Endogenous biological rhythms regulate multiple physiological and behavioral processes in mammals that are essential for proper reproduction; their misalignment may end up in reproductive disorders. Endogenous circadian rhythms have been identified for melatonin, cortisol, thyroid-stimulating hormone (TSH), and to a lesser extent prolactin. Clock genes within the suprachiasmatic nucleus (SCN) of the hypothalamus control such rhythms. Approximately 10 genes have been identified that are fundamental to cellular rhythmicity: PER1, PER2, PER3, CLOCK, BMAL1, CRY1, CRY2, DEC1, DEC2, and RevErb-α, with BMAL1 and CLOCK being the central genes. In the main loop, the transcription factors CLOCK and BMAL1 stimulate the expressions of PER1–3 and CRY1-2, which in turn suppress CLOCK and BMAL1 transcription and expression and shut down their own transcription. CLOCK/BMAL1 also controls the rhythmic expression of RevErb-α, a transcription factor sensitive to redox changes since it is regulated by heme, the prostetic group of hemoglobin and other hemoproteins.
CLOCK genes exist in central and peripheral reproductive tissues and temporal coordination within and between tissues is vital for reproduction. Circadian rhythms are found in almost all organisms with sensitivity to light. Problems with circadian rhythms in humans are related to diseases such as high blood pressure (hypertension), diabetes, insomnia and anxiety. Both shift workers and older people have increased the risk of these diseases due to changes in their circadian clock. However, a very little information is available to scientists about the connection of circadian rhythmes and oxidative stress, given the fact that the latter is a central regulator for animal and human longevity. Some knowledge was acquired a couple of years ago, when researchers at the University of Tokyo identified three new genes associated with the regulation of circadian rhythms. The new discovery reveals for the first time that circadian regulation can be directly linked to cellular stress.
Scientists used cells and mice lacking three genes: apoptosis-signaling kinases 1, 2 and 3 (ASK1, ASK2 and ASK3). In both cell and mouse results, ASK genes were necessary to respond to both sudden changes in the environment and gradual changes over time. Cells without ASK genes have not shown changes to their circadian rhythm, which occur in normal cells that grow in environments with too high or too low salt or sugar concentrations. Cells without ASK genes were also impermeable to expected changes after cells accumulated too much oxidative stress. One of the genes responsible for controlling circadian rhythms is CLOCK: it produces a protein that binds DNA together with its partner PER. The team of researchers has seen that the ASK kinases modify the CLOCK protein, varying the affinity for its partner PER. The results in the cells were further supported by observations on mouse behavior. Normal mice can change their wake-up time the next morning after unexpected exposure to light at night, as measured by their activity running on a wheel.
Mice without ASK genes have less ability to synchronize their circadian clock with changes in environmental light-darkness cycles. Many researchers in this field have long suspected that oxidative stress and circadian rhythms are somehow linked, due to the photosynthesis cycles and DNA replication that we also see in ancient organisms. Photosynthesis requires sunlight and creates free radicals that they could damage DNA, so cells postpone DNA replication and cell division until nightfall when photosynthesis stops. Uncontrolled oxidative stress creates potentially toxic environments within cells, due to changes in chemical equilibrium. Besides, ASK proteins are involved in a form of cellular death called apoptosis. They are activated when intracellular ROS increase inappropriately. One ROS sensor is thioredoxin-1 (Trx-1), a small protein that becomes oxidized in attempt to buffer ROS. Once oxidized, it becomes a partner for ASK-1, leading to its activation. ASK-1, in turn triggers a cascade that drives cell death (apoptosis).
Since a chronic lack of sleep has been linked to cognitive decline in the late age, it would be a good hypothesis to test if sleep deprivation would kill brain neurons by the cascade oxidative stress-ASK proteins. Neuronal loss would, in time, compromise higher functions like memory, mood and cognition. A more recent stud in mice reached similar conlcusion in other ways. Mice undergoing disruption in light-dark cycles (higher esposure either to dark or kight) had altered expression of antioxidant enzymes (catalase, SOD1, GSH peroxidase) in some tissues, principally in testes. Biomarkers for oxidateive stress (like MDA) were riduced by prolonged light exposure and substantially inhibited by prolonged dark exposure. Nitric oxide follwed a more linear pattern: it was significantly elevated by prolonged light exposure and markedly reduced by prolonged dark exposure. Also proteliferation markers in reproductive cells (nuclear PCNA) has a similar behavior. This means that circadian changes start to affect fertility.
Given the similarities of this genetic systems between rat, mouse and primates up to man, it is reasonable that such conditionings would happen in humans as well. It is as well noteworthy as melatonin, one of the keymaster regulator of circadian rhythms, has been always deemed to be provided with antioxidant properties. Many years ago it was speculated that it could have direct ROS scavenging activity, a notion that spawned many controverisal toughts. When extracellular (MT1R and MT2R) and intracellular (RZR-alpha) receptors binding melatonin were discovered, it was speculated that its antioxidant actions could have been mediated by a competent gene expression. However, later analyses got to prove that melatonin may actually scavenge at least some kind of ROS. Beside, connections between all the biochemical systems mentioned are recognized from some time now: melatonin, a natural regulator of female period, it is an antiproliferative agent that decreases PCNA expression in a hormone-dependent manner in vivo and in vitro in female rat ovarian cells and in mice prostate tumors.
Just a couple of weeks ago it has been demnostrated that melatonin acts on a chrono-disrupted mice model by restoring oxidative stress and modulating cellular autophagy in the brain tissue, especially in the cortex and hippocampus. By reducing secretion of inflammatory cytokine s (IL-6 and TNF-alpha) and modulating cellular remodeling proteins (ATG3, Beclin, Ngb), melatonin confirmed itself as either an effective biological regulator and a natural and possibly safe option to treat medical conditions dependent on circadian imbalances. Among these mood disturbances, insomnia, diabetes, atherosclerosis and hypertension, all constituting a big burden as a matter of public health.
- edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
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