Abstract
Glucocorticoid (GC) production is physiologically regulated through a negative feedback loop mediated by the GC, which appear disrupted in several pathological conditions. The inability to perform negative feedback of the hypothalamus-pituitary-adrenal (HPA) axis in several diseases is associated with an overproduction of reactive oxygen species (ROS); however, nothing is known about the effects of ROS on the functionality of the HPA axis during homeostasis. This study analyzed the putative impact of antioxidants on the HPA axis activity and GC-mediated negative feedback upon the HPA cascade. Male Wistar rats were orally treated with N-acetylcysteine (NAC) or vitamin E for 18 consecutive days. NAC-treated rats were then subjected to a daily treatment with dexamethasone, which covered the last 5 days of the antioxidant therapy. We found that NAC and vitamin E induced an increase in plasma corticosterone levels. NAC intensified MC2R and StAR expressions in the adrenal and reduced GR and MR expressions in the pituitary. NAC also prevented the dexamethasone-induced reduction in plasma corticosterone levels. Furthermore, NAC decreased HO-1 and Nrf2 expression in the pituitary. These findings show that antioxidants induce hyperactivity of the HPA axis via upregulation of MC2R expression in the adrenal and downregulation of GR and MR in the pituitary.4. Discussion
This study investigated the role of antioxidants on the modulation of endogenous glucocorticoid levels. We found that treatment with antioxidants either NAC or vitamin E increases the plasma levels of corticosterone in rats, in association with an overexpression of ACTH receptor and the steroidogenic enzyme StAR in the adrenal glands. NAC also induces a drop in HO-1 and Nrf2 expression in the pituitary and blocked the ability of dexamethasone to perform negative feedback of the HPA axis by decreasing the expression of glucocorticoid receptors in the pituitary. Our findings suggest that antioxidants cause a hyperactivation of the HPA axis with a clear dependency of upregulation of ACTH receptor in adrenals and downregulation of glucocorticoid receptors in the pituitary.In this study, we showed that both NAC and vitamin E increase circulating levels of corticosterone in rats. NAC and vitamin E are antioxidants that act through distinct mechanisms of action. While NAC provides cysteine, which is a precursor for reduced glutathione production, and scavenges oxidants directly, including hydroxyl radical, −OH, and hypochlorous acid [8]; vitamin E is a peroxyl radical scavenger and due to its lipid solubility plays an important role in maintaining integrity of long-chain polyunsaturated fatty acids in the membranes of cells [6, 7]. The fact that prolonged treatment with two antioxidants with different mechanisms of action can increase circulating levels of corticosterone indicates that this is not an epiphenomenon, but suggests that inhibition of physiological levels of ROS in the HPA axis is responsible for its hyperactivity.
The HPA axis is the main neuroendocrine system that regulates responses to stress [8]. It is well known that the production of high levels of ROS into the glands that comprise the HPA axis is associated with the activation of a stress-response system [13, 14]. Therewith, antioxidant treatment reduces corticosterone levels in several models of diseases, including brain oxidative stress induced by lipopolysaccharide [26]. However, although accumulation of oxidative damage in cellular macromolecules is immensely toxic, ROS products by normal cell metabolism are vital to cell homeostasis maintenance [15, 16]. Our hypothesis is that physiological levels of ROS have a fundamental role in maintaining the homeostasis of the HPA axis. In fact, treatment with NAC in normal rats effectively reduced ROS levels in chondrocytes and Lin−CD45+AnV− marrow cells [27], suggesting that in our model, the antioxidant therapy probably reduces intracellular ROS content in the adrenal and pituitary glands. Thereby, we strongly suggest that any imbalance in the redox system in glands which comprise the HPA axis culminates in its hyperactivation.
In an attempt to elucidate how antioxidants induce the production of glucocorticoids by the HPA axis, we analyzed the expression of adrenal MC2R. Prolonged treatment with NAC increases adrenal MC2R expression. This higher adrenal MC2R expression after treatment can explain, at least partially, the capacity of NAC to increase circulating glucocorticoid levels. Increased expression of MC2R may lead to high activation of this receptor by ACTH and induction of the transcription of several key genes of enzymes involved in steroidogenesis, including StAR [28]. In fact, we showed that NAC induces an upregulation in the expression of StAR into the adrenal glands. StAR rapidly transports cholesterol to the inner mitochondrial membrane, where the conversion of this steroid precursor into pregnenolone, a precursor of steroid hormones, occurs [29]. This metabolic step is crucial to rapid glucocorticoid production into the adrenals in a stress stimulus, once steroidogenic cells store very little amount of glucocorticoids [10].
Although the increased expression of MC2R and StAR alone may explain the increase in glucocorticoids levels, other molecular alterations can also participate in the HPA axis hyperactivity noted after antioxidant treatment. Once the HPA axis is finely regulated by a negative feedback response on the hypothalamus and/or pituitary that normalizes circulating corticosterone levels, we hypothesized that antioxidants could induce a defect in the negative feedback regulation in the HPA axis. We observed that treatment with NAC downregulated the expression of Nrf2 in the anterior pituitary gland of rats. Nrf2 is a transcription factor that regulates expression of several antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase, and HO-1 [30]. Although NAC can induce upregulation of Nrf2 expression in phosgene-induced acute lung injury [31], our data is in accordance with others which described that NAC inhibited Nrf2 expression in lymphoid malignant cell lines stimulated with honokiol [32], suggesting that the effect of antioxidant NAC on Nrf2 expression depends on the cell type and condition of the study. Furthermore, we noted that NAC also decreased the expression of HO-1 in the anterior pituitary of rats. The drop in HO-1 levels after treatment with NAC indicates that the low content of Nrf2 is associated with a reduced ability of this transcription factor to induce production of antioxidant enzymes by pituitary cells. We suppose that the downregulation of Nrf2 expression is a strategy of the organism to maintain homeostasis in rats treated for several days with NAC. In fact, it has been shown that exogenous antioxidants can reduce the expression and/or activity of endogenous antioxidant enzymes [33, 34]. These data indicate that the pituitary as well as adrenals is also a direct target of antioxidant drug effects.
Our next approach was to investigate the sensitivity of the HPA axis to negative feedback induced by synthetic glucocorticoid in NAC-treated rats. Dexamethasone decreased plasma corticosterone levels in control rats; however, it did not alter circulating glucocorticoid amount in rats treated with NAC, showing that antioxidants abolish the ability of glucocorticoids to perform negative feedback of the HPA axis. NAC treatment also decreased expression of both GR and MR in the pituitary of rats, indicating that a reduction in glucocorticoid receptor expression in the pituitary of rats can explain the inability of dexamethasone to induce negative feedback of the HPA axis in NAC-treated animals. Our data confirmed the capacity of NAC in inhibiting GR expression, once NAC decreases GR protein levels in the hypothalamus of mice fed with a high-cholesterol diet [35].
Currently, many people consume dietary supplementation with antioxidants to combat diseases associated with aging [36]; however, several clinical trials testing benefits and harms of antioxidant supplements found that antioxidants have been unable to demonstrate beneficial effects and pointed that they seemed to cause an increase in all-cause mortality [37–40]. Once antioxidants induce a HPA axis dysfunction with concomitant increased levels of circulating glucocorticoids, this food supplement is shown as a risk to human health. This occurs because the hyperactivation of the HPA axis, and consequently the glucocorticoid signaling system, may alter the epigenetic landscape and influence genomic regulation and function conducting to the development of aging-related diseases [41]. Some harmful effects of hypercorticoidism, which can culminate with aging-related diseases, are deleterious effects on the central nervous system (CNS), including neuroinflammatory environment, loss of neuronal function, and apoptosis of neuronal cells, causing a decrease in hippocampal neurogenesis and an increase in neuroinflammation and neurodegeneration [42–44]. These deleterious effects of hypercorticoidism on the CNS can lead to the development of a variety of progressive neurodegenerative and psychiatric diseases, including schizophrenia, dementia, depression, Huntington's disease, and Alzheimer's disease [45–49]. Although we showed that antioxidants can induce high production of glucocorticoids, it is well known that chronic stress promotes redox imbalance throughout the body [50], as in blood of humans [51–53], such as in several structures of the CNS of rats including the frontal cortex, hypothalamus, and hippocampus [54]. These data suggest that chronic stress accelerates cellular aging through inducing increased levels of ROS [50]. These observations are conflicting with ours, once we show that antioxidants decreased the expression of Nrf2 and HO-1. However, the induction of ROS production is not the only mechanism related to stress-induced cellular aging. Chronic stress reduces brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex and increases neuroinflammation, an alteration noted in the formation of depression [44]. Furthermore, chronic stress induces prolonged periods of glutamate release in the hippocampus and decreases the ability to clear extracellular glutamate. These alterations in the glutamate transmission may be related to the impairments in the spatial and contextual memory performance and stress-associated psychiatric disorders, including mood and anxiety [55].
In addition, hypercorticoidism can also increase susceptibility to cancer [56], one of the most important aging-related diseases. Although ROS can cause oncogenic mutations and activate oncogenic pathways [57], dietary supplementation with antioxidants promotes increased incidence and death from lung and prostate cancer [58]. Furthermore, antioxidants induce melanoma progression by promoting metastasis [59]. One possibility is that the high incidence of cancer in people which use dietary supplementation with antioxidants can be related to hyperactivation of the HPA axis.
In summary, our results indicate that antioxidant therapy can induce an activation of the HPA axis, with an increase in the levels of systemic glucocorticoids by upregulating ACTH receptor in the adrenal and downregulating glucocorticoid receptors in the pituitary. Thereby, indiscriminate use of antioxidant supplements can be a risk to develop several morbidities related to persistent hypercorticoidism, as observed in Cushing's disease.