Aging is an unavoidable fact of life. Aging increases the vulnerability of cells to environmental insults, which leads to progressive, global impairment in tissue function, and increases the risks of disease and death [1]. Antioxidant enzymes, such as, glutathione peroxidase (GPX), catalase (CAT), and manganese superoxide dismutase (Mn-SOD), protect against the harmful effects of reactive oxygen species (ROS) by reducing them to hydrogen peroxide (H₂O₂) and finally to water [2]. Aerodigestive organs are particularly susceptible to ROS damage, because they are continuously exposed to atmospheric oxygen [3]. However, only a few studies have addressed the subject of ROS-induced insults in tongues [4].

In a previous study, we showed the activities of total SOD, GPx, and Cu/Zn-SOD were lower in the tongues of aged rats than in those of young rats, and that their protein expressions were down-regulated in old rats [5]. On the other hand, although the protein expressions of Mn-SOD and CAT were lower in old rats, their enzyme activities were not [5]. Aging-associated impaired mitochondrial biogenesis has been associated with reduced levels and activities of nuclear factor erythroid 2-related factor 2 (Nrf2) [6]. In particular, the electron transport chain (ETC) in the inner membrane of mitochondria generates most ROS as a byproduct of the respiratory chain. Thus, mitochondria are exposed to the greatest amount of oxidative stress [7], which is defined as an imbalance between ROS production and antioxidant response [8].

The aim of this study was to document tongue Nrf2, ROS, and mitochondrial morphologic age-associated damage profiles.

Nrf2 is a redox-sensitive transcription factor that functions in antioxidant defense, and typically is activated by xenobiotics [9], and may be involved in the pathogenesis of cell and tissue damage. In particular, Nrf2 plays a crucial role in protecting cells by inducing the expressions of many cytoprotective genes following exposure to electrophilic or oxidative stress [10]. Keap1 is essential for Nrf2 activity regulation [11]. Normally, Nrf2 interacts with Keap1 in cytoplasm, and this interaction causes Nrf2 retention in cytoplasm and renders it liable to ubiquitination and proteasome degradation. However, Keap1 is inactivated when cells are exposed to ROS or electrophiles. This inactivation disrupts the Keap1/Nrf2 interaction and causes the release of Nrf2, which then translocate to the nucleus. In the nucleus Nrf2 activates the transcriptions of many genes that encode for enzymes and proteins that have detoxifying and antioxidant effects. In particular, the genes targeted by Nrf2 play roles in the synthesis of glutathione, ROS elimination, metabolism of xenobiotics, and protection against xenobiotic and oxidative stresses [12]. Ohkoshi et al. [13] reported Nrf2-knockout mice had a greater susceptibility to 4-nitroquinoline-1-oxide-induced carcinogenesis of the tongue and esophagus than wild-type mice.

In the present study, Western blotting showed that Nrf2 expression was suppressed in the tongue tissues of aged rats, which is in-line with our previous observation that antioxidant enzyme levels were diminished in aged rat tongues [5]. Smith et al. [14] focused on miRNA-146a, the expression of which leads to age-associated reductions in Nrf2 expression. In addition, miRNA-146a has been shown to block infections and treat inflammation, and functionally its effects are not diminished by aging. However, in aged rat’s liver, it has been reported that the translocation of Nrf2 was reduced by 55%. Additional studies are needed to measure miRNA-146a levels in tongues and to determine the influence of the interaction between miRNA-146a and Nrf2 on age-related degenerative changes observed in mitochondria.

Bone growth in rats tapers at 7 months, whereas human growth plates close in the 20’s. Thus, in the present study, we defined a 7-month-old rat to be equivalent to a 20-year-old human. Female rats reach reproductive senescence at 15 to 25 months of age (average, 20 months) while in humans the average menopausal age is 51 years, which indicates a 22-month-old rat is equivalent to a 55–60-year-old human (adult phase: 11.8 rat days=1 human year) [15]. Accordingly, in the present study, we defined 7- and 22-month-old rats as young and old rats, respectively.

Cell differentiation and proliferation can be promoted by a moderate increase in ROS level [16], whereas extreme increases can cause irreversible damage to bio-macromolecules and lead to apoptosis and cell death [17]. Therefore, the preservation of ROS balance is crucial for cellular health and survival. Furthermore, moderate ROS enhancement has been linked to cancer growth and the disruption of redox homeostasis [17]. Harris et al. [18] reported that normal epithelial cells exposed long-term to low amounts of exogenous oxidants became resistant to subsequent oxidative insults, which suggests cells are able to adapt and survive in the presence of oxidative stress, and that surviving cells have acquired mechanisms to counteract ROS toxicity and/or promote cell-survival pathways [16]. Oxidative damage by oxygen-derived free radicals has also been reported to be a causative factor of high mitochondrial DNA mutation rates in aged tissues [19]. Further studies are needed to assess whether aged tongue tissues are prone to certain mitochondrial DNA mutations and to identify the factors that contribute to such mutations. In the present study, ROS were found to be up-regulated in an aged rat tongues, and based on our previous finding that antioxidant enzyme levels were reduced in aged rat tongues [5], we suggest the up-regulation of ROS was possibly caused by diminished antioxidant enzyme levels.

Antioxidant enzymes importantly protect the cells of aerobic organs from oxidative stress. In particular, SOD’s, such as, mitochondrial Mn-SOD and cytosolic Cu/Zn-SOD reduce the highly toxic radical O₂^– to less harmful H₂O₂, which is further reduced by CAT and GPx into water. However, if the levels of these antioxidant enzymes are diminished, ROS cannot be removed and its accumulation challenges nearby mitochondria [20]. In a previous study, our protein level and enzyme activity results indicated reduced antioxidant ability in the tongue tissues of older rats, despite similar enzyme Mn-SOD and CAT activities in old and young animals [5]. In another study, diminished glutathione (GSH) levels and elevated oxidized glutathione (GSSG) levels in the cardiac tissue of aged rats, caused CAT activity to increase as a compensatory mechanism to convert H₂O₂ to water [21]. We surmise this increase in CAT activity was higher in aged rats because metabolically active tissues are susceptible to aging-induced glutathione (GSH) depletion and redox state perturbations.

Mitochondria are known to be involved in the etiologies and pathogeneses of a variety of diseases and in the aging process [22,23]. Mitochondrial morphology may also change during aging in skeletal muscle, that is, some mitochondria become nonfunctional, depolarized, or enlarged [24]. Furthermore, as compared with young animals, which typically have elongated mitochondria, the mitochondria found in the skeletal muscle of aged animals are more rounded [25], which suggests the fusing ability of mitochondria may be compromised [2]. Aged mitochondria also become significantly swollen due to increased calcium retention [26]. In addition, in a previous study, deficient cristae formation was found to lead to the homogenization of constituents in mitochondrial compartments by transmission electron microscopy [27].

Degenerative changes in aged rat tongues have rarely have described in the literature. Barlagiannis et al. [28] reported that by 5 months, rat tongues already began to show evidence of degeneration, that is, moderate disruption of inter-epithelial junctions and lipid droplet formation, which concurs with our observations. Three 7-month-old rats exhibited moderate mitochondrial degeneration, whereas at 12 and 18 months, rat tongues showed profound degeneration, including edema of inter-epithelia, swelling of mitochondrial, lipid droplet formation, abnormal nuclear morphology, and cristae disruption. Takahashi [29] reported epithelium, propria mucosae, and muscle layers at the tongue apex became thin with aging, and that boundaries between these layers became unclear, which suggests some relationship between degenerative changes and motion dysfunction, pain, and paresthesia of the tongue. Choi et al. [30] reported age-related functional and structural changes of salivary glands, that is, acinar cell atrophy, cytoplasmic vacuolization, lymphocyte infiltration, smaller mucin component, and more periductal fibrosis in histomorphometric examinations of 90-week-old mice. Additional studies are needed to determine whether the tongue undergoes similar age-related structural changes, and to characterize changes at the mitochondrial level in major head and neck organs.

This study expands understanding of the mechanism responsible for aging of the tongue. In particular, in old rats, attenuation of the antioxidant defense system, probably because of Nrf2 deficiency, might have increased the release and accumulation of ROS and caused mitochondrial structural deficits in aging tongue tissues.