All-trans-retinoic acid induces apoptosis in Leydig cells via activation of the mitochondrial death pathway and antioxidant enzyme regulation Paola Tucci & Erika Cione & Mariarita Perri & Giuseppe Genchi Received: 15 May 2008 / Accepted: 3 June 2008 / Published online: 31 July 2008 # Springer Science + Business Media, LLC 2008 Abstract In addition to playing a fundamental role in diverse processes, such as vision, growth and differentia- tion, vitamin A and its main biologically active derivative, retinoic acid (RA), are clearly involved in the regulation of testicular functions. The present study was undertaken to examine the direct effect of RA treatment on Leydig (TM-3) cells. TM-3 cells were cultured and treated with varying concentrations of RA for 24h. High doses of RA (1–20μM) induced a decrease in cell vitality and an increase in lipid peroxidation. RA treatment also induced a corresponding increase in apoptosis in the same cells in a dose-dependent manner. Apoptosis proceeded via the mitochondrial depen- dent pathway, as demonstrated by the release of cytochrome c, caspase-3 enzymatic activation and DNA fragmentation. Conversely, at physiological doses (0.1–500nM) RA did not increase lipid peroxidation or cell death and resulted in an increase of antioxidant enzyme activity. Keywords Retinoic acid . Apoptosis . Leydig cells . Mitochondria . Antioxidant enzymes Abbreviations RA All-trans-retinoic acid CAT Catalase GST Glutathione S-transferase SOD Superoxide dismutase MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide MPMS 1-methoxyphenazine methosulfate HS Horse serum FCS Foetal calf serum PBS Phosphate buffered saline TBARS Thiobarbituric acid reactive species TBA Thiobarbituric acid TCA Trichloroacetic acid Introduction Retinoids are natural and synthetic substances structurally related to vitamin A (retinol). They exert antiproliferative and differentiation-inducing effects on cancer cells and are used in the prevention and treatment of certain types of human cancer and precancerous lesions (Gudas et al. 1994; Lotan 1996). Their action is mediated by two types of receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (Chambon 1996) belonging to the steroid/thyroid hormone receptors superfamily. There is substantial evidence in vitro that retinoids exert their effect through the induction of apoptosis in tumor cells including hepatoma, leukemia, breast cancer and embryonal carcino- ma cell lines (Nagy et al. 1995; Nakamura et al. 1995; Horn et al. 1996; Kim et al. 1996; Li et al. 1999). The action of retinoids in promoting apoptosis may explain the anti- carcinogenic properties of these compounds. Apoptosis, a specific form of programmed cell death, is a biological process that plays a crucial role in normal development and tissue homeostasis (Woodle and Kulkarni 1998). Apoptosis is characterized by morphological changes including progressive cell shrinkage with conden- sation, fragmentation of nuclear chromatin and membrane blebbing (Kerr et al. 1997). The ability of the cell to undergo or resist apoptosis is modulated by several factors J Bioenerg Biomembr (2008) 40:315–323 DOI 10.1007/s10863-008-9156-8 P. Tucci : E. Cione : M. Perri : G. Genchi (*) Dipartimento Farmaco-Biologico, Laboratorio di Biochimica, Edificio Polifunzionale, Università della Calabria, 87036 Rende, Cosenza, Italy e-mail: genchi@unical.it