CUTANEOUS BIOLOGY DOI 10.1111/j.1365-2133.2005.06804.x Early epidermal response after a single dose of c-rays in organotypic culture of human breast skin E. Donetti, M. Bedoni, E. Boschini, A.A.E. Bertelli, C. Sforza and N. Gagliano Department of Human Morphology, University of Milan, Via Mangiagalli 31, 20133 Milan, Italy Correspondence Elena Donetti. E-mail: elena.donetti@unimi.it Accepted for publication 23 March 2005 Key words: 5-bromo-2¢-deoxyuridine, cytokeratin 10, desmocollin 1, human skin, ionizing radiation, transforming growth factor-b1 Conflicts of interest: None declared. Summary Background Skin reaction is the most common side-effect of radiation therapy. Radiation-induced dermal fibrosis has been characterized histologically, but little is known about the epidermis overlying fibronecrotic lesions. Objectives To characterize the epidermal response 24 h after a single clinically rele- vant dose of c-rays in cultured human breast skin. Methods Biopsies obtained from cosmetic surgery (n ¼ 7) were placed epidermis upwards in a Transwell system, and were exposed to a single dose of c-irradi- ation (2 Gy). A parallel set of nonirradiated skin fragments was incubated under the same conditions. Both irradiated and nonirradiated fragments were harvested 24 h after irradiation and processed for light microscopy and molecular biology analysis. A quantitative analysis of cell proliferation was per- formed after 5-bromo-2¢-deoxyuridine incorporation. Cytokeratin 10 (CK10) and desmocollin 1 (Dsc1) expression was evaluated by immunofluorescence. Dsc1 and transforming growth factor (TGF)-b1 gene expression was measured by reverse transcriptase–polymerase chain reaction analysis. Results The mean percentage inhibition of epidermal proliferation in irradiated samples was 53Æ7% (P <0Æ01, paired Student’s t-test). The inhibition of cell pro- liferation was significant in five of seven samples (P <0Æ05, unpaired Student’s t-test). Normal cell architecture was found in irradiated samples. Throughout the epithelial compartment, the distribution patterns of CK10 and Dsc1 were com- parable in nonirradiated and irradiated fragments. Condensation of CK10 fila- ments suggested a cytoskeletal rearrangement in irradiated samples. Dsc1 and TGF-b1 mRNA levels were, respectively, reduced and unmodified 24 h after irradiation. Conclusions A perturbation of epidermal homeostasis occurs as early as 24 h after a single dose of c-rays. Our immunofluorescence observations indicate that kera- tinocyte terminal differentiation is not yet affected at the protein level 24 h after exposure to c-rays. The lack of an inverse relationship between TGF-b1 gene expression and epidermal proliferation, together with decreased Dsc1 gene expression, may represent the early molecular basis for the development of the late effects of radiotherapy observed many months ⁄ years after radiotherapy. Our findings set the stage for further investigation of the best time to begin topical treatment at the start of radiation therapy. Radiotherapy is the main nonsurgical approach for cancer, but skin reaction is the most common side-effect during such a treatment. Up to 95% of patients receiving chest wall radio- therapy experience some dose-dependent degree of reaction. 1 Early skin response occurs during the first 70 days and begins with erythema, followed, as the dose increases, by dry and moist desquamation. 2–4 These side-effects induce discomfort ranging from mild irritation to severe pain, and may require a temporary or permanent cessation of the therapy. 5 Late effects are represented by subcutaneous fibrosis and atrophy; telan- giectasia may then develop. These late responses may appear after months or years, with a time interval of varying length during which the skin seems ‘normal’. 2 Radiation-induced dermal fibrosis has been characterized histologically, 6 but little is known about the epithelial com- partment overlying fibronecrotic lesions, i.e. the epidermis. Ó 2005 British Association of Dermatologists • British Journal of Dermatology 2005 153, pp881–886 881