© 2007 The Authors. Journal compilation © 2007 ESVD and ACVD. 18; 287–293 287 Blackwell Publishing Ltd In vivo long-term effects of retinoic acid exposure in utero on induced hyperplastic epidermal foci in murine skin Rosa A. García-Fernández*, Claudia Pérez-Martínez†, Javier Espinosa-Álvarez† and Maria J. García-Iglesias† *Histology and Pathological Anatomy Section, Department of Animal Medicine and Surgery, Faculty of Veterinary Science, University of Madrid (UCM), Madrid, Spain †Histology and Pathological Anatomy Section, Department of Animal Health, Faculty of Veterinary Science, University of León, León, Spain Correspondence: Maria J. García-Iglesias, Histology and Pathological Anatomy Section, Department of Animal Health, Faculty of Veterinary Science, University of León, León, Spain. E-mail: mjgari@unileon.es What is known about the topic of this paper • Retinoic acid (RA) plays an important role in embryogenesis and is necessary to maintain the differentiation pathway of the epithelial tissues (skin, and other epithelia). • RA has been shown to inhibit the induction of cancer in various organs including the skin. • Retinoids are used to treat skin diseases such as psoriasis, actinic keratosis, squamous and basal cell carcinomas. What this paper adds to the field of veterinary dermatology • RA administered to pregnant mice shows an in vivo long- term action on development of hyperplastic lesions in adult skin of their offspring. • This retinoid inhibits the cell differentiation and stimulates the cell proliferation in those hyperplastic lesions. • The chemopreventive effect of RA is questioned using our experimental mouse model. Abstract Adult Naval Medical Research Institute (NMRI) mice, after prenatal exposure to retinoic acid (RA), were treated with a standard two-stage skin carcinogenesis regime to characterize hyperplastic epidermal foci that precede the appearance of cutaneous papillomas, and to investigate the in vivo long-term action of RA on adult mouse skin treated with DMBA (7,12 dimethyl benz[a]anthracene) and TPA (12-O-tetradecanoylphorbol 13-acetate). The results demonstrate that RA admin- istered to pregnant mice had a long-term inhibitory action on the cell differentiation and development of hyperplastic lesions occurring prior to cancer on the adult skin of their offspring as well as a stimulatory effect on cell proliferation of these hyperplastic lesions. Accepted 8 June 2007 Introduction Retinoic acid (RA), the most biologically active natural metabolite of circulating vitamin A, 1 plays an important role in embryogenesis 1 and is fundamental to maintenance of the differentiation pathway of epithelial tissues. 2 Previous studies have demonstrated that prenatal RA exposure induces earlier epidermal differentiation 3 and a stimulatory effect on cell proliferation of the developing epidermis 3 and hair follicles. 4 RA and its analogues (retinoids) are used in the therapy of dermatological 5,6 and neoplastic dis- eases, 7 although their clinical usefulness is limited by their proven teratogenic potential. 8–10 Retinoids have been shown to be effective in the prevention of skin cancer 7 in different animal models, following either topical application 11 or oral administration. 12,13 However, there appears to be nothing known about the in vivo long-term effects on skin carcino- genesis after prenatal RA exposure in mice. The effects of retinoids on skin carcinogenesis have been studied using a two-stage chemical model involving the application of 7,12 dimethyl benz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol 13-acetate (TPA) as a promoter. 7 Polymerase chain reaction analysis specific for the Ha-ras gene has provided molecular evidence that squamous cell hyperplastic foci are precursors of cutaneous papillomas. 14 In this way, it is possible to use squamous cell hyperplastic foci as the earliest indicator of potential mouse skin tumorigenicity. 14 Morphological modifications of the epidermis in both multistage mouse skin carcinogenesis 15,16 and topical RA treatment 17 have been associated with changes in keratin expression. As keratins are considered to be ‘permanent’ markers for keratinocytes 18 as well as useful tools for investigation of skin morphogenesis and differentiation, 19 the aim of this study was to investigate the in vivo long-term effects of prenatal RA exposure on hyperplastic epidermal foci (HEFs) induced by DMBA/TPA in NMRI adult mouse skin by quantification of the number of HEFs and analysis of their keratin expression. Materials and methods Animals Two-week-old albino NMRI mice (n = 56) were maintained in a cycle of 12 h light and 12 h dark, at a controlled temperature (21 ± 1 °C) and relative humidity (55 ± 10%) with food and water ad libitum. All experiments were performed following the guidelines of the European Law on Laboratory Animal Protection. 20 Twenty-eight of these mice had previous exposure to RA in utero and were obtained from nine pregnant females treated by oral admin- istration with 30 mg kg –1 body weight of all-trans-retinoic acid (RA) (Sigma Chemical Company, St. Louis, MO, USA) in corn oil on day