Long-term melatonin administration protects brain mitochondria from aging Introduction Aging is a multifactorial process that includes progressive cellular loss, endocrine and metabolic deficits, decreasing defense mechanisms and functional losses that increase the risk of death. Currently, the most tested theory that tries to explain aging is the free radical theory proposed by Harman [1]. A majority of the free radicals are produced in the mitochondria [2] because the aerobic respiration, so the hypothesis was refined and recently updated suggesting that mitochondria are the major target of free radical attack that leads to aging [3]. Free radical generation may increase as a consequence of normal and pathobiological aging. Additionally, anti- oxidative defenses may diminish in effectiveness during aging [4–6]. The result is an increase in free radicals in the intracellular environment that may accelerate cell damage; radicals attack DNA, proteins and lipids and oxidative damage accumulates over time [7, 8]. This destruction causes DNA alterations, the accumulation of protein oxidation and lipid peroxidation products [9–12], that lead to the pathophysiological changes associated with aging. The senescence-accelerated mouse (SAM) was developed by Takeda et al. [13] as a murine model of accelerated aging. SAM includes two strains, one prone to accelerated senescence (SAMP) and one resistant to accelerated senes- cence (SAMR). SAMP8, a substrain of SAMP, shows among other features, decreased learning ability, memory loss, loss of activity and early death as characteristics of accelerated aging. In the brain, SAMP8 mice have an increase in lipid peroxidation and superoxide dismutase (SOD) activity with age [14, 15]. One possible mechanism promoting accelerated aging in SAMP8 is the higher hyperoxidative status found in this strain compared with SAMR1 [16], which is used as a control for SAMP8. Brain tissue is very vulnerable to free radical damage because of its high oxygen utilization (20% of the total oxygen inspired), high concentrations of polyunsaturated fatty acids [17] and transition metals such as iron, which is involved in the generation of the hydroxyl radical [18], and low concentrations of cytosolic antioxidants [4, 5, 19]. Oxidative modifications of mtDNA may be of central importance in normal and pathobiological aging [20]. mtDNA encodes for 13 hydrophobic polypeptide chains of four enzyme complexes of the inner mitochondrial electron transport chain involved in oxidative phosphory- lation [21]. A persistent attack by toxic oxygen and nitrogen-based species leads to an accumulation of mutations in mtDNA, which is relatively unprotected in Abstract: We tested whether chronic melatonin administration in the drinking water would reduce the brain mitochondrial impairment that accompanies aging. Brain mitochondria from male and female senescent prone (SAMP8) mice at 5 and 10 months of age were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation and nitrite, glutathione/glutathione disulfide ratio, and glutathione peroxidase and glutathione reductase activities. Electron transport chain activity and oxidative phosphorylation capability of mitochondria were also determined by measuring the activity of the respiratory chain complexes and the ATP content. The results support a significant age-dependent mitochondrial dysfunction with a diminished efficiency of the electron transport chain and reduced ATP production, accompanied by an increased oxidative/nitrosative stress. Melatonin administration between 1 and 10 months of age completely prevented the mitochondrial impairment, maintaining or even increasing ATP production. There were no major age- dependent differences between males in females, although female mice seemed to be somewhat more sensitive to melatonin treatment than males. Thus, melatonin administration as a single therapy maintained fully functioning brain mitochondria during aging, a finding with important consequences in the pathophysiology of brain aging. Miguel Carretero 1 , Germaine Escames 1,2 , Luis C. Lo ´ pez 1,2 , Carmen Venegas 1,2 , Jose ´ C. Dayoub 1,2 , L. Garcı ´a 1,2 and Darı ´o Acun ˜ a-Castroviejo 1,2,3 1 Centro de Investigacio ´n Biome ´ dica, Parque Tecnolo ´gico de Ciencias de la Salud, Universidad de Granada and RETICEF, Granada, Spain; 2 Departamento de Fisiologı ´a, Facultad de Medicina, Universidad de Granada, Granada, Spain; 3 Laboratorio de Ana ´ lisis Clı ´nicos, Hospital Universitario San Cecilio, Granada, Spain Key words: aging, brain, mitochondria, oxidative phosphorylation, oxidative stress, respiratory chain Address reprint requests to Darı ´o Acun ˜a- Castroviejo, MD, PhD, Centro de Investigacio ´n Biome ´ dica, Parque Tecnolo ´ gico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain. E-mail: dacuna@ugr.es Received May 15, 2009; accepted June 4, 2009. J. Pineal Res. 2009 Doi:10.1111/j.1600-079X.2009.00700.x Ó 2009 The Authors Journal compilation Ó 2009 John Wiley & Sons A/S Journal of Pineal Research 1 Molecular, Biological, Physiological and Clinical Aspects of Melatonin