was incubated with a pyrimidine dimer-specific Micrococcus luteus uv endonuclease, subjected to alkaline agarose gel electrophoresis, neutralized, and stained with ethidium bromide, and the DNA fluorescence was recorded either with a video camera or on photographic film. The slopes of the dose-response curves for the number of uv-endonuclease-sensitive sites per 10(3) bases (pyrimidine dimers) was 1.2 (+/- 0.1) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the video analysis and 1.3 (+/- 0.04) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the photographic analysis. Results for pyrimidine dimer determination by either method were statistically comparable.

EXCISION REPAIR OF UVR‐INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)‐induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica, using damage‐specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ‐2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10‐2 endonuclease‐sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m‐2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10‐2) in the DNA of skin of M. domestica in vivo. In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m‐2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10‐2) in the DNA of the cultured marsupial cell line Pt K2 (Potorous tridactylus).

Photoreactivation and Other Ultraviolet/Visible Light Effects on DNA in Human Skina

A computer-based system was devised for examining low levels (1 dimer/10/sup 8/ d) of DNA damage in human skin exposed to ultraviolet or visible radiation. The production of measurable levels of dimers was determined for light of wavelengths absorbed directly by DNA (290-320 nm). Exposure of skin to uv-A (320-380 nm) sources also produced significant numbers of endonuclease-sensitive sites. The immediate pigment darkening reaction (IPD) was tested to see whether IPD protects against uv-induced dimer formation. 29 references, 2 figures, 1 table. (ACR)

DNA damage and repair in human skin in situ

Understanding the molecular and cellular origins of sunlight-induced skin cancers in man requires knowledge of the damages inflicted on human skin during sunlight exposure, as well as the ability of cells in skin to repair or circumvent such damage. Although repair has been studied extensively in procaryotic and eucaryotic cells—including human cells in culture—there are important differences between repair by human skin cells in culture and human skin in situ: quantitative differences in rates of repair (B. Sutherland et al., 1980; D’Ambrosio et al., 1981a), as well as qualitative differences, including the presence or absence of repair mechanisms (B. Sutherland et al., 1980; D’Ambrosio et al., 1981b).