*3.3. Antioxidants*

As RONS and redox reactions appear to be pivotal for cellular health and function, mammalian cells require an inherent regulatory control mechanism to sustain redox homeostasis and prevent damage from oxidative stress. The antioxidant network of enzymatic and non-enzymatic antioxidants maintains RONS at physiological levels, thereby maintaining redox control. The principal antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [74]. Important non-enzymatic or dietary antioxidants include ascorbic acid (vitamin C), α-tocopherol (vitamin E), carotenoids, and flavonoids. In addition to the three main groups of enzymatic antioxidants (SODs, GPx, and Catalase), other accessory antioxidant enzymes help maintain redox balance. The Thioredoxin (Trx) antioxidant system comprising of Trx and the enzyme Trx reductase, serves as an electron donor to drive antioxidant systems and regulate proteins (in dithiol-disulfide exchange reactions) in response to a changing redox environment. This antioxidant assists in general metabolism (including DNA synthesis) and the prevention of deleterious levels of oxidative stress [75,76]. Peroxiredoxins (PRDXs) are a group of ubiquitous enzymes (PRDX1-6) that have emerged as important and widespread peroxide and ONOO− scavenging enzymes [77,78]. A growing body of evidence has begun to recognise the role of H2O2 in mediating redox signalling pathways during exercise, and thus it has been suggested that PRDX may be a salient regulator of exercise induced H2O2 levels (see review by Wadley et al. [79]).

#### **4. Circadian Rhythms, Oxidative Stress, and Redox Homeostasis**

Much is known about the cellular and molecular effects of RONS and circadian rhythms, respectively, but relatively less attention has been paid to the cross-talk and integration between the two and if/how these complex interactions affect physiological processes in health and disease. Current investigations have thus begun to focus on the molecular mechanisms linking RONS/oxidative stress and dysregulated circadian rhythms, sustaining the basis of a seemingly vital biological connection [80,81]—please see Figure 3. For instance, evidence now depicts a circadian influence on the immune system and this extends to the regulation of inflammatory and oxidative stress responses (especially in the ageing context—see review by [16]). It is now known that macrophages possess a molecular clock potentially able to impose temporal fluctuations in immune function [82]. RONS are integral to the proper functioning of the immune system and, interestingly, their production may be regulated by BMAL-1. In an elegant study by Early and colleagues [83] conducted on mice, ROS accumulation (detected by fluorescent probes) was increased in *BMAL-1*−*/*<sup>−</sup> macrophages, and related to decreased activity of Nrf2 in cells, resulting in a diminished antioxidant response (including a reduced synthesis of glutathione) and increased production of the proinflammatory cytokine, IL-1β. Conversely, the redox milieu may also modulate core clock regulation as the DNA binding activity of the CLOCK: BMAL1 heterodimer is dependent on the prevailing cellular redox status (as evidenced in the NADH/NAD+ and NADPH/NADP ratios), whereby binding is enhanced under reducing conditions [84]. Moreover, oxidative activation of Nrf2 via H2O2 has been shown to regulate core clock function by altering clock gene expression (*Per3*, *Nr1d1*, *Nr1d2*, *Dbp*, and *Tef*) and circadian function in mice in a comparable manner to overexpression or elimination of Nrf2 conditions [85].

**Figure 3.** The interplay between Circadian and Redox Biology: Oxidative Stress and Disrupted Circadian Rhythms. A proposed bidirectional relationship exists between oxidative stress and circadian dysregulation whereby external factors and behaviours that disrupt circadian rhythms (jet lag, the ageing process, and regular consumption of certain foods) may also induce oxidative stress and, likely vice versa. The result is a deterioration of normal physiological functions (DNA damage, increased blood pressure, and insulin resistance) and control that increases the risk for chronic diseases.
