*4.1. Antioxidant Regulation and Control*

Insight into the cross-talk between the circadian clock and oxidative stress has been gleaned from studies illustrating temporal expression patterns for antioxidant enzymes and compounds. For instance, O'Neill and Reddy [86] observed a circadian rhythm in mouse PRDXs that was altered in Cry1/2 double knockout cells, suggesting it may be regulated by the molecular clock. Using human RBCs, they also showed a robust ∼24-h redox cycle for PRDXs to directly show rhythms persist even without active transcription [86]. Furthermore, deletion of core clock components leads to increased thiol oxidation and

protein carbonylation in flies and mice [87], and Per1-knockout flies exposed to oxidative stress display a shorter lifespan and increased oxidative damage compared to controls [88] which may, in part, be due to decreased activity of RONS scavenging enzymes. Given that RONS production can oscillate over the course of the day often due to normal, habitual behaviours, i.e., the by-products of metabolism, circadian control of antioxidants seems intuitive to maintain redox control [89]. Several studies have reported differences in DNA damage [90], lipid peroxidation [91], and protein oxidation [92] at different times of the day. These oscillations directly reflect the daily rhythm of antioxidant expression and protective enzyme activity levels. Those that peak in the morning includes: GPx; CAT; SOD; and PRDXs [6]. On the other hand, melatonin, plasma thiols, and ascorbic acid peak in the evening which also corresponds to the reported peaks for PERs1 and 2 and the CRYs (see [6]). Daily rhythmicity in SOD activity was first reported by Diaz-Munoz and colleagues in 1985. In contrast to humans, they found that in the rat cerebral cortex, SOD activity peaked in the dark phase, coinciding with the peak level of malondialdehyde (MDA), a marker of lipid peroxidation [93]. Thus, circadian oscillations in antioxidant expression seem to vary depending on the species investigated (i.e., nocturnal vs. diurnal), and this is most likely due to variations in sleep/wake cycles and the corresponding differences in their respective feeding patterns and light exposure [6].

The master regulator of antioxidant defence appears to be the transcription factor Nrf2, which translocates to the nucleus where it drives the expression of several antioxidant enzymes and is directly regulated by the expression of NRF through E box elements in the promoter [89]. As antioxidants are important for governing intracellular/local RONS levels, which, in turn, have been documented to impinge on the expression of clock genes, it appears that further appreciation and understanding of this cross-talk may yield purposeful applications for those interested in redox and chronobiology given how circadian rhythmicity and oxidative stress are central to many disease pathologies. For example, life-long administration of the antioxidant, N-acetyl-L-cysteine, has been shown to delay the onset of premature ageing induced by chronic oxidative stress in *BMAL-1*−*/*<sup>−</sup> mice [94].
