**1. Introduction**

A diverse array of physiological functions, human behaviours, and social connections are determined by the complex interactions between the environment (light/dark cycles, temperature variations, and seasonal food opportunities) and endogenous biological, or circadian drivers. Such an intricate controlling mechanism is susceptible to disruption and, in current modern society, misalignment between the circadian system and environmental cues is a frequent occurrence associated with negative health consequences [1]. Modern chronic diseases are linked to changes in human lifestyle compared to our hunter-gatherer ancestors [2] involving, but not exclusively limited to, low levels of physical activity (PA) and exercise; prolonged sitting; regular access to highly palatable and energy-dense foods; inadequate/disrupted sleep quality/duration; shift work; and social jetlag [3]. These changes in contemporary work and domestic habits have outpaced genome adaption and

**Citation:** McClean, C.; Davison, G.W. Circadian Clocks, Redox Homeostasis, and Exercise: Time to Connect the Dots? *Antioxidants* **2022**, *11*, 256. https://doi.org/10.3390/ antiox11020256

Academic Editor: Stanley Omaye

Received: 6 December 2021 Accepted: 18 January 2022 Published: 28 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the underlying circadian rhythms are thus exposed to dysregulation (or shifts) that can predispose to chronic disease [4].

Oxidative stress has long been implicated in the pathogenesis of several chronic lifestyle-related diseases (e.g., type 2 diabetes mellitus) [5] and the interplay between circadian clocks and oxidative stress is evident whereby: (i) disruption to circadian rhythms can alter redox homeostasis leading to oxidative stress and (ii) elevated production of reactive oxygen and nitrogen species (RONS) may induce circadian oscillations [6]. While still in its relative infancy, research on the effects of exercise on circadian rhythmicity seems to be encouraging; certainly not discouraging [7–9]. At the same time, certain exercise stimuli may evoke oxidative stress, but to the contrary, many exercise-mediated adaptations seem to occur via contractile-induced RONS signalling, acting in a manner that can be explained by the concept of hormesis and other multi-dimensional models [10]. Hormesis, in this context, is when a potentially harmful agent (e.g., exercise) provokes an adaptation to a damaging agent (such as RONS-disrupted signalling) to up-regulate, for example, enzymatic antioxidant capacity [11]. Hence, exercise appears to be a potent activator of oxidative stress, the circadian clock, and, at the correct dose (intensity and volume), elicits desirable health outcomes. As a clear bi-directional link has already been established between the circadian clock and redox homeostasis/metabolism, and that exercise appears to be a potent stimulus of both, we propose that further exploration of exercise, circadian rhythms, and redox biology combined may reveal further important insights in this intriguing field of investigation. In this perspective, the exercise paradigm may help unravel some emerging concepts such as the potential existence of an optimal time to exercise for health benefits. The positive effects of exercise on health may be partially mediated via changes in tissue molecular clocks and/or the outcomes may be modified depending on the timing (and intensity) when exercise is performed [12], but the role, whether directly or indirectly, that RONS and oxidative stress/redox signalling play in such responses remains largely unexplored. Therefore, given the emerging nexus of circadian and redox biology for health and disease prevention, this narrative review will examine the multifactorial interrelationship between circadian clocks and oxidative stress relevant to the important biological stressor, exercise.

### **2. Circadian Rhythms and Molecular Clock Control**

Apparent in virtually all forms of life, circadian rhythms are endogenous 24-h oscillations in behaviour and biological processes enabling organisms to physiologically adjust to the transitions between light and dark (day and night). The circadian clock drives fluctuations in a diverse set of biological processes including sleep, locomotor activity, blood pressure, body temperature, and blood hormone levels [13]. Driven by cellular clocks distributed across the body, these rhythms control mammalian adjustment by preparing the brain and other tissues to perform biologically appropriate functions relative to the anticipated day or night pattern [14,15]. The principal pacemaker of the circadian clock in humans, the so-called central clock, is located within the suprachiasmatic nucleus (SCN) region of the hypothalamus, functioning as an autonomous timekeeper that coordinates the activity of downstream peripheral tissue clocks [15]. Neurons of the SCN receive input from the retina, via the retino-hypothalamic tract, to ensure its molecular clocks are synchronised to light-dark cycles, known as photic entrainment [16]. The SCN can be stimulated by cues other than light, especially by serotonin and melatonin, although these are thought to be internal feedback regulators as opposed to primary circadian rhythm initiators [6]. Using neuro-endocrine actions involving melatonin, insulin, and glucocorticoids (via the HPA axis), as well as the autonomic nervous system [6,17], the SCN relays its coordinating prompts to peripheral clocks [12]. In turn, these peripheral clocks oversee localised, temporal gene and protein expression patterns required for several physiological processes and functions across the ~24-h period [15,17]. Beyond the autonomous cues from the SCN, mammals can also synchronise their inherent timing systems in response to fluctuations in other environmental cues (termed *zeitgebers*) such as changes in temperature and food

availability [18], but light remains the strongest zeitgeber [6]—illustrated in Figure 1. While this innate flexibility to varying environmental stimuli is unquestionably important for adaptation and survival, it may also expose the host to negative consequences, such as the onset of sleeping and metabolic disorders, when the rhythms become misaligned or dysregulated [19].

**Figure 1.** Circadian Rhythms in Humans. Crucial biological and physiological processes such as blood pressure control, antioxidant expression, body temperature, and immune function normally fluctuate in a circadian pattern (~24 h). These oscillations are coordinated by circadian clocks distributed in virtually every cell. The core molecular clock functions to direct a daily program of gene transcription and protein expression. The molecular clock involves a transcriptional–translational feedback loop (TTFL) of core clock genes (positive limb: *BMAL-1* and *CLOCK*; negative limb: *PERs* and *CRYs*) that act to modulate the gene expression of clock-controlled genes (CCG) which generate tissue-specific circadian rhythms in transcription and cellular function across the day, even in the absence of external cues. The principal pacemaker of the circadian clock in humans is located within the suprachiasmatic nucleus (SCN) region of the hypothalamus. This master clock coordinates the activity of downstream peripheral tissue clocks in response to several stimuli such as the day-night cycle, food ingestion, exercise, and sleep. Figure key = SCN: suprachiasmatic nucleus; HPA: hypothalamic-pituitary-adrenal axis; CCG: clock-controlled genes; E: E-box motif.

The core molecular clock is a self-sustaining, transcriptional–translational feedback loop (TTFL) that exists in virtually every human cell and functions to direct a daily program of gene transcription and protein formation, permitting rhythmic adjustments in response to specific entrainment signals [12,13]. This molecular clock is comprised of a positive transcriptional limb and a negative feedback limb [16]; interrogation of this regulatory mechanism was recognized in 2017 when Jeffery Hall, Michael Rosbash, and Michael Young were awarded the Nobel Prize in Physiology or Medicine [13]. Their work, initially in the *Drosophila* model, identified some of the most important clock genes involved in the negative limb of the TTFL such as *Period* (*PER 1-3*) and *Timeless* (*Tim*; or *Cryptochrome (CRY*) as it is known in mammals). These were later followed by the identification of the positive limb transcription factors, CLOCK and BMAL-1 [20]. The heterodimerization of BMAL-1 and CLOCK, or its homolog NPAS2, initiates the circadian cycle. Once dimerized, BMAL-1 and CLOCK bind to E-box motifs in the promoters of target genes, initiating the intracellular transcriptional processes of various rhythmic proteins [6,16]. Two groups of these transcriptional targets are the PERs and the CRYs. The PER and CRY proteins, through

the formation of a second heterodimer, translocate back into the nucleus, and interfere with the activity of BMAL1 and CLOCK at promoter sites, thus completing the cycle by inhibiting CLOCK/BMAL-1 transcriptional activity [6]. This core clock is refined to a 24-h period by the combined actions of several post-translational mechanisms (e.g., covalent histone modifications) carried out by a network of secondary clock proteins involving the kinase family [16,21]. An influential secondary loop driven by BMAL-1 activity involving the nuclear hormone receptors REVERB and ROR has also been identified. These proteins can inhibit and activate the transcription of the BMAL-1 gene (ARNTL), respectively, to ensure its rhythmic expression, and thus they can exert influence on circadian regulation [22]. In this loop, the expression of REVERB proteins serves to repress transcription in the promoter and enhancer regions of target genes, including ARNTL, whereas ROR competes to enhance ARNTL expression. A further CLOCK/BMAL1-driven sub-loop contains the PAR-bZip factors DBP, TEF, and HLF [23–34]. These compete with the repressor NFIL3 (or E4BP4), driven by the REV-ERB/ROR loop, to drive the expression of clock genes from D-box-containing promoters [22].

In addition to its timekeeping function via the TTFLs discussed above, the core molecular clock directly modulates the expression of over 4000 genes in 24-h cycles of transcription with diverse expression phases, resting with the combination of cis-elements (E-box, RORE, D-box) as the promoters and enhancers of specific clock-controlled genes (CCGs) [12,23,24]. These CCGs are expressed in virtually every cell and can generate tissue-specific circadian rhythms in transcription and cellular function, even in the absence of external cues. The circadian clock regulates between 10 and 50% of all transcripts in a cell, depending on tissue type, and influences critical processes such as cell cycle, redox homeostasis, inflammation, and metabolism [13]. The fact that nearly half of the mammalian protein-coding genome expresses tissue-specific circadian rhythmicity [13] underlines the importance of maintaining these oscillations and the seemingly critical role played by circadian rhythms in optimising cell and tissue function for normal health. In fact, cardiovascular function, energy metabolism, fluid balance, inflammation/immune function, cognition/neurological responses, and some of their biological drivers (CCGs and endocrine hormones) all appear to exhibit, either directly or indirectly, some degree of rhythmicity [25,26]. Beyond the central or core factors that directly control the core molecular clock, external factors can regulate the stability, phase, or function of core molecular clock proteins. Hypoxia (and fluctuating oxygen levels) acts on circadian rhythms (body temperature, metabolic rate, cortisol, and melatonin release in humans) through a number of mechanisms involving HIF-1α, with mounting evidence showing cross-talk between the HIF pathway and the circadian clock [27].

Disruption of the circadian system has direct consequences for human health by the uncoupling of multiple physiological processes and the disturbance of normal homeostasis, thus increasing the risk of several disorders and diseases [28]. In addition to genetic disruption to the clock circuit (via specific mutations), humans encounter several potent zeitgebers, often in simultaneous exposures, which hinder circadian regulation such as shift work, excessive artificial/night-time light exposure, disrupted eating patterns (often as a result of shift work), and international travel across several time zones (inducing jet lag).

The current understanding of the effects of circadian disruption has relied mainly on observational studies exploring occupational practices. Jet lagged cabin crew demonstrated temporary cognitive defects and structural brain changes following trans-meridian flights (across at least seven times zones) with five-day recovery intervals when compared to colleagues with fourteen-day recovery intervals [29]. Shift work and social jetlag also disrupt the circadian system and are associated with an increased risk of multiple diseases such as neurological disorders, diabetes, cancer, and cardiovascular disorders [30–32]. In addition to occupational requirements, recreational activities may also affect normal circadian rhythms. For example, the common use of light-emitting (via so-called 'blue light') electronic devices for reading, communication, and entertainment inhibits melatonin production which poses an increased risk for several circadian-related disorders when such

devices are used in the evening [33]. As a consequence of modern living per se, many people are no longer exposed to the natural light/dark cycle of our ancestors. Humans are now inclined to be more active later into the night-time hours, often accompanied by eating and alcohol consumption, leading to a delayed bedtime but rising earlier than our natural rhythm would instigate. This is arguably leading to a chronic state of circadian disruption and associated risks [34].

#### **3. Oxidative Stress and Redox Homeostasis**

Humans are continually exposed to RONS from both endogenous (e.g., from aerobic metabolism and the immune response) and exogenous sources (e.g., tobacco smoke, UV light, etc.) and thus, antioxidants function to help prevent/curb high levels of oxidative stress and maintain redox homeostasis. Accordingly, aerobic organisms possess an intricate antioxidant defence system [35] that comprises an orchestrated synergism between several endogenous and exogenous antioxidants attempting to control the RONS produced in cells and tissues [36,37] which arise as a consequence of everyday activities such as the food we eat [38]. Oxidative stress is defined as 'an imbalance between oxidants and antioxidants in favor (sic) of the oxidants, leading to a disruption of redox signaling (sic) and control' [39]. Thus, the biological (physiological or pathological) effects of RONS depend critically on the amounts and location of production, relative to local antioxidant defences. For instance, when formed in low/moderate amounts, they can act as crucial mediators of signal transduction pathways e.g., in the growth of vascular smooth muscle cells (VSMC) and fibroblasts [40] and for exosome control and myokine release [41]. Yet, excessive RONS may cause widespread cellular toxicity [6,42]. Indeed, work from several labs, including our own, has demonstrated how RONS instigate oxidative damage by altering susceptible lipids, proteins, and DNA in human volunteers [43–45]. This can lead to impaired transcription and translational processes, altered protein function, and production of secondary by-products and metabolites which can further RONS production and/or cellular damage [46]. Although still an area of some discourse, RONS and other redox-active species have increasingly been acknowledged as fundamental regulators of genes, proteins, and the associated molecular signalling pathways integral to the regulation of many biological processes and functions [46,47].

#### *3.1. Oxidative Stress, Redox Homeostasis, and Exercise*

Multiple studies have now established a link between exercise (especially strenuous and/or exhaustive exercise) and oxidative stress [10,48]. The production of RONS in response to exercise, especially acutely, has long interested researchers and offers an intriguing model to examine the dynamic role of RONS from both the physiological and pathological perspectives—as depicted in Figure 2. The latter is important to note as evidence suggests that exhaustive (long duration) and/or strenuous exercise (highintensity maximal exercise, marathons, triathlons, and overtraining) can induce detrimental, oxidative DNA alterations if left unrepaired [49,50]. Yet, during low or moderate-intensity exercise, the generated RONS may serve to act as signalling molecules responsible for the initiation of exercise and skeletal muscle adaptation [50–52], including accentuation in antioxidant enzymes and drivers of mitochondrial biogenesis [53]. In fact, the observation that low doses of stressors can exert beneficial effects, but when in excess can be toxic, forms the fundamental basis of the hitherto mentioned hormesis theory. This paradoxical effect has led to the extension of the hormesis theory to exercise induced RONS formation: transient increases in RONS can alter signalling pathways and/or cause molecular damage that induces adaptive responses that protect against subsequent stronger stress [54] such as that evident in human volunteers for the repeated bout effect following eccentric muscle contractions [55]. Moreover, when a single bout of exhaustive exercise is performed by well-trained human volunteers, a large elevation in oxidative damage is not observed [56]. Mounting evidence suggests that exercise-induced RONS are essential upstream signals for the activation of redox-sensitive transcription factors (e.g., Nrf2, AP1) and the induction

of gene expression associated with exercise. As such, redox processes are increasingly recognized as an integral part of normal human biology and especially exercise (for further reading see the review by Margaritelis et al. [57]).

**Figure 2.** Exercise-induced RONS, Oxidative Stress, and Hormesis. The fundamental basis of the hormesis theory can be applied to exercise-induced RONS formation: low or transient increases in RONS, from moderate-intensity and regular exercise, activate signalling pathways that induce adaptive and protective responses. Whereas inactivity and/or sporadic strenuous/high-intensity exercise can lead to a RONS load that overwhelms antioxidant defences leading to oxidative stress, impaired physiological function, and an increased risk for chronic disease.

The adaptive potential of exercise and the extent of RONS production seems to be related to the exercise intensity of the bout or training stimulus [58]. High-intensity exercise produces higher concentrations of RONS than exercise at low/moderate-intensity aerobic exercise at <50% . VO2max [59]. While we have recently reported that acute intense/prolonged exercise can induce oxidative DNA damage in human volunteers, this damage does not persist indefinitely and is most likely repaired, at least in trained/experienced athletes [10]. This damage might act as a trigger for repair to prime the cell for further, subsequent damage and thus underpin the adaptive process and promote longevity. For instance, master endurance athletes are shown to have a longer telomere length (TL), a marker of biological age, than non-athlete, age-matched controls [60,61]. In addition, three weeks of highintensity interval training (HIIT) in humans improves plasma antioxidant capacity [62]. Furthermore, recent evidence in animal models has reported reductions in maximal exercise capacity, several exercise-responsive proteins, and mitochondrial network adaptations in mice lacking a functional NOX2 complex following a HIIT programme [62]. Thus, the fine balance between redox signalling and oxidative stress in the response to exercise requires further elucidation. A growing appreciation for the role of redox homeostasis has allowed researchers to understand that RONS can offer distinct signalling roles depending on where they originate. The following is a summary of the main sources of RONS

production in contracting muscle and the corresponding antioxidants that converge to support redox balance.

#### *3.2. Skeletal Muscle as a Biological Source of RONS*

Several RONS are generated continuously in, or very close to, skeletal muscle both at rest and during exercising contractions [63]. Of these, O2 •− is the primary species. Early work revealed mitochondria as a source of free radical production at rest [64], but it appears mitochondria are not a major source of RONS generation in exercising muscle [65]. A number of other potential sources for primary and subsequent RONS (e.g., H2O2) have been identified and include NADPH oxidases (NOX; which is active across several cellular organelles), xanthine oxidases (XO), and phospholipase A2 (PLA2) [49]. It should also be noted that nitric oxide (NO•) production occurs both at rest and during/following exercise in muscle. Interestingly, via the convergence of NOX4 and two forms of neuronal NOS in mouse models, the resultant NO• yields peroxynitrite (ONOO−) proposed to be involved in the signal transduction pathways for mTORC1 integral to muscle hypertrophy associated with strength/resistance training [66–69]. NO• itself has also been postulated to confer an intrinsic effect on the regulation of insulin secretion, and glucose transport and metabolism, which are also accentuated via exercise [70,71].

The NOX enzymes are viewed as an important source of RONS in skeletal muscle, particularly the NOX2 and NOX4 isoforms [72]. NOX2 is located within the sarcolemma and T-tubule, whereas NOX4 is located in both the sarcoplasmic reticulum and the mitochondria [73]. NOX4 is constitutively expressed and postulated to be involved in basal RONS production, whereas NOX2, sensitive to several stimuli, is believed to be the primary source of NOX-mediated RONS production in contracting muscle [11,72]. Whether or not NOX enzymes are the major source of muscular RONS is debatable as complexities remain in measuring their activity [49]; although recent advances using genetically encoded redox probes offer attractive possibilities in this respect [11,63], further scrutiny is beyond the scope of this narrative.
