Len Roza

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER‐ CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse‐plasmacytoma cells and spleen cells from mice immunized with a thymine dimer‐containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme‐linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single‐stranded DNA or poly(dT), but do not bind UV‐irradiated d(TpC)5. Less binding is observed with short thymine dimer‐containing sequences. In vitro treatment of UV‐irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV‐endonuclease results in disappearance of antigenicity. Antibody‐binding to DNA isolated from UV‐irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post‐irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m2). The induction of thymine dimers in human skin irradiated with low doses of UV‐B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.

Biological consequences of cyclobutane pyrimidine dimers

In the skin many molecules may absorb ultraviolet (UV) radiation upon exposure. In particular, cellular DNA strongly absorbs shorter wavelength solar UV radiation, resulting in various types of DNA damage. Among the DNA photoproducts produced the cyclobutane pyrimidine dimers (CPDs) are predominant. Although these lesions are efficiently repaired in the skin, this CPD formation results in various acute effects (erythema, inflammatory responses), transient effects (suppression of immune function), and chronic effects (mutation induction and skin cancer). The relationships between the presence of CPD in skin cells and the subsequent biological consequences are the subject of the present review.

Melanin Offers Protection Against Induction of Cyclobutane Pyrimidine Dimers and 6–4 Photoproducts by UVB in Cultured Human Melanocytes¶

Abstract The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6–4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type–I and skin type–VI melanocytes, congenital nevus (CN)-derived cells and skin type–II melanocytes from a multiple-melanoma patient were grown in media with low or high l-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.

Correlation of UVC and UVB cytotoxicity with the induction of specific photoproducts in T-lymphocytes and fibroblasts from normal human donors.

Abstract— By using specific monoclonal antibodies in situ and a computer‐assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad‐spectrum UVB and narrow‐spectrum UVB. The lamps produced these lesions in different proportions, with broad‐spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow‐spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad‐ or narrow‐spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad‐spectrum or narrow‐spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T‐lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T‐lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.

Determination of the photoprotective efficacy of a topical sunscreen against UVB-induced DNA damage in human epidermis

The ability of a chemical sunscreen with a sun protection factor of ten to protect human skin in situ against UVB-induced DNA damage (cyclobutyl thymine dimers) was evaluated. Biopsies were taken from the left buttock of ten human volunteers prior to UVB (280-315 nm) exposure. Subsequently, a sunscreen (n = 6) or vehicle (n = 4) was applied to a delineated area on the right buttock. After a period of 30 min, the entire buttock area was irradiated in a UVB cabin with one minimal erythema dose. Immediately after irradiation, biopsy specimens were obtained from the UVB-exposed sunscreen- or vehicle-treated right buttock and from the non-treated UVB-exposed left buttock. Dimers were assayed in skin sections by immunofluorescence microscopy with a monoclonal antibody against the cyclobutyl thymine dimer. The dimer-specific fluorescence from the epidermal cell nuclei, identified by counterstaining with propidium iodide, was quantified through computer-mediated image processing and analysis in skin sections of one sunscreen-treated and one vehicle-treated volunteer. After a single dose of UVB, significant dimer-specific nuclear fluorescence was observed and measured in the non-treated biopsy specimens. No nuclear fluorescence was observed and very little could be measured in the non-UVB-exposed skin and in the sunscreen-treated UVB-exposed skin respectively, indicating that the sunscreen offered good protection against the induction of cyclobutyl thymine dimers by UVB. This visual scoring is in general semiquantitative, but quantification through computer-mediated image processing was performed in one case for sunscreen-treated skin and in one case for vehicle-treated skin. Both assessments resulted in similar conclusions.(ABSTRACT TRUNCATED AT 250 WORDS)

Removal of UV-induced DNA lesions in mouse epidermis soon after irradiation

Induction and removal of cyclobutane thymine dimers and (6-4)photoproducts were studied in epidermal DNA isolated from UV-exposed hairless mice. For the detection of DNA damage, lesion-specific monoclonal antibodies were used in an immunoslotblot assay. Following the exposure of mice to 3.0 kJ m-2 UV-B, substantial removal of both thymine dimers (66%) and (6-4)photoproducts (77%) was observed at 24 h after irradiation. No removal, however, was detected at 4 h after irradiation. In contrast, immunofluorescence data obtained previously showed a rapid initial dimer removal after irradiation with 1.0 kJ m-2 UV-B (A.A. Vink, R.J.W. Berg, F.R. De Gruijl, L. Roza and R.A. Baan, Carcinogenesis, 12 (1991) 861-864). Reinvestigation of the removal of dimers and (6-4)-photoproducts shortly after three different UV doses showed a rapid decreases of both lesions at 2 h after irradiation with 1.0 kJ m-2. The results obtained after irradiations with 2.0 and 3.0 kJ m-2 UV-B suggest a saturation of repair already at 2.0 kJ m-2. Cyclobutane dimers were found to be removed at a lower rate than (6-4)photoproducts.

UVA hazards in skin associated with the use of tanning equipment

This article was commissioned in response to increasing concern in the photobiological/dermatological community about the possible long-term hazards of UVA (315-400 nm) radiation from the human use of tanning equipment. The use of such equipment has increased considerably in Western societies over the last decade

Cellular target of UVB-induced DNA damage resulting in local suppression of contact hypersensitivity

Experimental data are reviewed that lend support to the hypothesis that formation of DNA damage is the initiation event of local suppression of contact hypersensitivity (CHS) after exposure to ultraviolet (UV) radiation and that the antigen-presenting cell (APC) is an important target for this DNA damage.

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.

The Role of DNA Damage and Repair in Ultraviolet B Radiation‐Induced Immunomodulation: Relevance for Human Photocarcinogenesis

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