Epigenetics is an important tool for understanding the relation between environmental exposures and cellular functions, including metabolic and proliferative responses. At our research center, we have devolved a mouse model for characterizing the relation between exposure to artificial light at night (ALAN) and both global DNA methylation (GDM) and breast cancer. Generally, the model describes a close association between ALAN and cancer responses. Cancer responses are eminent at all light spectra, with the prevalent manifestation at the shorter end of the visible spectrum. ALAN-induced pineal melatonin suppression is the principal candidate mechanism mediating the environmental exposure at the molecular level by eliciting aberrant GDM modifications. The carcinogenic potential of ALAN can be ameliorated in mice by exogenous melatonin treatment. In contrast to BALB/c mice, humans are diurnal species, and thus, it is of great interest to evaluate the ALAN-melatonin-GDM nexus also in a diurnal mouse model. The fat sand rat (Psammomys obesus) provides an appropriate model as its responses to photoperiod are comparable to humans. Interestingly, melatonin and thyroxin have opposite effects on GDM levels in P. obesus. Melatonin, GDM levels, and even thyroxin may be utilized as novel biomarkers for detection, staging, therapy, and prevention of breast cancer progression.
- global DNA methylation
- diurnal species
- breast cancer
Since the invention of electrical light in 1879 by Thomas Alva Edison, artificial light at night (ALAN) has become a definitive feature of human development with accelerated increase concurrent with urbanization and industrialization. The light emitted from the original bulb of Edison known as incandescent bulb was weak, with a dominant long wavelength emission above 560 nm. Most of the incandescent electrical energy is dissipated as heat energy, thus making this type of illumination energetically inefficient. Therefore, new illumination technologies were developed, in order to discover efficient bulbs that transfer most of the electrical energy into light. White fluorescent and light-emitting diodes (LED) are examples of energy efficient bulbs developed to decrease carbon dioxide production from electric power plants, thus lessening the greenhouse effect. One of the adverse outcomes of using efficient illumination at night time is the emission of shorter wavelengths (SWLs) that further exacerbate the health and ecological problems associated with a new source of environmental pollution currently known as ALAN [1, 2, 3]. Light pollution is increasing rapidly, resulting in a more illuminated world, where outdoor and indoor illumination sources are increasing ALAN in developed and developing countries [4, 5].
From an anthropological perspective, electric light has brought pronounced benefits including advancing urbanization and industrialization by increasing productivity, but we are also increasingly being aware of serious public health and ecological negative impacts emerging from disrupting the adaptive temporal organization of biological responses [6, 7, 8]. Certainly, multiple studies have shown the effects of light pollution on social, behavioral, physiological, and molecular responses in many different taxa, including insects , fishes , amphibians , reptiles , birds , and mammals , as well as plants . Some of the most disturbing effects of ALAN on health are metabolic dysfunction and cancer progression [2, 16]. In mice and humans, several lines of evidence suggest a close association between ALAN levels and both obesity and breast cancer progression [17, 18, 19]. Here, we focus on ALAN as a novel environmental polluter that disrupts biological timing (temporal organization) and consequently may provoke severe health risk, particularly breast cancer development through epigenetic modifications. First, the mammalian photoperiodic system is reviewed in relation to light perception and downstream endocrine responses for timing biological rhythms. Thereafter, we discuss the sensitivity of the photoperiodic system to the spectral composition of ALAN, particularly SWL illuminations. We further discuss the ALAN signal transduction pathway involved in melatonin suppression and aberrant epigenetic modifications in breast cancer progression. Therefore, melatonin and epigenetics are suggested as new biomarkers for breast cancer prevention. Finally, melatonin and thyroxin treatments in the diurnal fat sand rat (
2. The mammalian photoperiodic system
In an early study, it has been demonstrated that the blind mole rat (
First, photoperiodic signals are perceived by ipRGCs that express the photo-pigment melanopsin . The detected environmental light signal by the ipRGCs synchronizes the master circadian clock located in the mammalian hypothalamic suprachiasmatic nucleus (SCN) by the retinohypothalamic tract (RHT). The SCN regulates the synthesis and release of the hormone melatonin by the pineal gland through multiunit sympathetic nerves from the superior cervical ganglion (SCG). The SCG presynaptic sympathetic terminals release noradrenalin that interacts with postsynaptic α- and β-adrenergic receptors to regulate synthesis and release of pineal melatonin . In mammals, the activity of the adrenergic SCG terminals that innervate the pineal gland is stimulated by darkness and inhibited by light . Under dark conditions, stimulation of the pineal adrenergic receptors increases cellular cAMP levels leading to the activation of aryl-alkyl-amine-N-acetyltransferase (AA-NAT), a key enzyme in melatonin synthesis . The nocturnal increase in the enzymatic activity of AA-NAT is strongly inhibited by light exposure, consequently leading to a rapid decrease in nocturnal melatonin levels . The pinealocytes are the primary neuroendocrine cells that synthesis melatonin by sequential hydroxylation and decarboxylation of its precursor tryptophan to serotonin. Thereafter, serotonin is acetylated by the rate-limiting enzyme AA-NAT and methylated by the enzyme hydroxyindole-O-methyltransferase (HIOMT) to the final product of melatonin [28, 30]. Finally, the activity of both AA-NAT and HIOMT is under photoperiodic control at the transcriptional level showing distinct diurnal rhythms with peak levels during night and nadir levels during the day .
3. Melatonin suppression as an indicator of SWL pollution
In most mammals, no level of light exposure is powerless regarding melatonin suppression and even low intensity and short-term exposures can reduce its production and lead to decreased circulating levels [32, 33]. Nonetheless, melatonin suppression is strongly wavelength- and irradiance-dependent, with faster and more robust response at the SWL end of the visible spectrum below 500 nm [19, 34, 35]. A large-scale study comparing the effect of different light technologies on melatonin production in humans demonstrated that the strongest suppression occurred in response to 4000 and 5000 K LED lights compared with incandescent, halogen, and fluorescent counterpart lightening systems . Narrow bandwidth blue LED exposure (λ = 469 nm, ½ peak bandwidth = 26 nm) decreased melatonin levels in an irradiance dose-dependent manner, and this light was more effective in decreasing the hormone levels compared with that of 4000 K of white fluorescent at twice the energy of the latter . In horses, 1 h exposure of 3 lux SWL blue light (468 nm) administered only to one eye was sufficient to decrease melatonin levels compared with control animals .
Furthermore, blue LED pulses (2-s pulse every 1 min for 1 h, λ = 450 nm) administrated through closed human eyelids markedly suppressed nocturnal melatonin levels and delayed the melatonin onset phase [39, 40, 41]. While the eyelids can weaken irradiance and wavelength (, light signals can still penetrate them, be detected by the retinal photoreceptors, and affect circadian regulation . In humans, blue LED exposure (40 lux, 470 nm) emitted from display screens (tablets and computers), suppressed nocturnal melatonin in a duration-dependent manner [44, 45] and melatonin suppression showed higher sensitivity to wavelength compared with intensity manipulations .
Together, it is clear that the adverse effects of light pollution are strongly manifested by the SWL portion of the spectrum. As the LED illumination is becoming ubiquitous in every aspect of our modern life, the expected increase in light pollution may exacerbate the problem since higher irradiance and shorter wavelengths would be emitted by the energy efficient technology [47, 48]. Accordingly, the American Medical Association  passed a resolution in 2016 calling upon communities in the USA to avoid using LED lighting in public domains as it is enriched with SWL . In summary, SWL-ALAN is a source of pollution and should be removed from public spaces through legislation.
4. ALAN as an environmental change and a model for studying epigenetic modifications
The flexibility and the sensitivity of the endocrine system play an adaptive role in determining the success and survival of organisms under contentiously changing environmental conditions in their habitat . As the endocrine system regulates several functions, it is expected to be the first system to respond to environmental changes such as ALAN by coordinating body functions to maintain homeostasis during the exposure. The core stimulus-response of the endocrine system to ALAN relies on four main components, including the pineal gland, the hypothalamic-pituitary-gonadal (HPG) axis, the hypothalamic-pituitary-thyroid axis (HPT), and the hypothalamic-pituitary-adrenal (HPA) axis . The elaborated hormonal responses generated by these axes to ALAN exposure might be mediated by transcriptional regulation of gene expression
The incidences of breast and prostate cancers show close association with light pollution particularly in urbanized and industrialized regions [2, 54]. Several epidemiological studies have found direct association between light pollution and incidence of breast cancer in women as well as prostate cancer in men [18, 55, 56]. Furthermore, the strong association between light pollution and cancer incidences displays divergent spatial disruption with higher incidences in urban compared with rural regions [57, 58]. Evidence for direct association between ALAN and cancer development comes also from animal studies.
In rats, ALAN exposure accelerated the growth rates of induced-tumors, including mammary cancer [59, 60, 61, 62]. Studies under control conditions demonstrated that 30-min ALAN per midnight emitted from either white fluorescent or blue LED illuminations can accelerate tumor growth and lung metastatic activity in female BALB/c mice inoculated with 4T1 mammary carcinoma [63, 64]. Indeed, the effects of ALAN on tumor growth have been demonstrated at different spectral compositions with markedly higher cancer burden in response to lighting exposure lower than 500 nm .
These studies have related the increased cancer burden to aberrant epigenetic modifications, particularly advanced global DNA hypo-methylation. Promoter hyper-methylation of cancer suppresser genes and global DNA hypo-methylation are characterizing epigenetic patterns in breast cancer cells [65, 66]. These aberrant epigenetic modifications may contribute to increase cancer burden by eliciting genomic instability and activation of both oncogenes and metastatic related genes, as well as silencing tumor suppressor genes. Generally, prominent decreased methylation in repetitive DAN elements is a common trait in most cancer cells . Demethylation of pro-metastatic genes is normally suppressed by DNA methylation and might advance gene overexpression leading to genetic instability that increases the risk of developing cancer [68, 69]. DNA hypomethylation can be detected at an early stage of breast cancer and is correlated with the degree of tumor differentiation [70, 71]. Altogether, the close association between aberrant DAN hypomethylation and tumorigenesis, particularly of breast cancer, is well-established, but the underlying mechanism remains poorly understood, especially how the adverse ALAN effects are mediated.
5. Melatonin as a mediating signal linking ALAN and epigenetic-induced cancer
Since the melatonin hypothesis was first proposed during the late twentieth century by Stevens , multiple studies in human and nonhuman animals have provided direct and indirect evidence that melatonin suppression by ALAN could impose health risks, including metabolic disorders and cancer progression [2, 54]. The importance of melatonin in the regulation of several biological functions depends heavily on its lipophilic and hydrophilic traits that make it omnipresent in all cell compartments, principally in the nucleus . Indeed, low levels of 6-sulfatoxymelatonin (6-SMT), the major metabolites of the hormone in urine , have been demonstrated to correlate with increased risk of breast cancer in postmenopausal women [75, 76, 77]. Furthermore, women with blindness or long sleep duration (elevated melatonin levels) present reduced breast cancer risk relative to normal women [78, 79].
Physiological blood concentration of melatonin blocked human leiomyosarcoma (soft tissue sarcoma) proliferation by inhibiting tumor metabolic and genetic pathways presumable by suppression of cellular cAMP levels
Melatonin could mediate its effects of cancer development
The strong association between ALAN, DNA hypo-methylation, and melatonin suppression may be of significant clinical importance. DNA methylation and melatonin can be utilized as biomarkers for detecting and preventing breast cancer development. The traditional diagnosis method for breast cancer is scanning by mammography, which is a useful technique to identify the growth of cancer. The mammography cannot predict risk for breast cancer as it indicates its existence, but trends in melatonin suppression and DNA methylation can provide a simple, noninvasive, and reliable tool for predicting cancer risk, particularly among a group of high-risk individuals for developing the disease such as night shift workers. Bearing in mind that epigenetic modifications are reversible , early treatment by melatonin or any other analogs  for individuals at high risk can be very effective in preventing breast cancer. We are aware today, that genetics factors such as breast cancer genes are not the major causes of the malignancy and other external factors are heavily involved. Therefore, much more attention should be given to environmental changes that link endocrinology with epigenetic modifications.
Collectively, in diurnal humans, circadian disruption enforced by activity impinging on the inactive period during the nighttime is recurrently associated with a number of health problems. However, a direct link between ALAN-induced circadian disruption and health risks is still difficult to clearly establish as most data are derived from epidemiological and nocturnal animal studies . Therefore, integrating diurnal animal models of chronodisruption with epidemiological and nocturnal model studies would add a significant value in defining potential direct signal transduction pathways mediating the environmental exposure impacts on physiology and health. Consequently, we conducted a preliminary study to investigate the effects of hormonal manipulations in diurnal species on physiological and epigenetic regulations. This preliminary study is a first step in a large-scale study using diurnal mouse model to elucidate the association between ALAN-induced circadian disruption and the development of health problems at the behavioral, physiological, and molecular levels.
6. Physiological and epigenetic responses to melatonin and thyroxin in diurnal species
Bearing in mind that humans are diurnal, understanding the physiological and epigenetic response to ALAN in human disease can benefit significantly from using a diurnal species such as the fat sand rat (
The results showed that melatonin alone significantly increased Wb from day 1 compared with controls, but with a decreasing magnitude with time (Figure 1A). Mass gain on day 1 was approximately 1.5-fold higher compared with that at the last. T4 also increased Wb from day 1 to day 5 compared with controls, but with significantly lesser effect compared with melatonin. Thereafter, mass was decreased showing a moderate mass loss from day 13 to day 21 compared with controls. Thyroxin and melatonin in combination markedly decreased Wb with time compared with all other groups. Mass gain decreased from 0.46 ± 0.88% at day 1 to −20.21 ± 2.56% at day 21. Thyroxin can regulate Wb by increasing heat production through nonshivering thermogenesis by changing membrane permeability to sodium, increasing the pump activity to maintain cell homeostasis in brown adipose tissue, resulting in higher body temperature values and loss in Wb .
Melatonin may operate through increasing the amount of brown adipose tissue, thus increasing heat production by increasing energy expenditure. Melatonin and thyroxin in combination provoked considerably more mass loss than melatonin alone, suggesting that melatonin may act synergistically with thyroxin to evoke mass loss in rats, due to the combined effect of increasing energy expenditure.
Body temperature rhythms were notably altered only in response to T4 treatment, while melatonin alone and in combination with thyroxin had no effect on body temperature compared with controls (Figure 1B). Furthermore, the significant decrease in body temperature following treatments with thyroxin and melatonin in combination, compared with T4 alone, suggests that melatonin and thyroxin exert a significant antagonistic effect on body temperature.
Thyroxin treatment had no effect on mean 6-SMT levels but altered the daily rhythms with higher amplitude and delayed acrophase by approximately 2 h (Figure 2A). Finally, melatonin treatment elicited hypomethylation while thyroxin alone or thyroxin and melatonin in combination exerted comparable effects on GDM levels showing marked hypermethylation compared with control levels (Figure 2B). Similar to Wb, thyroxin and melatonin may have exerted synergistic effects on promoting DNA hypermethylation, but this effect did not reach statistical significance.
These results suggest that melatonin and thyroxin have a role in the regulation of body temperature and apparently metabolism, in which the former may attenuate metabolism and the latter may accelerate it. Both hormones exerted inverse effects on global DNA levels, suggesting that different transduction pathways are involved in the circadian regulation of body temperature in
However, in humans, melatonin may interact with the HPT axis to modulate the circadian rhythm of body temperature . In mammals, the HPT axis plays a major role in several adaptive functions such as growth, development, metabolic rate, thermogenesis, heart rate, immune, and reproductive responses . The HPT releasing and stimulating hormones as well as the thyroid hormones (T4 and T3) are under photoperiodic control presumably by the pars tuberalis of the adenohypophysis [106, 107]. In rats, T3 and T4 concentrations exhibit significant circadian rhythms with elevated levels during the dark period compared with the counterpart light period . The nocturnal increase in the thyroid hormones was reported also in the rat pineal gland following an increase in type I 5′-iodothyronine deiodinase activity, which catalyzes the conversion of T4 to T3 . Furthermore, the thyroid hormones are crucial photoperiodic regulators of several physiological processes including energy metabolism and reproduction [110, 111]. While the relation between the HPT axis and the photoperiodic system are well-characterized, there are limited studies on the effect of ALAN on the HPT axis. However, due to the link with the photoperiodic system, environmental perturbation of the circadian clock by ALAN is expected to alter the activity of the HPT axis, including the thyroid hormones. In hamsters under short-day photoperiod, low levels of ALAN elevated the levels of thyroid-stimulating-hormone (TSH) receptors causing advanced Wb and gonadal growth . Continuous exposure to ALAN decreased TSH, but increased both T3 and T4 in mice . In birds, long-term exposure to ALAN increased both the blood levels of the thyroid hormones and Wb . Overall, ALAN may induce aberrant epigenetic modifications by disrupting endocrine axes such as HPT axis that interacts with melatonin to manifest the adverse effects of the environmental exposure. However, the exact mechanism of action by which HPT axis may directly, or
Currently, it is clear that electric light not only has remarkable anthropological advantages, but also severe adverse ecological and public health concerns. One of the most alerting impacts of ALAN on public health is the potential association between SWL exposure and cancer development, particularly in urbanized regions worldwide. ALAN effects are suggested to be mediated at the cellular level by inducing epigenetic modifications
These effects are presumably mediated by aberrant epigenetic modifications. Therefore, DNA methylations, which are a reversible modification in genes, triggered by melatonin, are a promising mechanism linking between environmental exposures like ALAN and hormonal/cellular pathway mediating carcinogenic activities like metastasis activity, tumor cell proliferation, and estrogen-related responses . Melatonin may affect DNA methylation by modulating the activity of DNA methyltransferases involved in the regulation of gene expression by changing DNA methylation patterns. The well-established fact that different tissues present specific patterns of epigenetic modifications  may account for the observed tissue-specific effects of ALAN and melatonin on DNA methyl-transferase activity and GDM levels. Tissue differential effects on the activity of DNA methyl-transferases and GDM levels in response to ALAN exposure may present tissue-specific responses to genes that are involved in circadian regulation of several transduction pathways including cancer cell proliferation and metastatic activity. Since humans are diurnal species and most studies have been conducted on nocturnal animals, a diurnal experimental model should be of a great clinical interest.
The authors of this chapter would like to thank the Vice President and Dean of Research at the University of Haifa, Prof. Ido Izhaki for allocating the funding to the publication fee.
This chapter is dedicated to the memory of Professor Abraham Haim, who passed away before publication of this work. His contribution was foremost among the authors of this chapter.
|ALAN||artificial light at night|
|GDM||global DNA methylation|
|SCN||hypothalamic suprachiasmatic nucleus|
|ipRGCs||intrinsically photosensitive retinal ganglion cells|
|SCG||superior cervical ganglion|