*4.2. Circadian and Oxidative Influence on Cardiovascular Physiology and Disease*

Despite considerable progress in understanding, preventing, and treating cardiovascular diseases (CVD), ischaemic heart disease and strokes remain a major source of global morbidity and mortality [95]. Clock mechanisms are integral to normal cardiovascular function by coordinating rhythms in blood pressure, heart rate, and cardiac muscle contractility. However, circadian dysregulation increases cardiovascular risk with strong data suggesting adverse cardiovascular events such as sudden cardiac death [96] and myocardial infarction [97] are more likely to occur in the morning after awakening. Notably, a recent meta-analysis reported how the risk of acute myocardial infarction (AMI) increases after daylight saving transitions to emphasise the precarity of the circadian system [98]. The complete mechanism(s) surrounding such phenomena are unclear but may be linked to disruptions to redox control as RONS and free radical biology have been implicated in both the pathophysiology and treatment of heart disease [25]. For instance, we have reported increased peripheral arterial stiffness in healthy volunteers following the ingestion of a high-fat meal which was associated with augmented lipid hydroperoxides, while decreases in SOD were also observed [38]. We believe the high-fat meal increased O2 •− production that was subsequently able to react with endothelium-derived NO• (via eNOS) to impair blood vessel function. The consequences of this (and similar reactions that elevate vascular RONS) may be detrimental to cardiovascular function by reducing NO• bioactivity and increasing the formation of the ONOO− [37], possibly leading to endothelial dysfunction and a pro-atherogenic environment [5]. ONOO− interacts with lipids, DNA, and proteins via direct or indirect radical mechanisms, and its generation has been cited in the pathogenesis of stroke, myocardial infarction, atherosclerosis, circulatory shock, and chronic inflammatory diseases [99,100]. These RONS-instigated reactions trigger cellular responses

ranging from subtle modulations of cell signalling to oxidative injury, committing cells to necrosis and apoptosis [100]. Of note, Man et al. [101] reported how the peripheral circadian clock can regulate eNOS and NO• production (which is lower during the morning) and that lipid metabolism also displays circadian oscillations. Combined, the misalignment of the circadian clock with these parameters could lead to the development/progression of atherosclerosis, which may be heightened with frequent exposure to conditions that amplify RONS production such as high-fat meal ingestion, as discussed above. Support for a circadian-redox mechanism is further evident from a study in middle-aged adults where vascular endothelial function, as measured by flow-mediated dilation (FMD), was impaired across the night and into the morning period and was accompanied by a pronounced rise in plasma MDA and a concomitant augmentation in the vasoconstrictor, endothelin-1 (ET-1) [102]. Such interactions are clearly pertinent to those already predisposed to increased risk for cardiovascular events, and other diseases like cancer, such as shift workers.

#### *4.3. Shift Work: DNA Damage and Repair*

In a recent cross-sectional study, higher levels of H2O2 and lower SOD and catalase were observed in night workers when compared to day workers [103]. In fact, the circadian clock was first implicated as a factor in various diseases as epidemiological studies reported an increased incidence of cancers in long-term shift workers [19,104,105]. Those working night shifts also appear to have increased DNA damage that is linked to lower melatonin induced by night working [106]. In strict laboratory conditions, when compared to simulated day shifts for three consecutive days, simulated night shifts of the same duration also caused circadian dysregulation of genes involved in key DNA repair pathways. Moreover, the percentage of cells with BRCA1 and γH2AX foci (representing DNA damage biomarkers using immunofluorescent microscopy) was significantly higher in the night shift condition, whereas the effectiveness of the processes to repair leukocyte DNA damage from both endogenous and exogenous sources were compromised in samples from the night shift volunteers [107]. Given that it exerts antioxidant and DNA repair properties (via the nucleotide excision repair (*NER*) pathways), melatonin supplementation, acting as a so-called chronobiotic [32], has been suggested to reduce the potentially damaging effects of shift work [108]. The studies and data summarised in this section are undoubtedly useful for helping tie the molecular and biochemical connections between shift workers and elevated cancer risk, but much remains open for further discovery.
