Morphofunctional Evidence of Changes in Principal and Mitochondria-Rich Cells in the Epidermis of the Frog Rana kl. esculenta Living in a Polluted Habitat C. Fenoglio, 1 A. Grosso, 1 E. Boncompagni, 1 G. Milanesi, 1 C. Gandini, 2 S. Barni 1 1 Lab. Anatomia Comparata, Dipartimento di Biologia Animale, Università di Pavia, Piazza Botta 10, 27100 Pavia, Italy 2 Dipartimento di Chimica Farmaceutica, Università di Pavia, Viale Taramelli 10, 27100 Pavia, Italy Received: 13 November 2005 /Accepted: 31 March 2006 Abstract. The epidermis of vertebrates is the bodyÕs principal barrier against environment and its possible contaminants. The presence of keratins, as well as specific detoxifying molecules or enzyme activities, in the various epidermis layers is be- lieved to be involved in providing protection from harmful environmental influences. Anuran integument is poorly hor- nified and thus permeable to some endogenous and exogenous compounds and thus serves as a good bioindicator of overall environmental conditions. In the present investigation, we studied the epidermis of Rana kl. esculenta adult specimens collected at two different rice fields, relatively unpolluted and heavily polluted, respectively. Environmental pollution was assayed by chemical analysis performed on both sediments and animals. We evaluated the structural aspects of the epidermis at both light and electron microscopy levels and the pattern of keratinization by immunohistochemistry. Furthermore, we studied the activities of some enzymes (acid and alkaline phosphatase, nitric oxide synthase–related nicotinamide ade- nine dinucleotide phosphate dehydrogenase, glucose-6-phos- phate dehydrogenase, catalase, nonspecific esterases, and succinic dehydrogenase) involved mainly in membrane trans- port, xenobiotics, and oxidative metabolism. Compared with controls, in polluted animals we found the following results: (1) an increase in pollutant levels (i.e., cadmium, mercury, and lead); (2) less keratinized superficial cells in the epidermis; and (3) changes in most enzyme activities in keratinocytes and mitochondria-rich cells (particularly glucose-6-phosphate dehydrogenase and esterases, both important to counteract oxidative and toxic stress). Taken as a whole, the present data indicate the morphofunctional plasticity of the frog epidermis in response to environmental contamination. Anuran skin, which undergoes drastic structural changes dur- ing metamorphosis, exhibits a complex cellular composition and has several functions (i.e., mechanical protection, ion and water transport, respiration, sensory perception). The general structure of the epidermis, including its cell types and keratin contents, differs in tadpoles and adults. Larval epidermis is composed of three cell types: apical cells, skein cells (exclusive in tadpoles), and basal cells, which have been described in premetamorphosing larvae and are considered precursors of adult germinative cells (Fox 1986; Robinson and Heintzelman 1987; Izutsu et al. 1993). Skein cells are characterized by the presence of bundles of tonofil- aments—the figures of Eberth—which morphologically ex- press larval keratins (Ellison et al. 1985; Fox 1992; Spies 1997; Suzuki et al. 2001). The stratified epidermis of adult anurans consists of several layers: germinative (or basal), spinous (or spinosum), granu- lous (or granulosum), and hornified (or corneum), all con- sisting of principal cells or keratinocytes showing a peculiar morphologic aspect depending on the layers (Fox 1986; Lindeman and Voute 1976). Flask-shaped mitochondria-rich cells (MRCs) are characteristic of anuran skin. They are intermingled in between keratinocytes and extend their necks into the interstice beneath the horny layer (Amstrup et al. 2001). They are variable in number in different species and in Rana esculenta they are more numerous in ventral than in dorsal skin (Ehrenfeld et al. 1976). Within the epidermis, leukocytes, melanocytes, macro- phages, and Merkel cells are also present. Compared with amniotes, anuran epidermis shows some structural differences; in particular, it exhibits a relatively thin hornified surface composed of one or two layers of flattened cells that retain their nuclei (see Alibardi 2003 for a review). Based on these struc- tural traits, frogs can use the epidermis for respiratory gas (mainly carbon dioxide) and ion exchange. In fact, being ex- posed to both air and pond water, the skin of the frog represents one of the principal organs for body-fluid homeostasis. Several studies have suggested that in frogs, epidermal principal cells are mainly involved in active transport, whereas MRCs are specialized in proton and bicarbonate secretion (Lindeman and Voute l976; Fox 1986; Ehrenfeld and Klein 1997). Hence, numerous investigations on frog skin have focused mainly on morphologic and ion-transport changes under both natural and experimental conditions (i.e., seasonality or different ion concentration) (Barni et al. 1987; De Piceis Polver et al. 1988; Vanatta and Frazier 1989; Malvin and Correspondence to: C. Fenoglio; email: fenoglio@unipv.it Arch. Environ. Contam. Toxicol. 51, 690–702 (2006) DOI: 10.1007/s00244-005-0245-y