Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away and Epigenetic Modifications

*Abraham Haim, Sinam Boynao and Abed Elsalam Zubidat*

## **Abstract**

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.

**Keywords:** melatonin, thyroxin, light-at-night, global DNA methylation, diurnal species, breast cancer, biomarkers

#### **1. Introduction**

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

#### *Epigenetics*

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 [1–3]. 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 [6–8]. Certainly, multiple studies have shown the effects of light pollution on social, behavioral, physiological, and molecular responses in many different taxa, including insects [9], fishes [10], amphibians [11], reptiles [12], birds [13], and mammals [14], as well as plants [15]. 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 [17–19]. 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 (*Psammomys obesus*) are discussed in relation to their potential role in mediating the environmental exposures at the molecular level *via* epigenetic modifications, particularly global DNA methylation (GDM).

### **2. The mammalian photoperiodic system**

In an early study, it has been demonstrated that the blind mole rat (*Spalax ehrenbergi*) responded differently to short and long photoperiod manipulations in regard to its capability to cope with low ambient temperature exposure [20]. Results of a more recent study on *S. ehrenbergi* manifested robust and differential responses in metabolism, stress, and melatonin levels to ALAN of different spectral compositions and acclimation duration [21]. These results suggested that the vestigial retina of this species still expresses photoreceptors that are involved mainly in nonvisual response. Currently, the mammalian eye is described as a dual-function organ, expressing photoreceptors for both visual and nonvisual responses [22]. The visual response is mediated by two distinct photoreceptor types, rods and cones, which control scotopic vision and photopic vision, respectively [23]. The nonvisual responses are mainly mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the photopigment melanopsin. Even though the ipRGCs are connected with rods and cones by bipolar cells, they mediate nonvisual responses including photo-entrainment of biological rhythms [24].

First, photoperiodic signals are perceived by ipRGCs that express the photopigment melanopsin [25]. 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

**45**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

**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 [36]. 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 [37]. In horses, 1 h exposure of 3 lux SWL blue light (468 nm) administered only to one eye was sufficient to decrease melatonin levels

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 [39–41]. While the eyelids can weaken irradiance and wavelength ([42], light signals can still penetrate them, be detected by the retinal photoreceptors, and affect circadian regulation [43]. 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

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 [49] passed a resolution in 2016 calling upon

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 [26]. In mammals, the activity of the adrenergic SCG terminals that innervate the pineal gland is stimulated by darkness and inhibited by light [27]. 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 [28]. 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 [29]. 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

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

levels during the day [31].

compared with control animals [38].

compared with intensity manipulations [46].

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

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 [26]. In mammals, the activity of the adrenergic SCG terminals that innervate the pineal gland is stimulated by darkness and inhibited by light [27]. 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 [28]. 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 [29]. 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 [31].

### **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 [36]. 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 [37]. 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 [38].

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 [39–41]. While the eyelids can weaken irradiance and wavelength ([42], light signals can still penetrate them, be detected by the retinal photoreceptors, and affect circadian regulation [43]. 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 [46].

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 [49] passed a resolution in 2016 calling upon

*Epigenetics*

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 [1–3]. Light pollution is increasing rapidly, resulting in a more illuminated world, where outdoor and indoor illumination

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 [6–8]. Certainly, multiple studies have shown the effects of light pollution on social, behavioral, physiological, and molecular responses in many different taxa, including insects [9], fishes [10], amphibians [11], reptiles [12], birds [13], and mammals [14], as well as plants [15]. 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 [17–19]. 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 (*Psammomys obesus*) are discussed in relation to their potential role in mediating the environmental exposures at the molecular level *via* epigenetic modifications, particularly global

In an early study, it has been demonstrated that the blind mole rat (*Spalax ehrenbergi*) responded differently to short and long photoperiod manipulations in regard to its capability to cope with low ambient temperature exposure [20]. Results of a more recent study on *S. ehrenbergi* manifested robust and differential responses in metabolism, stress, and melatonin levels to ALAN of different spectral compositions and acclimation duration [21]. These results suggested that the vestigial retina of this species still expresses photoreceptors that are involved mainly in nonvisual response. Currently, the mammalian eye is described as a dual-function organ, expressing photoreceptors for both visual and nonvisual responses [22]. The visual response is mediated by two distinct photoreceptor types, rods and cones, which control scotopic vision and photopic vision, respectively [23]. The nonvisual responses are mainly mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the photopigment melanopsin. Even though the ipRGCs are connected with rods and cones by bipolar cells, they mediate nonvisual responses

First, photoperiodic signals are perceived by ipRGCs that express the photopigment melanopsin [25]. 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

sources are increasing ALAN in developed and developing countries [4, 5].

**44**

DNA methylation (GDM).

**2. The mammalian photoperiodic system**

including photo-entrainment of biological rhythms [24].

communities in the USA to avoid using LED lighting in public domains as it is enriched with SWL [49]. 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 [50]. 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-pituitarythyroid axis (HPT), and the hypothalamic-pituitary-adrenal (HPA) axis [51]. The elaborated hormonal responses generated by these axes to ALAN exposure might be mediated by transcriptional regulation of gene expression *via* epigenetic modifications [52]. Therefore, epigenetic-elicited alteration in gene expression is a potential transduction pathway by which hormonal responses (e.g., melatonin) may mediate environmental exposures (e.g., ALAN). Conversely, the ALANinduced alteration in melatonin rhythms may also exert endocrine responses *via* epigenetic modifications [53].

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 [59–62]. 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 [19].

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 [67]. 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

**47**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

DAN hypomethylation and tumorigenesis, particularly of breast cancer, is wellestablished, but the underlying mechanism remains poorly understood, especially

**5. Melatonin as a mediating signal linking ALAN and epigenetic-induced** 

Since the melatonin hypothesis was first proposed during the late twentieth century by Stevens [72], 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 [73]. Indeed, low levels of 6-sulfatoxymelatonin (6-SMT), the major metabolites of the hormone in urine [74], have been demonstrated to correlate with increased risk of breast cancer in postmenopausal women [75–77]. Furthermore, women with blindness or long sleep duration (elevated melatonin levels)

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 *via* melatonin receptor [80]. In hepatocellular carcinoma-induced mice, melatonin treatment suppressed tumor cell proliferation through arresting the cell cycle [81]. The metastatic activity of oral squamous cell carcinoma was notably reduced by melatonin-mediated inhibition of tumor-associated neutrophils [82], inflammatory cells involved in promoting several solid tumors [83]. Similarly, the anti-oncogenic property of melatonin has been demonstrated also in other cancer types, including lung [84],

gastric [85], ovarian [86], and colon [87], as well as breast cancers [88].

Melatonin could mediate its effects of cancer development *via* epigenetic modifications, particularly GDM [89]. Melatonin treatment to MCF-7 cell lines significantly increased DNA methylation that was associated with increased transcriptional levels of the tumor metastasis suppressor gene glypican-3 and decreased expression levels of the oncogenes EGR3 and POU4F2/Brn-3b [90]. In estrogenreceptor-related breast cancer, melatonin may decrease transcriptional levels of the aromatase gene (involved in the regulation of estrogen synthesis) by either methylation of the gene or deacetylation of the promoter gene [91]. Additionally, nocturnal melatonin treatment can rectify the induced DNA demethylation, tumor growth, and metastatic activity by both blue LED and fluorescent ALAN in 4T1 mammary cancer cell-inoculated female BALB/c mice [63, 64]. In a more recent study that evaluated the effects of ALAN and melatonin treatment at different spectral compositions in 4T1-inoculated BALB/c mice, a tissue-specific response in GDM was detected [19]. In this study, the tumor tissue manifested the most prominent changes in GDM showing an inverse wavelength-dependent correlation that was reversed by melatonin. Conversely, other tissues (e.g., lung, liver, and spleen) showed mixed results of positive, negative, or indifferent correlation between methylation levels and both wavelength and melatonin treatments [19]. Largely, melatonin may regulate epigenetic modifications in a number of tumor-related genes mainly by DNA methylation, but other modifications are also possible. 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

present reduced breast cancer risk relative to normal women [78, 79].

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

how the adverse ALAN effects are mediated.

**cancer**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

DAN hypomethylation and tumorigenesis, particularly of breast cancer, is wellestablished, 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 [72], 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 [73]. Indeed, low levels of 6-sulfatoxymelatonin (6-SMT), the major metabolites of the hormone in urine [74], have been demonstrated to correlate with increased risk of breast cancer in postmenopausal women [75–77]. 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 *via* melatonin receptor [80]. In hepatocellular carcinoma-induced mice, melatonin treatment suppressed tumor cell proliferation through arresting the cell cycle [81]. The metastatic activity of oral squamous cell carcinoma was notably reduced by melatonin-mediated inhibition of tumor-associated neutrophils [82], inflammatory cells involved in promoting several solid tumors [83]. Similarly, the anti-oncogenic property of melatonin has been demonstrated also in other cancer types, including lung [84], gastric [85], ovarian [86], and colon [87], as well as breast cancers [88].

Melatonin could mediate its effects of cancer development *via* epigenetic modifications, particularly GDM [89]. Melatonin treatment to MCF-7 cell lines significantly increased DNA methylation that was associated with increased transcriptional levels of the tumor metastasis suppressor gene glypican-3 and decreased expression levels of the oncogenes EGR3 and POU4F2/Brn-3b [90]. In estrogenreceptor-related breast cancer, melatonin may decrease transcriptional levels of the aromatase gene (involved in the regulation of estrogen synthesis) by either methylation of the gene or deacetylation of the promoter gene [91]. Additionally, nocturnal melatonin treatment can rectify the induced DNA demethylation, tumor growth, and metastatic activity by both blue LED and fluorescent ALAN in 4T1 mammary cancer cell-inoculated female BALB/c mice [63, 64]. In a more recent study that evaluated the effects of ALAN and melatonin treatment at different spectral compositions in 4T1-inoculated BALB/c mice, a tissue-specific response in GDM was detected [19]. In this study, the tumor tissue manifested the most prominent changes in GDM showing an inverse wavelength-dependent correlation that was reversed by melatonin. Conversely, other tissues (e.g., lung, liver, and spleen) showed mixed results of positive, negative, or indifferent correlation between methylation levels and both wavelength and melatonin treatments [19]. Largely, melatonin may regulate epigenetic modifications in a number of tumor-related genes mainly by DNA methylation, but other modifications are also possible.

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

*Epigenetics*

communities in the USA to avoid using LED lighting in public domains as it is enriched with SWL [49]. In summary, SWL-ALAN is a source of pollution and

**4. ALAN as an environmental change and a model for studying** 

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 [50]. 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-pituitarythyroid axis (HPT), and the hypothalamic-pituitary-adrenal (HPA) axis [51]. The elaborated hormonal responses generated by these axes to ALAN exposure might be mediated by transcriptional regulation of gene expression *via* epigenetic modifications [52]. Therefore, epigenetic-elicited alteration in gene expression is a potential transduction pathway by which hormonal responses (e.g., melatonin) may mediate environmental exposures (e.g., ALAN). Conversely, the ALANinduced alteration in melatonin rhythms may also exert endocrine responses *via*

The incidences of breast and prostate cancers show close association with light

In rats, ALAN exposure accelerated the growth rates of induced-tumors, including mammary cancer [59–62]. 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

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 [67]. 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

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

should be removed from public spaces through legislation.

**epigenetic modifications**

epigenetic modifications [53].

than 500 nm [19].

cancer development comes also from animal studies.

**46**

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 [92], early treatment by melatonin or any other analogs [93] 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 [94]. 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 (*Psammomys obesus*). This species is a good model because it is a photoperiodic species that responds to photoperiod with robust daily rhythms in a number of physiological functions, including body temperature, melatonin levels, and AA-NAT activity [95, 96]. Furthermore, *P. obesus* is a useful model for studying human health and diseases such as metabolic disorders, obesity, diabetes, inflammation, and cardiovascular impairment [97–100]. Since most previous studies on photoperiodic responses were conducted on nocturnal species, in our research center at the University of Haifa, we use *P. obesus* as a model for studying photoperiodic and hormonal manipulations. In *P. obesus*, melatonin and body temperature rhythms were diminished in response to constant dim blue light exposure, while melatonin treatment restored the disrupted rhythms [63]. Although the previous studies have clearly indicated that as a diurnal species, *P. obesus* can respond to photoperiod and light manipulations, the underling mechanism mediating the effect of the environmental changes remains unknown. An unanswered question is how melatonin and thyroxin interact to mediated environmental-induced epigenetic modifications. To answer this question, male *P. obesus* were acclimated to a long photoperiod cycle of 16L:8D at an ambient temperature of 24 ± 1°C and humidity of 45 ± 2%. Lights during the day were emitted from cool fluorescent lamps at 470 lux and 470 nm. Rats were caged individually and provided with ad libitum tap water and low energy diet. At the end of 3-week acclimation period, rats were either untreated, *i.p.* injected

**49**

cal significance.

the other hormone.

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

with melatonin, thyroxin, or melatonin and thyroxin in combination 3 h after the dark period onset (01:00 h). Hormones were daily administered for 3 weeks at a dose of 50 μg/kg for melatonin and 2 mg/kg for thyroxin. During the experimental period, body mass (Wb) was monitored every other day and urine samples were collected by a noninvasive method [19] at 4 h intervals over a 28 h period. Urine samples were used to measure the major metabolite of melatonin in urine, 6-SMT [101]. The urinary metabolite concentrations were assayed by enzymelinked immunosorbent assay utilizing a commercial IBL kit (RE54031) following the manufacture's protocol. Finally, digit tips were collected from rats at the end of urine collection for DNA isolation (High pure PCR Template Preparation Kit, Roche) and subsequently for GDM analysis (MethylFlash™ Methylated DNA Quantification Kit, Epigentek). All experimental procedures were performed with the approval from the Ethics and Animal Care Committee of the University

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

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

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 statisti-

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 *P. obesus*. The results suggest also that change in body temperature is more sensitive to thyroxin treatment than melatonin, as the effect of the latter was masked in the combined treatment with

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

higher body temperature values and loss in Wb [102].

exert a significant antagonistic effect on body temperature.

of Haifa.

#### *Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

with melatonin, thyroxin, or melatonin and thyroxin in combination 3 h after the dark period onset (01:00 h). Hormones were daily administered for 3 weeks at a dose of 50 μg/kg for melatonin and 2 mg/kg for thyroxin. During the experimental period, body mass (Wb) was monitored every other day and urine samples were collected by a noninvasive method [19] at 4 h intervals over a 28 h period. Urine samples were used to measure the major metabolite of melatonin in urine, 6-SMT [101]. The urinary metabolite concentrations were assayed by enzymelinked immunosorbent assay utilizing a commercial IBL kit (RE54031) following the manufacture's protocol. Finally, digit tips were collected from rats at the end of urine collection for DNA isolation (High pure PCR Template Preparation Kit, Roche) and subsequently for GDM analysis (MethylFlash™ Methylated DNA Quantification Kit, Epigentek). All experimental procedures were performed with the approval from the Ethics and Animal Care Committee of the University of Haifa.

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 [102].

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 *P. obesus*. The results suggest also that change in body temperature is more sensitive to thyroxin treatment than melatonin, as the effect of the latter was masked in the combined treatment with the other hormone.

*Epigenetics*

physiological, and molecular levels.

**diurnal species**

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 [92], early treatment by melatonin or any other analogs [93] 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 [94]. 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,

**6. Physiological and epigenetic responses to melatonin and thyroxin in** 

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 (*Psammomys obesus*). This species is a good model because it is a photoperiodic species that responds to photoperiod with robust daily rhythms in a number of physiological functions, including body temperature, melatonin levels, and AA-NAT activity [95, 96]. Furthermore, *P. obesus* is a useful model for studying human health and diseases such as metabolic disorders, obesity, diabetes, inflammation, and cardiovascular impairment [97–100]. Since most previous studies on photoperiodic responses were conducted on nocturnal species, in our research center at the University of Haifa, we use *P. obesus* as a model for studying photoperiodic and hormonal manipulations. In *P. obesus*, melatonin and body temperature rhythms were diminished in response to constant dim blue light exposure, while melatonin treatment restored the disrupted rhythms [63]. Although the previous studies have clearly indicated that as a diurnal species, *P. obesus* can respond to photoperiod and light manipulations, the underling mechanism mediating the effect of the environmental changes remains unknown. An unanswered question is how melatonin and thyroxin interact to mediated environmental-induced epigenetic modifications. To answer this question, male *P. obesus* were acclimated to a long photoperiod cycle of 16L:8D at an ambient temperature of 24 ± 1°C and humidity of 45 ± 2%. Lights during the day were emitted from cool fluorescent lamps at 470 lux and 470 nm. Rats were caged individually and provided with ad libitum tap water and low energy diet. At the end of 3-week acclimation period, rats were either untreated, *i.p.* injected

**48**

#### **Figure 1.**

*Percentage change in body mass (A) and body temperature (B) in long-day acclimated P. obesus under four conditions: control no treatments, thyroxin (T4) treatment, melatonin (MLT) treatment, and combined treatment with T4 + MLT. Data are presented as mean ± standard error of nine animals. Different letters represent statistically significant difference among groups (Bonferroni, P < 0.01). # vs. day 21 (Bonferroni, P < 0.02).*

However, in humans, melatonin may interact with the HPT axis to modulate the circadian rhythm of body temperature [104]. 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 [105]. 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 [108]. 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 [109]. Furthermore, the thyroid hormones are crucial photoperiodic regulators of several physiological

**51**

**Figure 2.**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

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 [112]. Continuous exposure to ALAN decreased

*Daily rhythms of urinary 6-sulfatoxymelatonin (A) and global DNA methylation (B) levels in long-day acclimated P. obesus under four conditions: control no treatments, thyroxin (T4) treatment, melatonin (MLT) treatment, and combined treatment with T4 + MLT. In panel A, the best-fitted cosine curve (black and gray lines) and Cosinor estimates (period, P-value, and percentage of the rhythm [PR]) are depicted [103]. The gray area in each plot represents the length of the dark period. Data are presented as mean ± standard error of seven to nine animals. Different letters represent statistically significant difference among groups (Bonferroni, P < 0.01).*

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

#### **Figure 2.**

*Epigenetics*

**50**

**Figure 1.**

However, in humans, melatonin may interact with the HPT axis to modulate the circadian rhythm of body temperature [104]. 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 [105]. 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 [108]. 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 [109]. Furthermore, the thyroid hormones are crucial photoperiodic regulators of several physiological

*Percentage change in body mass (A) and body temperature (B) in long-day acclimated P. obesus under four conditions: control no treatments, thyroxin (T4) treatment, melatonin (MLT) treatment, and combined treatment with T4 + MLT. Data are presented as mean ± standard error of nine animals. Different letters represent statistically significant difference among groups (Bonferroni, P < 0.01). # vs. day 21 (Bonferroni, P < 0.02).*

*Daily rhythms of urinary 6-sulfatoxymelatonin (A) and global DNA methylation (B) levels in long-day acclimated P. obesus under four conditions: control no treatments, thyroxin (T4) treatment, melatonin (MLT) treatment, and combined treatment with T4 + MLT. In panel A, the best-fitted cosine curve (black and gray lines) and Cosinor estimates (period, P-value, and percentage of the rhythm [PR]) are depicted [103]. The gray area in each plot represents the length of the dark period. Data are presented as mean ± standard error of seven to nine animals. Different letters represent statistically significant difference among groups (Bonferroni, P < 0.01).*

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 [112]. Continuous exposure to ALAN decreased

TSH, but increased both T3 and T4 in mice [113]. In birds, long-term exposure to ALAN increased both the blood levels of the thyroid hormones and Wb [114]. 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 *via* melatonin, mediate the disruption effects of ALAN on the circadian system and promote downstream health risk is still unclear, and further efforts are warranted for elucidating it.

## **7. Conclusions**

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 *via* nocturnal melatonin suppression. A schematic of ALAN-induced adverse effects is presented in **Figure 3**. Accordingly, light signals including ALAN are detected by ipRGCs and conveyed to the SCN by RHT. During a normal light dark cycle, melatonin is synthesized and secreted to the blood during the night, where it entrains central and peripheral oscillators to regulate normal physiological responses. Conversely, ALAN suppresses melatonin levels causing chronodisruption and misalignment in central and peripheral oscillators resulting in impaired physiological responses. The central and peripheral oscillators can be regulated directly by the melatonin signal or indirectly by modifying the body temperature rhythms [115]. In mice, daily variations in body temperature rhythms have been demonstrated to synchronize circadian gene expressions [116] and these central-controlled variations can be utilized to regulate variant peripheral circadian clocks in mammals [117]. Consequently, in diurnal species, thyroxin as an endocrine pathway is presumably involved in center circadian regulation of peripheral clocks by modifying body temperature daily rhythms.

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 [89]. 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 [118] 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. *P. obesus* may be very useful as a diurnal animal model for understanding the physiological and molecular effects of light pollution on public health. Melatonin suppression, GDM, and even thyroxin levels may present a significant clinical importance as a biomarker for early detection of cancer, particularly in individuals who are at increased risk of developing cancer by circadian disruption induced by excessive ALAN exposures. As epigenetic modifications are revisable, these biomarkers retain therapeutic value

**53**

**Figure 3.**

*by inducing aberrant epigenetic modifications.*

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

for ALAN-induced cancer by gene demethylation. Finally, the accumulating data regarding the adverse effects of light pollution on ecology and heath compel us to take drastic and rapid measures to reduce light pollution by extreme regulation or at

*Schematic representation of the mechanism of ALAN in eliciting adverse health effects. Light signal, including short wavelength ALAN (SWL-ALAN), is detected by intrinsically photosensitive retinal ganglion cells (ipRGCs) that propagate it to the SCN via the retinohypothalamic tract (RHT). Thereafter, the signal is transmitted to the pineal gland (PG) via superior cervical ganglion (SCG). Finally, melatonin is synthesized and secreted to circulation by the PG during the night, where it synchronizes peripheral clocks with the ambient photoperiod. Generally, ALAN suppresses nocturnal melatonin, in which adverse health impacts are generated* 

least reducing SWL emission by developing safe lightning technology.

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

#### **Figure 3.**

*Epigenetics*

**7. Conclusions**

TSH, but increased both T3 and T4 in mice [113]. In birds, long-term exposure to ALAN increased both the blood levels of the thyroid hormones and Wb [114]. 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 *via* melatonin, mediate the disruption effects of ALAN on the circadian system and promote downstream health risk is still unclear,

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 *via* nocturnal melatonin suppression. A schematic of ALAN-induced adverse effects is presented in **Figure 3**. Accordingly, light signals including ALAN are detected by ipRGCs and conveyed to the SCN by RHT. During a normal light dark cycle, melatonin is synthesized and secreted to the blood during the night, where it entrains central and peripheral oscillators to regulate normal physiological responses. Conversely, ALAN suppresses melatonin levels causing chronodisruption and misalignment in central and peripheral oscillators resulting in impaired physiological responses. The central and peripheral oscillators can be regulated directly by the melatonin signal or indirectly by modifying the body temperature rhythms [115]. In mice, daily variations in body temperature rhythms have been demonstrated to synchronize circadian gene expressions [116] and these central-controlled variations can be utilized to regulate variant peripheral circadian clocks in mammals [117]. Consequently, in diurnal species, thyroxin as an endocrine pathway is presumably involved in center circadian regulation of peripheral clocks by modifying body temperature daily rhythms. 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 [89]. 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 [118] 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. *P. obesus* may be very useful as a diurnal animal model for understanding the physiological and molecular effects of light pollution on public health. Melatonin suppression, GDM, and even thyroxin levels may present a significant clinical importance as a biomarker for early detection of cancer, particularly in individuals who are at increased risk of developing cancer by circadian disruption induced by excessive ALAN exposures. As epigenetic modifications are revisable, these biomarkers retain therapeutic value

and further efforts are warranted for elucidating it.

**52**

*Schematic representation of the mechanism of ALAN in eliciting adverse health effects. Light signal, including short wavelength ALAN (SWL-ALAN), is detected by intrinsically photosensitive retinal ganglion cells (ipRGCs) that propagate it to the SCN via the retinohypothalamic tract (RHT). Thereafter, the signal is transmitted to the pineal gland (PG) via superior cervical ganglion (SCG). Finally, melatonin is synthesized and secreted to circulation by the PG during the night, where it synchronizes peripheral clocks with the ambient photoperiod. Generally, ALAN suppresses nocturnal melatonin, in which adverse health impacts are generated by inducing aberrant epigenetic modifications.*

for ALAN-induced cancer by gene demethylation. Finally, the accumulating data regarding the adverse effects of light pollution on ecology and heath compel us to take drastic and rapid measures to reduce light pollution by extreme regulation or at least reducing SWL emission by developing safe lightning technology.

## **Acknowledgements**

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.

## **Dedication**

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.

## **Abbreviations**


## **Author details**

Abraham Haim, Sinam Boynao and Abed Elsalam Zubidat\* The Israeli Center for Interdisciplinary Research in Chronobiology, University of Haifa, Haifa, Israel

\*Address all correspondence to: zubidat3@013.net.il

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**55**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

disruptor. Physiology and Behavior. 2018;**190**:82-89. DOI: 10.1016/j.

[9] Owens ACS, Lewis SM. The impact of artificial light at night on nocturnal insects: A review and synthesis. Ecology and Evolution. 2018;**8**(22):11337-11358.

[10] Pulgar J, Zeballos D, Vargas J, Aldana M, Manriquez PH, Manriquez K,

et al. Endogenous cycles, activity patterns and energy expenditure of an intertidal fish is modified by artificial light pollution at night (ALAN). Environmental

[11] Gastón MS, Pereyra LC, Vaira M. Artificial light at night and captivity induces differential effects on leukocyte profile, body condition, and erythrocyte size of a diurnal toad. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology. 2019;**331**(2):93-102. DOI: 10.1002/

[12] Hu Z, Hu H, Huang Y. Association between nighttime artificial light pollution and sea turtle nest density along Florida coast: A geospatial study using VIIRS remote sensing data.

Environmental Pollution. 2018;**239**:30-42. DOI: 10.1016/j.envpol.2018.04.021

Bellingham M, O'Shaughnessy P, van Oers K, Robinson J, et al. Dose-response effects of light at night on the reproductive physiology of great tits (Parus major): Integrating morphological analyses with candidate gene expression. Journal of Experimental Zoology Part A: Ecological

[13] Dominoni DM, de Jong M,

and Integrative Physiology. 2018; **329**(8-9):473-487. DOI: 10.1002/jez.2214

[14] Hoffmann J, Palme R, Eccard JA. Long-term dim light during nighttime changes activity patterns and space

Pollution. 2019;**244**:361-366. DOI: 10.1016/j.envpol.2018.10.063

jez.2240

physbeh.2017.08.029

DOI: 10.1002/ece3.4557

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

[1] Haim A, Portnov BA. LAN and breast cancer risk: Can we see a forest through the trees?—Response to "measurements of light at night (LAN) for a sample of female school teachers" by M. S. Rea, J. A. Brons, and M. G. Figueiro. Chronobiology International. 2011;**28**(8):734-736. DOI:

[2] Zubidat AE, Haim A. Artificial lightat-night—A novel lifestyle risk factor for metabolic disorder and cancer morbidity. Journal of Basic and Clinical Physiology and Pharmacology. 2017;**28**(4):295-313.

[3] Haim A, Scantlebury DM, Zubidat AE.

emerging from artificial light at night. Chronobiology International. 2018;**19**:1- 3. DOI: 10.1080/07420528.2018.1534122

[4] Cinzano P, Falchi F, Elvidge CD. The first world atlas of the artificial night sky brightness. Monthly Notices of the Royal Astronomical Society. 2001;**328**(3):689-707. DOI: 10.1046/j.1365-8711.2001.04882.x

[5] Hölker F, Moss T, Griefahn B, Kloas W, Voigt CC, Henckel D, et al. The dark side of light: A transdisciplinary research agenda for light pollution policy. Ecology and Society. 2010;**15**:13.

ecologyandsociety.org/vol15/iss4/art13/

[7] Dominoni DM, Borniger JC, Nelson RJ. Light at night, clocks and health: From humans to wild organisms. Biological Letters. 2016;**12**(2):20160015. DOI:

[8] Russart KLG, Nelson RJ. Light at night as an environmental endocrine

[6] Navara KJ, Nelson RJ. The dark side of light at night: Physiological, epidemiological, and ecological consequences. Journal of Pineal Research. 2007;**43**(3):215-224

Available from: http://www.

10.1098/rsbl.2016.0015

10.3109/07420528.2011.604591

**References**

DOI: 10.1515/jbcpp-2016-0116

The loss of ecosystem-services

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

## **References**

*Epigenetics*

**Acknowledgements**

the authors of this chapter.

Wb body mass

ALAN artificial light at night

GDM global DNA methylation

LED light-emitting diodes RHT retinohypothalamic tract SWLs short wavelengths

SCG superior cervical ganglion TSH thyroid-stimulating hormone

AA-NAT aryl-alkyl-amine-N-acetyltransferase

HIOMT hydroxyindole-O-methyltransferase SCN hypothalamic suprachiasmatic nucleus HPA hypothalamic-pituitary-adrenal HPG hypothalamic-pituitary-gonadal HPT hypothalamic-pituitary-thyroid axis

ipRGCs intrinsically photosensitive retinal ganglion cells

Abraham Haim, Sinam Boynao and Abed Elsalam Zubidat\*

\*Address all correspondence to: zubidat3@013.net.il

publication fee.

**Dedication**

**Abbreviations**

**Author details**

Haifa, Haifa, Israel

**54**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The Israeli Center for Interdisciplinary Research in Chronobiology, University of

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

This chapter is dedicated to the memory of Professor Abraham Haim, who passed away before publication of this work. His contribution was foremost among [1] Haim A, Portnov BA. LAN and breast cancer risk: Can we see a forest through the trees?—Response to "measurements of light at night (LAN) for a sample of female school teachers" by M. S. Rea, J. A. Brons, and M. G. Figueiro. Chronobiology International. 2011;**28**(8):734-736. DOI: 10.3109/07420528.2011.604591

[2] Zubidat AE, Haim A. Artificial lightat-night—A novel lifestyle risk factor for metabolic disorder and cancer morbidity. Journal of Basic and Clinical Physiology and Pharmacology. 2017;**28**(4):295-313. DOI: 10.1515/jbcpp-2016-0116

[3] Haim A, Scantlebury DM, Zubidat AE. The loss of ecosystem-services emerging from artificial light at night. Chronobiology International. 2018;**19**:1- 3. DOI: 10.1080/07420528.2018.1534122

[4] Cinzano P, Falchi F, Elvidge CD. The first world atlas of the artificial night sky brightness. Monthly Notices of the Royal Astronomical Society. 2001;**328**(3):689-707. DOI: 10.1046/j.1365-8711.2001.04882.x

[5] Hölker F, Moss T, Griefahn B, Kloas W, Voigt CC, Henckel D, et al. The dark side of light: A transdisciplinary research agenda for light pollution policy. Ecology and Society. 2010;**15**:13. Available from: http://www. ecologyandsociety.org/vol15/iss4/art13/

[6] Navara KJ, Nelson RJ. The dark side of light at night: Physiological, epidemiological, and ecological consequences. Journal of Pineal Research. 2007;**43**(3):215-224

[7] Dominoni DM, Borniger JC, Nelson RJ. Light at night, clocks and health: From humans to wild organisms. Biological Letters. 2016;**12**(2):20160015. DOI: 10.1098/rsbl.2016.0015

[8] Russart KLG, Nelson RJ. Light at night as an environmental endocrine disruptor. Physiology and Behavior. 2018;**190**:82-89. DOI: 10.1016/j. physbeh.2017.08.029

[9] Owens ACS, Lewis SM. The impact of artificial light at night on nocturnal insects: A review and synthesis. Ecology and Evolution. 2018;**8**(22):11337-11358. DOI: 10.1002/ece3.4557

[10] Pulgar J, Zeballos D, Vargas J, Aldana M, Manriquez PH, Manriquez K, et al. Endogenous cycles, activity patterns and energy expenditure of an intertidal fish is modified by artificial light pollution at night (ALAN). Environmental Pollution. 2019;**244**:361-366. DOI: 10.1016/j.envpol.2018.10.063

[11] Gastón MS, Pereyra LC, Vaira M. Artificial light at night and captivity induces differential effects on leukocyte profile, body condition, and erythrocyte size of a diurnal toad. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology. 2019;**331**(2):93-102. DOI: 10.1002/ jez.2240

[12] Hu Z, Hu H, Huang Y. Association between nighttime artificial light pollution and sea turtle nest density along Florida coast: A geospatial study using VIIRS remote sensing data. Environmental Pollution. 2018;**239**:30-42. DOI: 10.1016/j.envpol.2018.04.021

[13] Dominoni DM, de Jong M, Bellingham M, O'Shaughnessy P, van Oers K, Robinson J, et al. Dose-response effects of light at night on the reproductive physiology of great tits (Parus major): Integrating morphological analyses with candidate gene expression. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology. 2018; **329**(8-9):473-487. DOI: 10.1002/jez.2214

[14] Hoffmann J, Palme R, Eccard JA. Long-term dim light during nighttime changes activity patterns and space

use in experimental small mammal populations. Environmental Pollution. 2018l;**238**:844-851. DOI: 10.1016/j. envpol.2018.03.107

[15] Solano-Lamphar HA, Kocifaj M. Numerical research on the effects the skyglow could have in phytochromes and RQE photoreceptors of plants. Journal of Environmental Management. 2018;**209**:484-494. DOI: 10.1016/j. jenvman.2017.12.036

[16] Nelson RJ, Chbeir S. Dark matters: Effects of light at night on metabolism. Proceedings of the Nutrition Society. 2018;**77**(30):223-229. DOI: 10.1017/ S0029665118000198

[17] Abay KA, Amare M. Night light intensity and women's body weight: Evidence from Nigeria. Economics and Human Biology. 2018;**31**:238-248. DOI: 10.1016/j.ehb.2018.09.001

[18] Garcia-Saenz A, Sánchez de Miguel A, Espinosa A, Valentin A, Aragonés N, Llorca J, et al. Evaluating the association between artificial light-at-night exposure and breast and prostate cancer risk in Spain (MCC-Spain Study). Environmental Health Perspectives. 2018;**126**:047011. DOI: 10.1289/ EHP1837

[19] Zubidat AE, Fares B, Fares F, Haim A. Artificial light at night of different spectral compositions differentially affects tumor growth in mice: Interaction with melatonin and epigenetic pathways. Cancer Control. 2018;**25**(1):1073274818812908. DOI: 10.1177/1073274818812908

[20] Haim A, Heth G, Pratt H, Nevo E. Photoperiodic effects on thermoregulation in a 'blind' subterranean mammal. Journal Experimental Biology. 1983; **107**:59-64

[21] Zubidat AE, Nelson RJ, Haim A. Spectral and duration sensitivity to

light-at-night in 'blind' and sighted rodent species. Journal of Experimental Biology. 2011;**214**(Pt 19):3206-3217. DOI: 10.1242/jeb.058883

[22] Matynia A. Blurring the boundaries of vision: Novel functions of intrinsically photosensitive retinal ganglion cells. Journal of Experimental Neuroscience. 2013;**7**:43-50. DOI: 10.4137/JEN.S11267

[23] Collin SP, Davies WL, Hart NS, Hunt DM. The evolution of early vertebrate photoreceptors. Philosophical Transactions of The Royal Society B Biological Sciences. 2009;**364**(1531):2925-2940. DOI: 10.1098/rstb.2009.0099

[24] Detwiler PB. Phototransduction in retinal ganglion cells. Yale Journal of Biology and Medicine. 2018;**91**(1):49-52

[25] Hannibal J, Christiansen AT, Heegaard S, Fahrenkrug J, Kiilgaard JF. Melanopsin expressing human retinal ganglion cells: Subtypes, distribution, and intraretinal connectivity. Journal of Comparative Neurology. 2017;**525**(8):1934-1961. DOI: 10.1002/ cne.24181

[26] Moore RY. Neural control of the pineal gland. Behavioural Brain Research. 1996;**73**(1-2):125-130

[27] Perreau-Lenz S, Kalsbeek A, Van Der Vliet J, Pévet P, Buijs RM. In vivo evidence for a controlled offset of melatonin synthesis at dawn by the suprachiasmatic nucleus in the rat. Neuroscience. 2005;**130**(3):797-803

[28] Klein DC. Arylalkylamine N-acetyltransferase: "the Timezyme". Journal of Biological Chemistry. 2007;**282**(7):4233-4237

[29] Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA, et al. The melatonin rhythm-generating enzyme: Molecular regulation of

**57**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

suppression, photosynthesis, and star visibility. PLoS One. 2013;**8**:e67798. DOI: 10.1371/journal.pone.0067798

[37] West KE, Jablonski MR, Warfield B,

Cecil KS, James M, Ayers MA, et al. Blue light from light-emitting diodes elicits a dose-dependent suppression of melatonin in humans. Journal of Applied Physiology. 2011;**110**(3):619-626. DOI: 10.1152/

japplphysiol.01413.2009

10.1016/j.tvjl.2012.09.003

NSS.S52203

10.2147/NSS.S73856

**39**(1):22-25

[41] Haim A, Portnov BA. Light Pollution as a New Risk Factor for Human Breast and Prostate Cancers. Dordecht: Springer Science + Buisness Media; 2013. DOI: 10.1007/978-94-007-6220-6\_1

[42] Ando K, Kripke DF. Light attenuation by the human eyelid. Biological Psychiatry. 1996;

[43] Bierman A, Figueiro MG, Rea MS. Measuring and predicting eyelid spectral transmittance. Journal of Biomedical Optics. 2011;**16**:067011.

DOI: 10.1117/1.3593151

[38] Walsh CM, Prendergast RL,

Sheridan JT, Murphy BA. Blue light from light-emitting diodes directed at a single eye elicits a dose-dependent suppression of melatonin in horses. The Veterinary Journal. 2013;**196**(2):231-235. DOI:

[39] Figueiro MG, Bierman A, Rea MS. A train of blue light pulses delivered through closed eyelids suppresses melatonin and phase shifts the human circadian system. Nature and Science of Sleep. 2013;**5**:133-141. DOI: 10.2147/

[40] Figueiro MG, Plitnick B, Rea MS. Pulsing blue light through closed eyelids: Effects on acute melatonin suppression and phase shifting of dim light melatonin onset. Nature and Science of Sleep. 2014;**6**:149-156. DOI:

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

[31] Ribelayga C, Garidou ML, Malan A, Gauer F, Calgari C, Pévet P, et al. Photoperiodic control of the rat pineal arylalkylamine-N-acetyltransferase and hydroxyindole-O-methyltransferase gene expression and its effect on melatonin synthesis. Journal of

Biological Rhythms. 1999;**14**(2):105-115

[32] Thapan K, Arendt J, Skene DJ. An action spectrum for melatonin

suppression: Evidence for a novel nonrod, non-cone photoreceptor system in humans. Journal of Physiology.

[33] Cajochen C, Münch M, Kobialka S, Kräuchi K, Steiner R, Oelhafen P, et al. High sensitivity of human melatonin, alertness, thermoregulation, and heart rate to short wavelength light. Journal of Clinical Endocrinology and Metabolism.

[34] Brainard GC, Lewy AJ, Menaker M, Fredrickson RH, Miller LS, Weleber RG,

et al. Dose-response relationship between light irradiance and the suppression of plasma melatonin in human volunteers. Brain Research.

[35] Falchi F, Cinzano P, Elvidge CD, Keith DM, Haim A. Limiting the impact of light pollution on human health, environment and stellar visibility. Journal of Environmental Management. 2011;**92**(10):2714-2722. DOI: 10.1016/j.

2001;**535**(Pt 1):261-267

2005;**90**(3):1311-1316

1988;**454**(1-2):212-218

jenvman.2011.06.029

[36] Aubé M, Roby J, Kocifaj M. Evaluating potential spectral impacts of various artificial lights on melatonin

serotonin N-acetyltransferase in the pineal gland. Recent Progress in Hormone Research. 1997;**52**:307-357

[30] Maronde E, Stehle JH. The mammalian pineal gland: Known facts, unknown facets. Trends in Endocrinology and Metabolism.

2007;**18**(11):142-149

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

serotonin N-acetyltransferase in the pineal gland. Recent Progress in Hormone Research. 1997;**52**:307-357

*Epigenetics*

envpol.2018.03.107

jenvman.2017.12.036

S0029665118000198

use in experimental small mammal populations. Environmental Pollution. 2018l;**238**:844-851. DOI: 10.1016/j.

light-at-night in 'blind' and sighted rodent species. Journal of Experimental Biology. 2011;**214**(Pt 19):3206-3217.

boundaries of vision: Novel functions of intrinsically photosensitive retinal ganglion cells. Journal of Experimental

[23] Collin SP, Davies WL, Hart NS,

[24] Detwiler PB. Phototransduction in retinal ganglion cells. Yale Journal of Biology and Medicine. 2018;**91**(1):49-52

[25] Hannibal J, Christiansen AT, Heegaard S, Fahrenkrug J, Kiilgaard JF. Melanopsin expressing human retinal ganglion cells: Subtypes, distribution,

and intraretinal connectivity. Journal of Comparative Neurology. 2017;**525**(8):1934-1961. DOI: 10.1002/

[26] Moore RY. Neural control of the pineal gland. Behavioural Brain Research. 1996;**73**(1-2):125-130

[27] Perreau-Lenz S, Kalsbeek A, Van Der Vliet J, Pévet P, Buijs RM. In vivo evidence for a controlled offset of melatonin synthesis at dawn by the suprachiasmatic nucleus in the rat. Neuroscience. 2005;**130**(3):797-803

[28] Klein DC. Arylalkylamine N-acetyltransferase: "the Timezyme". Journal of Biological Chemistry.

[29] Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA, et al. The melatonin rhythm-generating enzyme: Molecular regulation of

2007;**282**(7):4233-4237

cne.24181

DOI: 10.1242/jeb.058883

[22] Matynia A. Blurring the

Neuroscience. 2013;**7**:43-50. DOI: 10.4137/JEN.S11267

Hunt DM. The evolution of early vertebrate photoreceptors. Philosophical Transactions of The Royal Society B Biological Sciences. 2009;**364**(1531):2925-2940. DOI:

10.1098/rstb.2009.0099

[15] Solano-Lamphar HA, Kocifaj M. Numerical research on the effects the skyglow could have in phytochromes and RQE photoreceptors of plants. Journal of Environmental Management. 2018;**209**:484-494. DOI: 10.1016/j.

[16] Nelson RJ, Chbeir S. Dark matters: Effects of light at night on metabolism. Proceedings of the Nutrition Society. 2018;**77**(30):223-229. DOI: 10.1017/

[17] Abay KA, Amare M. Night light intensity and women's body weight: Evidence from Nigeria. Economics and Human Biology. 2018;**31**:238-248. DOI: 10.1016/j.ehb.2018.09.001

[18] Garcia-Saenz A, Sánchez de Miguel A, Espinosa A, Valentin A, Aragonés N, Llorca J, et al. Evaluating the association

exposure and breast and prostate cancer risk in Spain (MCC-Spain Study). Environmental Health Perspectives. 2018;**126**:047011. DOI: 10.1289/

between artificial light-at-night

[19] Zubidat AE, Fares B, Fares F, Haim A. Artificial light at night of different spectral compositions differentially affects tumor growth in mice: Interaction with melatonin and epigenetic pathways. Cancer Control. 2018;**25**(1):1073274818812908. DOI:

10.1177/1073274818812908

[20] Haim A, Heth G, Pratt H, Nevo E. Photoperiodic effects on thermoregulation in a 'blind' subterranean mammal. Journal Experimental Biology. 1983;

[21] Zubidat AE, Nelson RJ, Haim A. Spectral and duration sensitivity to

EHP1837

**56**

**107**:59-64

[30] Maronde E, Stehle JH. The mammalian pineal gland: Known facts, unknown facets. Trends in Endocrinology and Metabolism. 2007;**18**(11):142-149

[31] Ribelayga C, Garidou ML, Malan A, Gauer F, Calgari C, Pévet P, et al. Photoperiodic control of the rat pineal arylalkylamine-N-acetyltransferase and hydroxyindole-O-methyltransferase gene expression and its effect on melatonin synthesis. Journal of Biological Rhythms. 1999;**14**(2):105-115

[32] Thapan K, Arendt J, Skene DJ. An action spectrum for melatonin suppression: Evidence for a novel nonrod, non-cone photoreceptor system in humans. Journal of Physiology. 2001;**535**(Pt 1):261-267

[33] Cajochen C, Münch M, Kobialka S, Kräuchi K, Steiner R, Oelhafen P, et al. High sensitivity of human melatonin, alertness, thermoregulation, and heart rate to short wavelength light. Journal of Clinical Endocrinology and Metabolism. 2005;**90**(3):1311-1316

[34] Brainard GC, Lewy AJ, Menaker M, Fredrickson RH, Miller LS, Weleber RG, et al. Dose-response relationship between light irradiance and the suppression of plasma melatonin in human volunteers. Brain Research. 1988;**454**(1-2):212-218

[35] Falchi F, Cinzano P, Elvidge CD, Keith DM, Haim A. Limiting the impact of light pollution on human health, environment and stellar visibility. Journal of Environmental Management. 2011;**92**(10):2714-2722. DOI: 10.1016/j. jenvman.2011.06.029

[36] Aubé M, Roby J, Kocifaj M. Evaluating potential spectral impacts of various artificial lights on melatonin suppression, photosynthesis, and star visibility. PLoS One. 2013;**8**:e67798. DOI: 10.1371/journal.pone.0067798

[37] West KE, Jablonski MR, Warfield B, Cecil KS, James M, Ayers MA, et al. Blue light from light-emitting diodes elicits a dose-dependent suppression of melatonin in humans. Journal of Applied Physiology. 2011;**110**(3):619-626. DOI: 10.1152/ japplphysiol.01413.2009

[38] Walsh CM, Prendergast RL, Sheridan JT, Murphy BA. Blue light from light-emitting diodes directed at a single eye elicits a dose-dependent suppression of melatonin in horses. The Veterinary Journal. 2013;**196**(2):231-235. DOI: 10.1016/j.tvjl.2012.09.003

[39] Figueiro MG, Bierman A, Rea MS. A train of blue light pulses delivered through closed eyelids suppresses melatonin and phase shifts the human circadian system. Nature and Science of Sleep. 2013;**5**:133-141. DOI: 10.2147/ NSS.S52203

[40] Figueiro MG, Plitnick B, Rea MS. Pulsing blue light through closed eyelids: Effects on acute melatonin suppression and phase shifting of dim light melatonin onset. Nature and Science of Sleep. 2014;**6**:149-156. DOI: 10.2147/NSS.S73856

[41] Haim A, Portnov BA. Light Pollution as a New Risk Factor for Human Breast and Prostate Cancers. Dordecht: Springer Science + Buisness Media; 2013. DOI: 10.1007/978-94-007-6220-6\_1

[42] Ando K, Kripke DF. Light attenuation by the human eyelid. Biological Psychiatry. 1996; **39**(1):22-25

[43] Bierman A, Figueiro MG, Rea MS. Measuring and predicting eyelid spectral transmittance. Journal of Biomedical Optics. 2011;**16**:067011. DOI: 10.1117/1.3593151

[44] Figueiro MG, Wood B, Plitnick B, Rea MS. The impact of light from computer monitors on melatonin levels in college students. Neuro Endocrinology Letters. 2011;**32**(2):158-163

[45] Wood B, Rea MS, Plitnick B, Figueiro MG. Light level and duration of exposure determine the impact of self-luminous tablets on melatonin suppression. Applied Ergonomics. 2013;**44**(2):237-240. DOI: 10.1016/j. apergo.2012.07.008

[46] Green A, Cohen-Zion M, Haim A, Dagan Y. Evening light exposure to computer screens disrupts human sleep, biological rhythms, and attention abilities. Chronobiology International. 2017;**34**(7):855-865. DOI: 10.1080/07420528.2017.1324878

[47] Gaston KJ, Duffy JP, Gaston S, Bennie J, Davies TW. Human alteration of natural light cycles: Causes and ecological consequences. Oecologia. 2014;**76**(4):917-931. DOI: 10.1007/ s00442-014-3088-2

[48] Kyba CCM, Kuester T, Sánchez de Miguel A, Baugh K, Jechow A, Hölker F, et al. Artificially lit surface of Earth at night increasing in radiance and extent. Science Advances. 2017;**3**:e1701528. DOI: 10.1126/sciadv.1701528

[49] AMA. Human and environment effects of light emitting diode (LED). Community Lighting. 2016. Available from: https://circadianlight.com/ images/pdfs/newscience/American-Medical-Association-2016-Health-Effects-of-LED-Street-Lighting [Accessed: 28 December 2019]

[50] Wingfield JC. Environmental endocrinology: Insights into the diversity of regulatory mechanisms in life cycles. Integrative and Comparative Biology. 2018;**58**(4):790-799. DOI: 10.1093/icb/icy081

[51] Ouyang JQ , Davies S, Dominoni D. Hormonally mediated effects of artificial light at night on behavior and fitness: linking endocrine mechanisms with function. Journal of Experimental Biology. 2018;**221**(Pt 6):1-11. pii: jeb156893. DOI: 10.1242/jeb.156893

[52] Zhang X, Ho SM. Epigenetics meets endocrinology. Journal of Molecular Endocrinology. 2011;**46**(1):R11-R32

[53] Fleisch AF, Wright RO, Baccarelli AA. Environmental epigenetics: A role in endocrine disease? Journal of Molecular Endocrinology. 2012;**49**(2):R61-R67. DOI: 10.1530/JME-12-0066

[54] Touitou Y, Reinberg A, Touitou D. Association between light at night, melatonin secretion, sleep deprivation, and the internal clock: Health impacts and mechanisms of circadian disruption. Life Science. 2017;**173**: 94-106. DOI: 10.1016/j.lfs.2017.02.008

[55] Kloog I, Haim A, Stevens RG, Portnov BA. Global co-distribution of light at night (LAN) and cancers of prostate, colon, and lung in men. Chronobiology International. 2009;**26**(1):108-125. DOI: 10.1080/07420520802694020

[56] Kloog I, Stevens RG, Haim A, Portnov BA. Nighttime light level co-distributes with breast cancer incidence worldwide. Cancer Causes & Control. 2010;**21**(12):2059-2068. DOI: 10.1007/s10552-010-9624-4

[57] Kim YJ, Park MS, Lee E, Choi JW. High incidence of breast cancer in lightpolluted areas with spatial effects in Korea. Asian Pacific Journal of Cancer Prevention. 2016;**17**(1):361-367

[58] Keshet-Sitton A, Or-Chen K, Yitzhak S, Tzabary I, Haim A. Light and the City: Breast cancer risk factors differ between urban and rural women in Israel. Integrative Cancer Therapies.

**59**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

et al. Epigenetic mechanisms of breast cancer: An update of the current knowledge. Epigenomics. 2014;**6**(6): 651-664. DOI: 10.2217/epi.14.59

[66] Xiang TX, Yuan Y, Li LL, Wang ZH,

Dan LY, Chen Y, et al. Aberrant promoter CpG methylation and its translational applications in breast cancer. Chinse Journal Cancer. 2013;**32**(10):12-20. DOI: 10.5732/

[67] Ross JP, Rand KN, Molloy PL. Hypomethylation of repeated DNA sequences in cancer. Epigenomics. 2010;**2**(2):245-269. DOI: 10.2217/epi.10.2

[68] Ogishima T, Shiina H, Breault JE, Tabatabai L, Bassett WW, Enokida H, et al. Increased heparanase expression is caused by promoter hypomethylation and up-regulation of transcriptional factor early growth response-1 in human prostate cancer. Clinical Cancer

Research. 2005;**11**(3):1028-1036

DOI: 10.4161/epi.29628

1999;**85**(1):112-118

2004;**3**(12):1225-1231

[70] Soares J, Pinto AE, Cunha CV, André S, Barão I, Sousa JM, et al. Global DNA hypomethylation in breast carcinoma: Correlation with prognostic factors and tumor progression. Cancer.

[71] Jackson K, Yu MC, Arakawa K, Fiala E, Youn B, Fiegl H, et al. DNA hypomethylation is prevalent even in low-grade breast cancers. Cancer Biology and Therapy.

[72] Stevens RG. Electric power use and breast cancer: A hypothesis.

[69] Loriot A, Van Tongelen A, Blanco J, Klaessens S, Cannuyer J, van Baren N, et al. A novel cancer-germline transcript carrying pro-metastatic miR-105 and TET-targeting miR-767 induced by DNA hypomethylation in tumors. Epigenetics. 2014;**9**(8):1163-1171.

cjc.011.10344

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

[59] Anderson LE, Morris JE, Sasser LB, Stevens RG. Effect of constant light on DMBA mammary tumorigenesis in rats. Cancer Letters. 2000;**148**(2):121-126

2017;**16**(2):176-187. DOI: 10.1177/

[60] Blask DE, Dauchy RT, Sauer LA, Krause JA, Brainard GC. Growth and fatty acid metabolism of human breast cancer (MCF-7) xenografts in nude rats: Impact of constant light-induced nocturnal melatonin suppression. Breast Cancer Research and

Treatment. 2003;**79**(3):313-320

2006;**235**(2):266-271

2010;**2**(2):82-92

[61] Cos S, Mediavilla D, Martínez-Campa C, González A, Alonso-González C, Sánchez-Barceló EJ. Exposure to light-at-night increases the growth of DMBA-induced mammary adenocarcinomas in rats. Cancer Letters.

[62] Vinogradova IA, Anisimov VN, Bukalev AV, Ilyukha VA, Khizhkin EA, Lotosh TA, et al. Circadian disruption induced by light-at-night accelerates aging and promotes tumorigenesis in young but not in old rats. Aging (Albany NY).

[63] Schwimmer H, Metzer A, Pilosof Y, Szyf M, Machnes ZM, Fares F, et al. Light at night and melatonin have opposite effects on breast cancer tumors in mice assessed by growth rates and global DNA methylation. Chronobiology International. 2014;**31**(1):144-150. DOI:

[64] Zubidat AE, Fares B, Faras F, Haim A. Melatonin functioning through DNA methylation to constrict breast cancer growth accelerated by blue LED light at night in 4T1 tumor bearing mice. Journal of Cancer Biology and Therapeutics. 2015;**1**(2):57-73. DOI: 10.18314/gjct.v1i2.35

[65] Karsli-Ceppioglu S, Dagdemir A, Judes G, Ngollo M, Penault-Llorca F, Pajon A,

10.3109/07420528.2013.842925

1534735416660194

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

2017;**16**(2):176-187. DOI: 10.1177/ 1534735416660194

*Epigenetics*

[44] Figueiro MG, Wood B, Plitnick B, Rea MS. The impact of light from computer monitors on melatonin levels in college students. Neuro Endocrinology Letters.

[51] Ouyang JQ , Davies S, Dominoni D. Hormonally mediated effects of artificial light at night on behavior and fitness: linking endocrine mechanisms with function. Journal of Experimental Biology. 2018;**221**(Pt 6):1-11. pii: jeb156893. DOI: 10.1242/jeb.156893

[52] Zhang X, Ho SM. Epigenetics meets endocrinology. Journal of Molecular Endocrinology. 2011;**46**(1):R11-R32

[53] Fleisch AF, Wright RO, Baccarelli AA. Environmental epigenetics: A role in endocrine disease? Journal of Molecular Endocrinology. 2012;**49**(2):R61-R67.

[54] Touitou Y, Reinberg A, Touitou D. Association between light at night, melatonin secretion, sleep deprivation,

impacts and mechanisms of circadian disruption. Life Science. 2017;**173**: 94-106. DOI: 10.1016/j.lfs.2017.02.008

[55] Kloog I, Haim A, Stevens RG, Portnov BA. Global co-distribution of light at night (LAN) and cancers of prostate, colon, and lung in men. Chronobiology International. 2009;**26**(1):108-125. DOI:

DOI: 10.1530/JME-12-0066

and the internal clock: Health

10.1080/07420520802694020

[56] Kloog I, Stevens RG, Haim A, Portnov BA. Nighttime light level co-distributes with breast cancer incidence worldwide. Cancer Causes & Control. 2010;**21**(12):2059-2068. DOI: 10.1007/s10552-010-9624-4

[57] Kim YJ, Park MS, Lee E, Choi JW. High incidence of breast cancer in lightpolluted areas with spatial effects in Korea. Asian Pacific Journal of Cancer Prevention. 2016;**17**(1):361-367

[58] Keshet-Sitton A, Or-Chen K, Yitzhak S, Tzabary I, Haim A. Light and the City: Breast cancer risk factors differ between urban and rural women in Israel. Integrative Cancer Therapies.

[45] Wood B, Rea MS, Plitnick B, Figueiro MG. Light level and duration of exposure determine the impact of self-luminous tablets on melatonin suppression. Applied Ergonomics. 2013;**44**(2):237-240. DOI: 10.1016/j.

[46] Green A, Cohen-Zion M, Haim A, Dagan Y. Evening light exposure to computer screens disrupts human sleep, biological rhythms, and attention abilities. Chronobiology International. 2017;**34**(7):855-865. DOI:

10.1080/07420528.2017.1324878

[47] Gaston KJ, Duffy JP, Gaston S, Bennie J, Davies TW. Human alteration of natural light cycles: Causes and ecological consequences. Oecologia. 2014;**76**(4):917-931. DOI: 10.1007/

[48] Kyba CCM, Kuester T, Sánchez de Miguel A, Baugh K, Jechow A, Hölker F, et al. Artificially lit surface of Earth at night increasing in radiance and extent. Science Advances. 2017;**3**:e1701528.

[49] AMA. Human and environment effects of light emitting diode (LED). Community Lighting. 2016. Available from: https://circadianlight.com/ images/pdfs/newscience/American-Medical-Association-2016-Health-Effects-of-LED-Street-Lighting [Accessed: 28 December 2019]

[50] Wingfield JC. Environmental endocrinology: Insights into the diversity of regulatory mechanisms in life cycles. Integrative and Comparative

Biology. 2018;**58**(4):790-799. DOI: 10.1093/icb/icy081

DOI: 10.1126/sciadv.1701528

2011;**32**(2):158-163

apergo.2012.07.008

s00442-014-3088-2

**58**

[59] Anderson LE, Morris JE, Sasser LB, Stevens RG. Effect of constant light on DMBA mammary tumorigenesis in rats. Cancer Letters. 2000;**148**(2):121-126

[60] Blask DE, Dauchy RT, Sauer LA, Krause JA, Brainard GC. Growth and fatty acid metabolism of human breast cancer (MCF-7) xenografts in nude rats: Impact of constant light-induced nocturnal melatonin suppression. Breast Cancer Research and Treatment. 2003;**79**(3):313-320

[61] Cos S, Mediavilla D, Martínez-Campa C, González A, Alonso-González C, Sánchez-Barceló EJ. Exposure to light-at-night increases the growth of DMBA-induced mammary adenocarcinomas in rats. Cancer Letters. 2006;**235**(2):266-271

[62] Vinogradova IA, Anisimov VN, Bukalev AV, Ilyukha VA, Khizhkin EA, Lotosh TA, et al. Circadian disruption induced by light-at-night accelerates aging and promotes tumorigenesis in young but not in old rats. Aging (Albany NY). 2010;**2**(2):82-92

[63] Schwimmer H, Metzer A, Pilosof Y, Szyf M, Machnes ZM, Fares F, et al. Light at night and melatonin have opposite effects on breast cancer tumors in mice assessed by growth rates and global DNA methylation. Chronobiology International. 2014;**31**(1):144-150. DOI: 10.3109/07420528.2013.842925

[64] Zubidat AE, Fares B, Faras F, Haim A. Melatonin functioning through DNA methylation to constrict breast cancer growth accelerated by blue LED light at night in 4T1 tumor bearing mice. Journal of Cancer Biology and Therapeutics. 2015;**1**(2):57-73. DOI: 10.18314/gjct.v1i2.35

[65] Karsli-Ceppioglu S, Dagdemir A, Judes G, Ngollo M, Penault-Llorca F, Pajon A,

et al. Epigenetic mechanisms of breast cancer: An update of the current knowledge. Epigenomics. 2014;**6**(6): 651-664. DOI: 10.2217/epi.14.59

[66] Xiang TX, Yuan Y, Li LL, Wang ZH, Dan LY, Chen Y, et al. Aberrant promoter CpG methylation and its translational applications in breast cancer. Chinse Journal Cancer. 2013;**32**(10):12-20. DOI: 10.5732/ cjc.011.10344

[67] Ross JP, Rand KN, Molloy PL. Hypomethylation of repeated DNA sequences in cancer. Epigenomics. 2010;**2**(2):245-269. DOI: 10.2217/epi.10.2

[68] Ogishima T, Shiina H, Breault JE, Tabatabai L, Bassett WW, Enokida H, et al. Increased heparanase expression is caused by promoter hypomethylation and up-regulation of transcriptional factor early growth response-1 in human prostate cancer. Clinical Cancer Research. 2005;**11**(3):1028-1036

[69] Loriot A, Van Tongelen A, Blanco J, Klaessens S, Cannuyer J, van Baren N, et al. A novel cancer-germline transcript carrying pro-metastatic miR-105 and TET-targeting miR-767 induced by DNA hypomethylation in tumors. Epigenetics. 2014;**9**(8):1163-1171. DOI: 10.4161/epi.29628

[70] Soares J, Pinto AE, Cunha CV, André S, Barão I, Sousa JM, et al. Global DNA hypomethylation in breast carcinoma: Correlation with prognostic factors and tumor progression. Cancer. 1999;**85**(1):112-118

[71] Jackson K, Yu MC, Arakawa K, Fiala E, Youn B, Fiegl H, et al. DNA hypomethylation is prevalent even in low-grade breast cancers. Cancer Biology and Therapy. 2004;**3**(12):1225-1231

[72] Stevens RG. Electric power use and breast cancer: A hypothesis.

American Journal of Epidemiology. 1987;**125**(4):556-561

[73] Reiter RJ. Functional pleiotropy of the neurohormone melatonin: Antioxidant protection and neuroendocrine regulation. Frontiers in Neuroendocrinology. 1995;**16**(4):383-415

[74] Middleton B. Measurement of melatonin and 6-sulphatoxymelatonin. Methods in Molecular Biology. 2013;**1065**:171-199. DOI: 10.1007/978-1-62703-616-0\_11

[75] Schernhammer ES, Hankinson SE. Urinary melatonin levels and postmenopausal breast cancer risk in the Nurses' Health Study cohort. Cancer Epidemiology, Biomarkers and Prevention. 2009;**18**(1):74-79. DOI: 10.1158/1055-9965.EPI-08-0637

[76] Basler M, Jetter A, Fink D, Seifert B, Kullak-Ublick GA, Trojan A. Urinary excretion of melatonin and association with breast cancer: Meta-analysis and review of the literature. Breast Care (Basel). 2014;**9**(3):182-187. DOI: 10.1159/000363426

[77] Brown SB, Hankinson SE, Eliassen AH, Reeves KW, Qian J, Arcaro KF, et al. Urinary melatonin concentration and the risk of breast cancer in Nurses' Health Study II. American Journal Epidemiology. 2015;**181**(3):155-162. DOI: 10.1093/aje/kwu261

[78] Kliukiene J, Tynes T, Andersen A. Risk of breast cancer among Norwegian women with visual impairment. British Journal of Cancer. 2001;**84**(3): 397-399

[79] Wu AH, Wang R, Koh WP, Stanczyk FZ, Lee HP, Yu MC. Sleep duration, melatonin and breast cancer among Chinese women in Singapore. Carcinogenesis. 2008;**29**(6): 1244-1248. DOI: 10.1093/carcin/bgn100 [80] Mao L, Dauchy RT, Blask DE, Dauchy EM, Slakey LM, Brimer S, et al. Melatonin suppression of aerobic glycolysis (Warburg effect), survival signalling and metastasis in human leiomyosarcoma. Journal Pineal Research. 2016;**60**(2):167-177. DOI: 10.1111/jpi.12298

[81] Sánchez DI, González-Fernández B, Crespo I, San-Miguel B, Álvarez M, González-Gallego J, et al. Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamineinduced hepatocellular carcinoma. Jornal of Pineal Research. 2018;**65**(3):e12506. DOI: 10.1111/jpi.12506

[82] Lu H, Wu B, Ma G, Zheng D, Song R, Huang E, Mao M, Lu B. Melatonin represses oral squamous cell carcinoma metastasis by inhibiting tumorassociated neutrophils. American Journal of Translational Research. 2017;**9**:5361-5374

[83] Hurt B, Schulick R, Edil B, El Kasmi KC, Barnett C Jr. Cancer-promoting mechanisms of tumor-associated neutrophils. The American Journal of Surgery. 2017;**214**(5):938-944. DOI: 10.1016/j.amjsurg.2017.08.003

[84] Fan C, Pan Y, Yang Y, Di S, Jiang S, Ma Z, et al. HDAC1 inhibition by melatonin leads to suppression of lung adenocarcinoma cells via induction of oxidative stress and activation of apoptotic pathways. Journal of Pineal Research. 2015;**59**(3):321-333. DOI: 10.1111/jpi.12261

[85] Li W, Fan M, Chen Y, Zhao Q, Song C, Yan Y, et al. Melatonin induces cell apoptosis in AGS cells through the activation of JNK and P38 MAPK and the suppression of nuclear factor-kappa B: A novel therapeutic implication for gastric cancer. Cellular Physiology and Biochemistry. 2015;**37**(6):2323-2338. DOI: 10.1159/000438587

**61**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

of Pharmacology and Toxicology. 2018;**58**:187-207. DOI: 10.1146/ annurev-pharmtox-010716-105106

[93] Gatti G, Lucini V, Dugnani S, Calastretti A, Spadoni G, Bedini A, Rivara S, Mor M, Canti G, Scaglione F, Bevilacqua A. Antiproliferative and pro-apoptotic activity of melatonin analogues on melanoma and breast cancer cells. Oncotarget. 2017; **8**:68338-68353. DOI: 10.18632/

[94] Nunez AA, Yan L, Smale L. The cost of activity during the rest phase: Animal models and theoretical

[95] Neuman A, Gothilf Y, Haim A, Ben-Aharon G, Zisapel N. Nocturnal patterns and up-regulated excretion of the melatonin metabolite 6-sulfatoxymelatonin in the diurnal rodent *Psammomys obesus* post-weaning under a short photoperiod. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology.

[96] Schwimmer H, Mursu N, Haim A. Effects of light and melatonin treatment on body temperature and melatonin secretion daily rhythms in a diurnal rodent, the fat sand rat. Chronobiology International. 2010;**27**(7):1401-1419. DOI: 10.3109/07420528.2010.505355

[97] Bouderba S, Sanchez-Martin C, Villanueva GR, Detaille D, Koceïr EA. Beneficial effects of silibinin against the progression of metabolic syndrome, increased oxidative stress, and liver steatosis in *Psammomys obesus*, a relevant animal model of human obesity and diabetes. Journal of Diabetes. 2014;**6**(2):184-192. DOI:

10.1111/1753-0407.12083

[98] Sihali-Beloui O, El-Aoufi S,

Maouche B, Marco S. *Psammomys obesus*,

perspectives. Frontiers in Endocrinology (Lausanne). 2018;**9**:72. DOI: 10.3389/

oncotarget.20124.

fendo.2018.00072

2005;**142**(3):297-307

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

[86] Akbarzadeh M, Movassaghpour AA, Ghanbari H, Kheirandish M, Fathi Maroufi N, Rahbarghazi R, et al. The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells. Scientific Reports. 2017;**7**:17062. DOI: 10.1038/

[87] Bakalova R, Zhelev Z, Shibata S, Nikolova B, Aoki I, Higashi T. Impressive suppression of colon cancer growth by triple combination SN38/EF24/melatonin: "oncogenic" versus "onco-suppressive" reactive oxygen species. Anticancer Research.

[88] Mao L, Summers W, Xiang S, Yuan L, Dauchy RT, Reynolds A, et al. Melatonin represses metastasis in Her2 postive human breast cancer cells by suppressing RSK2 expression. Molecular Cancer Research. 2016;**14**(11):1159-1169

[89] Haim A, Zubidat AE. Artificial light at night: Melatonin as a mediator

epigenome. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 2015;**370**(1667): 1-7. pii: 20140121. DOI: 10.1098/

[90] Lee SE, Kim SJ, Yoon HJ, Yu SY, Yang H, Jeong SI, et al. Genome-wide profiling in melatonin-exposed human breast cancer cell lines identifies differentially methylated genes involved in the

anticancer effect of melatonin. Journal of Pineal Research. 2013;**54**(1):80-88. DOI: 10.1111/j.1600-079X.2012.01027.x

[91] Korkmaz A, Sanchez-Barcelo EJ, Tan DX, Reiter RJ. Role of melatonin in the epigenetic regulation of breast cancer. Breast Cancer Research and Treatment. 2009;**115**(1):13-27. DOI:

[92] Bennett RL, Licht JD. Targeting epigenetics in cancer. Annual Review

10.1007/s10549-008-0103-5

between the environment and

rstb.2014.0121

s41598-017-16940-y

2017;**37**(10):5449-5458

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

[86] Akbarzadeh M, Movassaghpour AA, Ghanbari H, Kheirandish M, Fathi Maroufi N, Rahbarghazi R, et al. The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells. Scientific Reports. 2017;**7**:17062. DOI: 10.1038/ s41598-017-16940-y

*Epigenetics*

1987;**125**(4):556-561

1995;**16**(4):383-415

American Journal of Epidemiology.

[80] Mao L, Dauchy RT, Blask DE, Dauchy EM, Slakey LM, Brimer S, et al. Melatonin suppression of aerobic glycolysis (Warburg effect), survival signalling and metastasis in human leiomyosarcoma. Journal Pineal Research. 2016;**60**(2):167-177. DOI:

[81] Sánchez DI, González-Fernández B, Crespo I, San-Miguel B, Álvarez M, González-Gallego J, et al. Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamineinduced hepatocellular carcinoma. Jornal of Pineal Research. 2018;**65**(3):e12506.

[82] Lu H, Wu B, Ma G, Zheng D, Song R, Huang E, Mao M, Lu B. Melatonin represses oral squamous cell carcinoma

[83] Hurt B, Schulick R, Edil B, El Kasmi KC,

metastasis by inhibiting tumorassociated neutrophils. American Journal of Translational Research.

Barnett C Jr. Cancer-promoting mechanisms of tumor-associated neutrophils. The American Journal of Surgery. 2017;**214**(5):938-944. DOI: 10.1016/j.amjsurg.2017.08.003

[84] Fan C, Pan Y, Yang Y, Di S, Jiang S, Ma Z, et al. HDAC1 inhibition by melatonin leads to suppression of lung adenocarcinoma cells via induction of oxidative stress and activation of apoptotic pathways. Journal of Pineal Research. 2015;**59**(3):321-333. DOI:

[85] Li W, Fan M, Chen Y, Zhao Q, Song C, Yan Y, et al. Melatonin induces cell apoptosis in AGS cells through the activation of JNK and P38 MAPK and the suppression of nuclear factor-kappa B: A novel therapeutic implication for gastric cancer.

Cellular Physiology and Biochemistry.

10.1111/jpi.12298

DOI: 10.1111/jpi.12506

2017;**9**:5361-5374

10.1111/jpi.12261

2015;**37**(6):2323-2338. DOI: 10.1159/000438587

[73] Reiter RJ. Functional pleiotropy of the neurohormone melatonin: Antioxidant protection and neuroendocrine regulation. Frontiers in Neuroendocrinology.

[74] Middleton B. Measurement of melatonin and 6-sulphatoxymelatonin.

[75] Schernhammer ES, Hankinson SE.

[76] Basler M, Jetter A, Fink D, Seifert B, Kullak-Ublick GA, Trojan A. Urinary excretion of melatonin and association with breast cancer: Meta-analysis and review of the literature. Breast Care

[77] Brown SB, Hankinson SE, Eliassen AH, Reeves KW, Qian J, Arcaro KF, et al. Urinary melatonin concentration and the risk of breast cancer in Nurses' Health Study II. American Journal Epidemiology. 2015;**181**(3):155-162.

[78] Kliukiene J, Tynes T, Andersen A. Risk of breast cancer among Norwegian women with visual impairment. British

Methods in Molecular Biology. 2013;**1065**:171-199. DOI: 10.1007/978-1-62703-616-0\_11

Urinary melatonin levels and postmenopausal breast cancer risk in the Nurses' Health Study cohort. Cancer Epidemiology, Biomarkers and

Prevention. 2009;**18**(1):74-79. DOI: 10.1158/1055-9965.EPI-08-0637

(Basel). 2014;**9**(3):182-187. DOI: 10.1159/000363426

DOI: 10.1093/aje/kwu261

Journal of Cancer. 2001;**84**(3):

[79] Wu AH, Wang R, Koh WP, Stanczyk FZ, Lee HP, Yu MC. Sleep duration, melatonin and breast cancer among Chinese women in Singapore.

Carcinogenesis. 2008;**29**(6):

1244-1248. DOI: 10.1093/carcin/bgn100

**60**

397-399

[87] Bakalova R, Zhelev Z, Shibata S, Nikolova B, Aoki I, Higashi T. Impressive suppression of colon cancer growth by triple combination SN38/EF24/melatonin: "oncogenic" versus "onco-suppressive" reactive oxygen species. Anticancer Research. 2017;**37**(10):5449-5458

[88] Mao L, Summers W, Xiang S, Yuan L, Dauchy RT, Reynolds A, et al. Melatonin represses metastasis in Her2 postive human breast cancer cells by suppressing RSK2 expression. Molecular Cancer Research. 2016;**14**(11):1159-1169

[89] Haim A, Zubidat AE. Artificial light at night: Melatonin as a mediator between the environment and epigenome. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 2015;**370**(1667): 1-7. pii: 20140121. DOI: 10.1098/ rstb.2014.0121

[90] Lee SE, Kim SJ, Yoon HJ, Yu SY, Yang H, Jeong SI, et al. Genome-wide profiling in melatonin-exposed human breast cancer cell lines identifies differentially methylated genes involved in the anticancer effect of melatonin. Journal of Pineal Research. 2013;**54**(1):80-88. DOI: 10.1111/j.1600-079X.2012.01027.x

[91] Korkmaz A, Sanchez-Barcelo EJ, Tan DX, Reiter RJ. Role of melatonin in the epigenetic regulation of breast cancer. Breast Cancer Research and Treatment. 2009;**115**(1):13-27. DOI: 10.1007/s10549-008-0103-5

[92] Bennett RL, Licht JD. Targeting epigenetics in cancer. Annual Review of Pharmacology and Toxicology. 2018;**58**:187-207. DOI: 10.1146/ annurev-pharmtox-010716-105106

[93] Gatti G, Lucini V, Dugnani S, Calastretti A, Spadoni G, Bedini A, Rivara S, Mor M, Canti G, Scaglione F, Bevilacqua A. Antiproliferative and pro-apoptotic activity of melatonin analogues on melanoma and breast cancer cells. Oncotarget. 2017; **8**:68338-68353. DOI: 10.18632/ oncotarget.20124.

[94] Nunez AA, Yan L, Smale L. The cost of activity during the rest phase: Animal models and theoretical perspectives. Frontiers in Endocrinology (Lausanne). 2018;**9**:72. DOI: 10.3389/ fendo.2018.00072

[95] Neuman A, Gothilf Y, Haim A, Ben-Aharon G, Zisapel N. Nocturnal patterns and up-regulated excretion of the melatonin metabolite 6-sulfatoxymelatonin in the diurnal rodent *Psammomys obesus* post-weaning under a short photoperiod. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 2005;**142**(3):297-307

[96] Schwimmer H, Mursu N, Haim A. Effects of light and melatonin treatment on body temperature and melatonin secretion daily rhythms in a diurnal rodent, the fat sand rat. Chronobiology International. 2010;**27**(7):1401-1419. DOI: 10.3109/07420528.2010.505355

[97] Bouderba S, Sanchez-Martin C, Villanueva GR, Detaille D, Koceïr EA. Beneficial effects of silibinin against the progression of metabolic syndrome, increased oxidative stress, and liver steatosis in *Psammomys obesus*, a relevant animal model of human obesity and diabetes. Journal of Diabetes. 2014;**6**(2):184-192. DOI: 10.1111/1753-0407.12083

[98] Sihali-Beloui O, El-Aoufi S, Maouche B, Marco S. *Psammomys obesus*,

#### *Epigenetics*

a uniquemodel of metabolic syndrome, inflammation and autophagy in the pathologic development of hepatic steatosis. Comptes Rendus Biologies. 2016;**339**(11-12):475-486. DOI: 10.1016/j.crvi.2016.08.001

[99] Gouaref I, Detaille D, Wiernsperger N, Khan NA, Leverve X, Koceir EA. The desert gerbil *Psammomys obesus* as a model for metformin-sensitive nutritional type 2 diabetes to protect hepatocellular metabolic damage: Impact of mitochondrial redox state. PLoS One. 2017;**12**:e0172053. DOI: 10.1371/journal.pone.0172053

[100] Chaudhary R, Walder KR, Hagemeyer CE, Kanwar JR. *Psammomys obesus*: A atural diet-controlled model for diabetes and cardiovascular diseases. Current Atherosclerosis Reports. 2018;**20**(9):46. DOI: 10.1007/ s11883-018-0746-6

[101] de Almeida EA, Di Mascio P, Harumi T, Spence DW, Moscovitch A, Hardeland R, et al. Measurement of melatonin in body fluids: Standards, protocols and procedures. Child's Nervous System. 2011;**27**(6):879-891. DOI: 10.1007/s00381-010-1278-8

[102] Bianco AC, McAninch EA. The role of thyroid hormone and brown adipose tissue in energy homoeostasis. Lancet Diabetes and Endocrinology. 2013;**1**(3):250-258. DOI: 10.1016/ S2213-8587(13)70069-X

[103] Nelson W, Tong Y, Lee J, Halberg F. Methods for cosinor-rhythmometry. Chronobiologia. 1979;**6**(4): 305-323

[104] Mazzoccoli G, Giuliani A, Carughi S, De Cata A, Puzzolante F, La Viola M, et al. The hypothalamicpituitary-thyroid axis and melatonin in humans: Possible interactions in the control of body temperature. Neuro Endocrinology Letters. 2004;**25**(5):368-372

[105] Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical Reviews in Toxicology. 2007;**37**(1-2):11-53

[106] Dardente H, Hazlerigg DG, Ebling FJ. Thyroid hormone and seasonal rhythmicity. Frontiers Endocrinology (Lausanne). 2014;**5**:19. DOI: 10.3389/ fendo.2014.00019

[107] Korf HW. Signaling pathways to and from the hypophysial pars tuberalis, an important center for the control of seasonal rhythms. General and Comparative Endocrinology. 2018;**258**:236-243. DOI: 10.1016/j. ygcen.2017.05.011

[108] Campos-Barros A, Musa A, Flechner A, Hessenius C, Gaio U, Meinhold H, et al. Evidence for circadian variations of thyroid hormone concentrations and type II 5′-iodothyronine deiodinase activity in the rat central nervous system. Journal of Neurochemistry. 1997;**68**(2):795-803

[109] Soutto M, Guerrero JM, Osuna C, Molinero P. Nocturnal increases in the triiodothyronine/thyroxine ratio in the rat thymus and pineal gland follow increases of type II 5′-deiodinase activity. The International Journal of Biochemistry and Cell Biology. 1998;**30**(2):235-241

[110] Hut RA. Photoperiodism: Shall EYA compare thee to a summer's day? Current Biology. 2011;**21**(1):R22-R25. DOI: 10.1016/j.cub.2010.11.060

[111] Wood S, Loudon A. Clocks for all seasons: Unwinding the roles and mechanisms of circadian and interval timers in the hypothalamus and pituitary. Journal of Endocrinology. 2014;**222**(2):R39-R59. DOI: 10.1530/ JOE-14-0141

[112] Ikeno T, Weil ZM, Nelson RJ. Dim light at night disrupts the short-day

**63**

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away…*

*DOI: http://dx.doi.org/10.5772/intechopen.84789*

response in Siberian hamsters. General and Comparative Endocrinology. 2014;**197**:56-64. DOI: 10.1016/j.

[113] Maroni MJ, Capri KM, Cushman AV, Monteiro De Pina IK, Chasse MH, Seggio JA. Constant light alters serum hormone levels related to thyroid function in male CD-1 mice. Chronobiology International.

ygcen.2013.12.005

2018;**35**(10):1456-1463. DOI: 10.1080/07420528.2018

[114] Yang Y, Yu Y, Yang B, Zhou H, Pan J. Physiological responses to daily light exposure. Scientific Reports. 2016;**6**:24808. DOI: 10.1038/srep24808

[115] Borniger JC, Maurya SK, Periasamy M, Nelson RJ. Acute dim light at night increases body mass, alters metabolism, and shifts core body temperature circadian rhythms. Chronobiology International. 2014;**31**(8):917-925. DOI: 10.3109/07420528.2014.926911

[116] Saini C, Morf J, Stratmann M, Gos P, Schibler U. Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators. Genes and Development. 2012;**26**(6):567-580. DOI: 10.1101/

[117] Brown SA, Zumbrunn G, Fleury-Olela F, Preitner N, Schibler U. Rhythms of mammalian body temperature can sustain peripheral circadian clocks. Current Biology. 2002;**12**(18): 1574-1583. Available from: https:// linkinghub.elsevier.com/retrieve/pii/

[118] Zhang M, Xu C, von Wettstein D, Liu B. Tissue-specific differences in cytosine methylation and their association with differential gene expression in sorghum. Plant Physiology. 2011;**156**(4):1955-1966.

gad.183251.111

S0960-9822(02)01145-4

DOI: 10.1104/pp.111.176842

*Consequences of Artificial Light at Night: The Linkage between Chasing Darkness Away… DOI: http://dx.doi.org/10.5772/intechopen.84789*

response in Siberian hamsters. General and Comparative Endocrinology. 2014;**197**:56-64. DOI: 10.1016/j. ygcen.2013.12.005

*Epigenetics*

a uniquemodel of metabolic syndrome, inflammation and autophagy in the pathologic development of hepatic steatosis. Comptes Rendus Biologies. 2016;**339**(11-12):475-486. DOI: 10.1016/j.crvi.2016.08.001

[105] Zoeller RT, Tan SW, Tyl RW. General background on the hypothalamic-pituitary-thyroid (HPT) axis. Critical Reviews in Toxicology.

Thyroid hormone and seasonal rhythmicity. Frontiers Endocrinology (Lausanne). 2014;**5**:19. DOI: 10.3389/

[107] Korf HW. Signaling pathways to and from the hypophysial pars tuberalis, an important center for the control of seasonal rhythms. General and Comparative Endocrinology. 2018;**258**:236-243. DOI: 10.1016/j.

[108] Campos-Barros A, Musa A, Flechner A, Hessenius C, Gaio U, Meinhold H, et al. Evidence for circadian variations of thyroid hormone concentrations and type II 5′-iodothyronine deiodinase activity in the rat central nervous system. Journal of Neurochemistry. 1997;**68**(2):795-803

[109] Soutto M, Guerrero JM, Osuna C, Molinero P. Nocturnal increases in the triiodothyronine/thyroxine ratio in the rat thymus and pineal gland follow increases of type II 5′-deiodinase activity. The International Journal of Biochemistry and Cell Biology.

[110] Hut RA. Photoperiodism: Shall EYA compare thee to a summer's day? Current Biology. 2011;**21**(1):R22-R25. DOI: 10.1016/j.cub.2010.11.060

[111] Wood S, Loudon A. Clocks for all seasons: Unwinding the roles and mechanisms of circadian and interval timers in the hypothalamus and pituitary. Journal of Endocrinology. 2014;**222**(2):R39-R59. DOI: 10.1530/

[112] Ikeno T, Weil ZM, Nelson RJ. Dim light at night disrupts the short-day

[106] Dardente H, Hazlerigg DG, Ebling FJ.

2007;**37**(1-2):11-53

fendo.2014.00019

ygcen.2017.05.011

1998;**30**(2):235-241

JOE-14-0141

[99] Gouaref I, Detaille D, Wiernsperger N,

Khan NA, Leverve X, Koceir EA. The desert gerbil *Psammomys obesus* as a model for metformin-sensitive nutritional type 2 diabetes to protect hepatocellular metabolic damage: Impact of mitochondrial redox state. PLoS One. 2017;**12**:e0172053. DOI: 10.1371/journal.pone.0172053

[100] Chaudhary R, Walder KR, Hagemeyer CE, Kanwar JR. *Psammomys obesus*: A atural diet-controlled model for diabetes and cardiovascular diseases. Current Atherosclerosis Reports. 2018;**20**(9):46. DOI: 10.1007/

[101] de Almeida EA, Di Mascio P, Harumi T, Spence DW, Moscovitch A, Hardeland R, et al. Measurement of melatonin in body fluids: Standards, protocols and procedures. Child's Nervous System. 2011;**27**(6):879-891. DOI: 10.1007/s00381-010-1278-8

[102] Bianco AC, McAninch EA. The role of thyroid hormone and brown adipose tissue in energy homoeostasis. Lancet Diabetes and Endocrinology. 2013;**1**(3):250-258. DOI: 10.1016/

[103] Nelson W, Tong Y, Lee J, Halberg F. Methods for cosinor-rhythmometry.

S2213-8587(13)70069-X

Chronobiologia. 1979;**6**(4):

2004;**25**(5):368-372

[104] Mazzoccoli G, Giuliani A, Carughi S, De Cata A, Puzzolante F, La Viola M, et al. The hypothalamicpituitary-thyroid axis and melatonin in humans: Possible interactions in the control of body temperature. Neuro Endocrinology Letters.

s11883-018-0746-6

**62**

305-323

[113] Maroni MJ, Capri KM, Cushman AV, Monteiro De Pina IK, Chasse MH, Seggio JA. Constant light alters serum hormone levels related to thyroid function in male CD-1 mice. Chronobiology International. 2018;**35**(10):1456-1463. DOI: 10.1080/07420528.2018

[114] Yang Y, Yu Y, Yang B, Zhou H, Pan J. Physiological responses to daily light exposure. Scientific Reports. 2016;**6**:24808. DOI: 10.1038/srep24808

[115] Borniger JC, Maurya SK, Periasamy M, Nelson RJ. Acute dim light at night increases body mass, alters metabolism, and shifts core body temperature circadian rhythms. Chronobiology International. 2014;**31**(8):917-925. DOI: 10.3109/07420528.2014.926911

[116] Saini C, Morf J, Stratmann M, Gos P, Schibler U. Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators. Genes and Development. 2012;**26**(6):567-580. DOI: 10.1101/ gad.183251.111

[117] Brown SA, Zumbrunn G, Fleury-Olela F, Preitner N, Schibler U. Rhythms of mammalian body temperature can sustain peripheral circadian clocks. Current Biology. 2002;**12**(18): 1574-1583. Available from: https:// linkinghub.elsevier.com/retrieve/pii/ S0960-9822(02)01145-4

[118] Zhang M, Xu C, von Wettstein D, Liu B. Tissue-specific differences in cytosine methylation and their association with differential gene expression in sorghum. Plant Physiology. 2011;**156**(4):1955-1966. DOI: 10.1104/pp.111.176842

**65**

**Chapter 5**

*Apiwat Mutirangura*

deterioration in aging-associated NCDs.

**Keywords:** genome-wide hypomethylation, genomic instability, global hypomethylation, DNA damage, youth-associated genomic-stabilizing DNA gaps, youth-DNA-GAPs, physiological replication-independent endogenous DNA

double-strand breaks, RIND-EDSBs, Phy-RIND-EDSBs, aging

**Abstract**

A Hypothesis to Explain How

the DNA of Elderly People Is

Wide Hypomethylation Drives

by Reducing Youth-Associated

Gnome-Stabilizing DNA Gaps

Genomic Instability in the Elderly

Epigenetic changes are how the DNA of elderly people is prone to damage. One role of DNA methylation is to prevent DNA damage. In the elderly and those with aging-associated noncommunicable diseases (NCDs), DNA shows reduced methylation; consequently, the aging genome is unstable and accumulates DNA damage. While the DNA damage response (DDR) of the direct intracellular machinery repairs DNA lesions, too much DDR halts cell proliferation, and promotes senescence. Therefore, genome-wide hypomethylation drives genomic instability, causing aging-associated disease phenotypes. However, the mechanism is unknown. Independent of DNA replication, the eukaryotic genome retains a certain amount of endogenous DNA double-strand breaks (EDSBs), called physiologic replication-independent EDSBs (Phy-RIND-EDSBs), that possess physiological function. Phy-RIND-EDSBs are reduced in aging yeast, and low levels of Phy-RIND-EDSBs decrease cell viability and increase DNA damage. Thus, Phy-RIND-EDSBs have a biological role as youth-associated genomic-stabilizing DNA gaps. In humans, Phy-RIND-EDSBs are located in the hypermethylated genome. Because the genomes of aging people are hypomethylated, the elderly should also have a low level of Phy-RIND-EDSBs. Based on this evidence, I hypothesize that in the human Phy-RIND-EDSBs, reduction is a molecular process that mediates the genome-wide hypomethylation driving genomic instability, which is a nidus pathogenesis mechanism of human body

Prone to Damage: Genome-

## **Chapter 5**

A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide Hypomethylation Drives Genomic Instability in the Elderly by Reducing Youth-Associated Gnome-Stabilizing DNA Gaps

*Apiwat Mutirangura*

## **Abstract**

Epigenetic changes are how the DNA of elderly people is prone to damage. One role of DNA methylation is to prevent DNA damage. In the elderly and those with aging-associated noncommunicable diseases (NCDs), DNA shows reduced methylation; consequently, the aging genome is unstable and accumulates DNA damage. While the DNA damage response (DDR) of the direct intracellular machinery repairs DNA lesions, too much DDR halts cell proliferation, and promotes senescence. Therefore, genome-wide hypomethylation drives genomic instability, causing aging-associated disease phenotypes. However, the mechanism is unknown. Independent of DNA replication, the eukaryotic genome retains a certain amount of endogenous DNA double-strand breaks (EDSBs), called physiologic replication-independent EDSBs (Phy-RIND-EDSBs), that possess physiological function. Phy-RIND-EDSBs are reduced in aging yeast, and low levels of Phy-RIND-EDSBs decrease cell viability and increase DNA damage. Thus, Phy-RIND-EDSBs have a biological role as youth-associated genomic-stabilizing DNA gaps. In humans, Phy-RIND-EDSBs are located in the hypermethylated genome. Because the genomes of aging people are hypomethylated, the elderly should also have a low level of Phy-RIND-EDSBs. Based on this evidence, I hypothesize that in the human Phy-RIND-EDSBs, reduction is a molecular process that mediates the genome-wide hypomethylation driving genomic instability, which is a nidus pathogenesis mechanism of human body deterioration in aging-associated NCDs.

**Keywords:** genome-wide hypomethylation, genomic instability, global hypomethylation, DNA damage, youth-associated genomic-stabilizing DNA gaps, youth-DNA-GAPs, physiological replication-independent endogenous DNA double-strand breaks, RIND-EDSBs, Phy-RIND-EDSBs, aging

## **1. Introduction**

As people age, their bodies begin to deteriorate. Understanding how changes in the DNA of aging people affect cellular function will be an important clue for future prevention and treatment of age-associated noncommunicable diseases (NCDs). Genomic instability, a hallmark of cancer and aging, is defined as a high frequency of mutations within the genome [1, 2]. In cancer, the permanent alteration of the nucleotide sequence of DNA, or mutations, occurring in protooncogenes and tumor suppressor genes lead to cancer development and progression. In the aging process, however, the accumulation of DNA damage, which is an abnormal chemical structure in DNA and includes base modification, base loss, and DNA breaks (which are precursors of mutations), stimulates the DNA damage repair signal (DDR) to induce cells to repair DNA damage [3, 4]. Nevertheless, DDR arrests the cell cycle, rewires cellular metabolism, promotes senescence, and initiates programmed cell death. As a result, too much DDR drives the cellular aging process [3, 4]. Accumulation of DNA damage is found in the elderly and people with age-associated NCDs (**Figure 1**) [5]. Therefore, DNA damage accumulation is a crucial molecular pathogenic mechanism of the aging process. However, the mechanism by which DNA damage spontaneously accumulates in the aging genome remains to be explored.

Both epigenetic marks and DNA damage or lesions are temporary modifications of DNA. However, both are produced by different mechanisms and play roles in genomic instability in opposite directions. Epigenetic marks are produced by biological processes and possess physiological functions [6, 7]. For example, DNA methylation or methyl CpG is produced by DNA methyltransferase. The molecular function of methyl CpG is to interact with a protein such as methyl-CpG-binding protein. This interaction forms a cascade of molecular biological processes for gene regulation control and genomic stability. DNA lesions, on the other hand, are produced by endogenous or exogenous hazards [8]. For example, pyrimidine dimers, one type of DNA lesion, are formed via photochemical reactions such as exposure to UV light. DNA damage is converted into a mutation during subsequent replication, so accumulation of DNA damage leads to genomic instability. This chapter describes that genomic instability in the elderly should occur by the alteration of epigenetic marks leading to spontaneous accumulation of DNA damage.

Global DNA hypomethylation is an epigenetic change in the elderly and people with NCDs that promotes genomic instability [9–13]. However, the underlying mechanism of how the hypomethylated genome accumulates DNA damage is unknown [14]. In 2008, my group discovered an unprecedented type of endogenous DNA double-strand break (EDSB). These breaks are found in all cells, including nondividing cells, so we named them replication-independent EDSBs (RIND-EDSBs) [15]. RIND-EDSBs are located in hypermethylated DNA. Therefore, cells with global hypomethylation, such as cancer cells, have lower levels of RIND-EDSBs than noncancer cells [15]. After the discovery, we explored several characteristics of RIND-EDSBs and found that the majority of RIND-EDSBs possess physiological functions, namely, physiologic RIND-EDSBs (Phy-RIND-EDSBs), as epigenetic marks in maintaining genomic stability [16–19]. Interestingly, Phy-RIND-EDSBs in yeast decrease when yeast cells age [19]. So here I rename Phy-RIND-EDSBs in accordance with their role as youth-associated genomic-stabilizing DNA gaps (Youth-DNA-GAPs). In this chapter, we propose a hypothesis that the hypomethylated genome of the elderly reduces Phy-RIND-EDSBs and that this reduction causes DNA damage. The accumulation of DNA damage initiates DDR and consequently drives the cellular aging process (**Figure 1**).

**67**

**Figure 1.**

*arrest cells, causing metabolic rewiring and senescence.*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

In other words, the reduction in Phy-RIND-EDSBs by genome-wide hypomethylation is the underlying molecular pathogenesis mechanism of aging phenotypes.

*Genome-wide hypomethylation drives genomic instability in the elderly by reducing youth-associated genome-stabilizing DNA gaps: A hypothesis. DNA methylation in the elderly is generally reduced, genomewide hypomethylation. A reduction in DNA methylation leads to genomic instability, accumulation of endogenous DNA damage, and sensitivity to DNA-damaging agents. Here, we propose a hypothesis that global hypomethylation causes a reduction in Phy-RIND-EDSBs and that the reduction in Phy-RIND-EDSBs causes DNA damage. The accumulation of endogenous DNA damage will promote DDR, and too much DDR will* 

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

In other words, the reduction in Phy-RIND-EDSBs by genome-wide hypomethylation is the underlying molecular pathogenesis mechanism of aging phenotypes.

#### **Figure 1.**

*Epigenetics*

**1. Introduction**

remains to be explored.

of DNA damage.

As people age, their bodies begin to deteriorate. Understanding how changes in the DNA of aging people affect cellular function will be an important clue for future prevention and treatment of age-associated noncommunicable diseases (NCDs). Genomic instability, a hallmark of cancer and aging, is defined as a high frequency of mutations within the genome [1, 2]. In cancer, the permanent alteration of the nucleotide sequence of DNA, or mutations, occurring in protooncogenes and tumor suppressor genes lead to cancer development and progression. In the aging process, however, the accumulation of DNA damage, which is an abnormal chemical structure in DNA and includes base modification, base loss, and DNA breaks (which are precursors of mutations), stimulates the DNA damage repair signal (DDR) to induce cells to repair DNA damage [3, 4]. Nevertheless, DDR arrests the cell cycle, rewires cellular metabolism, promotes senescence, and initiates programmed cell death. As a result, too much DDR drives the cellular aging process [3, 4]. Accumulation of DNA damage is found in the elderly and people with age-associated NCDs (**Figure 1**) [5]. Therefore, DNA damage accumulation is a crucial molecular pathogenic mechanism of the aging process. However, the mechanism by which DNA damage spontaneously accumulates in the aging genome

Both epigenetic marks and DNA damage or lesions are temporary modifications of DNA. However, both are produced by different mechanisms and play roles in genomic instability in opposite directions. Epigenetic marks are produced by biological processes and possess physiological functions [6, 7]. For example, DNA methylation or methyl CpG is produced by DNA methyltransferase. The molecular function of methyl CpG is to interact with a protein such as methyl-CpG-binding protein. This interaction forms a cascade of molecular biological processes for gene regulation control and genomic stability. DNA lesions, on the other hand, are produced by endogenous or exogenous hazards [8]. For example, pyrimidine dimers, one type of DNA lesion, are formed via photochemical reactions such as exposure to UV light. DNA damage is converted into a mutation during subsequent replication, so accumulation of DNA damage leads to genomic instability. This chapter describes that genomic instability in the elderly should occur by the alteration of epigenetic marks leading to spontaneous accumulation

Global DNA hypomethylation is an epigenetic change in the elderly and people with NCDs that promotes genomic instability [9–13]. However, the underlying mechanism of how the hypomethylated genome accumulates DNA damage is unknown [14]. In 2008, my group discovered an unprecedented type of endogenous DNA double-strand break (EDSB). These breaks are found in all cells, including nondividing cells, so we named them replication-independent EDSBs (RIND-EDSBs) [15]. RIND-EDSBs are located in hypermethylated DNA. Therefore, cells with global hypomethylation, such as cancer cells, have lower levels of RIND-EDSBs than noncancer cells [15]. After the discovery, we explored several characteristics of RIND-EDSBs and found that the majority of RIND-EDSBs possess physiological functions, namely, physiologic RIND-EDSBs (Phy-RIND-EDSBs), as epigenetic marks in maintaining genomic stability [16–19]. Interestingly, Phy-RIND-EDSBs in yeast decrease when yeast cells age [19]. So here I rename Phy-RIND-EDSBs in accordance with their role as youth-associated genomic-stabilizing DNA gaps (Youth-DNA-GAPs). In this chapter, we propose a hypothesis that the hypomethylated genome of the elderly reduces Phy-RIND-EDSBs and that this reduction causes DNA damage. The accumulation of DNA damage initiates DDR and consequently drives the cellular aging process (**Figure 1**).

**66**

*Genome-wide hypomethylation drives genomic instability in the elderly by reducing youth-associated genome-stabilizing DNA gaps: A hypothesis. DNA methylation in the elderly is generally reduced, genomewide hypomethylation. A reduction in DNA methylation leads to genomic instability, accumulation of endogenous DNA damage, and sensitivity to DNA-damaging agents. Here, we propose a hypothesis that global hypomethylation causes a reduction in Phy-RIND-EDSBs and that the reduction in Phy-RIND-EDSBs causes DNA damage. The accumulation of endogenous DNA damage will promote DDR, and too much DDR will arrest cells, causing metabolic rewiring and senescence.*

## **2. Genome-wide hypomethylation**

Genome-wide hypomethylation reduces the DNA methylation level of the whole genome. DNA methylation possesses two basic roles, gene regulation and the prevention of genomic instability, which we emphasize here [20]. The majority of DNA methylation in the human genome is on interspersed repetitive sequences (IRSs). Genome-wide hypomethylation or global hypomethylation mostly reflects a decrease in the DNA methylation of IRSs [11, 21]. Here, I will describe how IRS methylation occurs, how hypomethylation occurs, and how hypomethylation drives genomic instability in the elderly.

## **2.1 Interspersed repetitive sequence methylation**

To evaluate the global methylation level, most recent studies have used PCR techniques to measure the DNA methylation level of each IRS, including Alu elements (Alu), long interspersed element-1s (LINE-1s), and several types of human endogenous retroviruses (HERVs). A reduction in Alu element methylation represents a genome-wide hypomethylation, driving genomic instability more than that of LINE-1 s and HERVs [11]. Throughout the human genome, there are over 1 million copies of Alu elements [22]. Although there is also a vast number of LINE-1 s, only approximately 3000 copies of LINE-1s contain a 5' UTR where LINE-1 methylation was usually measured [23, 24]. Because there are several classes of HERVs, each PCR measured DNA methylation of one class and as a result, covered a smaller percentage of the genome [25]. Furthermore, methylation of LINE-1 and HERV was reported to possess gene regulation functions [24, 26]. The tissue-specific methylation level of LINE-1 is locus dependent [27, 28]. In contrast, the global hypomethylation occurs as a generalized process [11, 28]. Therefore, methylation of LINE-1 and HERV represents global methylation in a lesser proportion than that of Alu elements.

## **2.2 Alu hypomethylation in aging and NCDs**

Although global hypomethylation has been reported in the elderly, not all IRSs are hypomethylated. We investigated Alu, LINE-1, and HERV-K and found Alu and HERV-K hypomethylation in aging but not LINE-1 [11]. Therefore, methylation of LINE-1 and Alu may possess different roles. Global hypomethylation is also associated with the aging phenotype. First, lower global DNA methylation is associated with higher cardiovascular risk in postmenopausal women [29]. Second, Alu hypomethylation was observed in individuals with lower bone mass, osteopenia, osteoporosis, and a high body mass index [12]. Finally, Alu hypomethylation was reported in diabetes mellitus patients and was directly correlated with high fasting blood sugar, HbA1C, and blood pressure [13]. Interestingly, the Alu methylation level was also high in catch-up growth in a 20-year-old offspring [30]. These studies indicated the positive role of Alu methylation in the human growth process and the role of Alu hypomethylation as an epigenetic cause of the human aging process.

## **2.3 Mechanism causing global hypomethylation**

The direct correlation between IRS methylation levels suggests that the mechanisms causing global hypomethylation in both aging cells and cancer are a generalizing process [11, 28]. The actual mechanism causing global hypomethylation in aging remains to be explored. Nevertheless, exposure to oxidative stress, benzene, air pollution, UV light, radiation, smoke, and folate deficiency facilitates genome-wide hypomethylation processes [31–37]. Therefore, the accumulation of

**69**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

DNA damage, oxidative stress, or a lack of DNA methylation precursors can lead to

Evidence suggests that DNA damage drives the demethylation process. DNA repair, which is how cells remove DNA lesions, is also a demethylation mechanism that directly removes 5-methylcytosine. Methylcytosine is a DNA base that is prone to be deaminated and must be fixed by base excision repair (BER) machinery to prevent cytosine-to-thymine substitution. However, BER replaces the DNA lesion with an unmethylated form of cytosine. As a result, the methylcytosine is demethylated. The other mechanism is to remove the entire DNA patch and refill with unmethylated nucleotides by nucleotide excision repair (NER) or mismatch repair

For oxidative stress, oxidation of 5-methylcytosine forms 5-hydroxymethylcytosine. There are several mechanisms for removing 5-hydroxymethylcytosines and replacing them with unmethylated forms, AID/APOBEC enzymes and TET enzymes followed by BER [39–44]. Alternatively, oxidative stress may interfere with the DNA methylation protein machinery. For example, oxidative stress depletes the synthesis of glutathione and decreases the availability of S-adenosylmethionine for DNA methylation [45]. This proposed mechanism is similar to DNA demethylation in depletion of the methyl pool in folate-deficient

The hypomethylated genome is prone to accumulating multiple kinds of DNA damage, which is an abnormal chemical structure in DNA and includes oxidative damage, depurination, depyrimidination, and pathologic EDSBs [10, 14]. Alu methylation levels in white blood cells were found to inversely correlate with 8-hydroxy-2′-deoxyguanosine (8-OHdG) oxidative damage and apurinic/apyrimidinic sites (AP sites) [37]. Transfection of cells with Alu small interfering RNA (Alu siRNA) increased Alu methylation and reduced endogenous 8-OHdG and AP sites [37]. Interestingly, Alu siRNA also increased cell division and resistance to DNA damage-causing agents [37]. This evidence indirectly suggests that Alu methylation stabilizes the human genome. DNA methylation also prevents pathologic EDSBs. The chromosomal rearrangements and deletions of DNA commonly found in cancer cells treated with DNA demethylating agents and DNA methyltransferase (DNMT) knockout mice and naturally occurring mutations in the cytosine DNA methyltransferase DNMT3B suggest that pathologic EDSBs are the intermediate

**2.4 Hypomethylation accumulates multiple kinds of DNA lesions**

products of hypomethylation that drive genomic instability [10, 48–50].

**2.5 DNA lesions as a molecular pathogenesis mechanism of the aging process and** 

A number of studies support the idea that accumulation of DNA damage drives

the aging process. First, congenital defects in DNA repair accelerate aging. For example, progeroid syndrome patients with ERCC4 mutations have premature aging of many organs. ERCC4 is a protein designated as the DNA repair endonuclease XPF that is critical for many DNA repair pathways, including NER [51]. Second, genotoxic agents accelerate the aging process in cancer survivor patients. For example, 50-year-old survivors of childhood cancer have an increased incidence of age-related diseases compared to their siblings [52]. Third, there is evidence of DNA damage accumulation when cells age. Pathologic EDSBs are accumulated in chronological aging yeast [17]. Many kinds of DNA damage from base modifications to γH2AX foci, representing pathologic EDSBs, have been reported in several organs

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

genome-wide hypomethylation.

(MMR) [38].

models [46, 47].

**NCDs**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

DNA damage, oxidative stress, or a lack of DNA methylation precursors can lead to genome-wide hypomethylation.

Evidence suggests that DNA damage drives the demethylation process. DNA repair, which is how cells remove DNA lesions, is also a demethylation mechanism that directly removes 5-methylcytosine. Methylcytosine is a DNA base that is prone to be deaminated and must be fixed by base excision repair (BER) machinery to prevent cytosine-to-thymine substitution. However, BER replaces the DNA lesion with an unmethylated form of cytosine. As a result, the methylcytosine is demethylated. The other mechanism is to remove the entire DNA patch and refill with unmethylated nucleotides by nucleotide excision repair (NER) or mismatch repair (MMR) [38].

For oxidative stress, oxidation of 5-methylcytosine forms 5-hydroxymethylcytosine. There are several mechanisms for removing 5-hydroxymethylcytosines and replacing them with unmethylated forms, AID/APOBEC enzymes and TET enzymes followed by BER [39–44]. Alternatively, oxidative stress may interfere with the DNA methylation protein machinery. For example, oxidative stress depletes the synthesis of glutathione and decreases the availability of S-adenosylmethionine for DNA methylation [45]. This proposed mechanism is similar to DNA demethylation in depletion of the methyl pool in folate-deficient models [46, 47].

#### **2.4 Hypomethylation accumulates multiple kinds of DNA lesions**

The hypomethylated genome is prone to accumulating multiple kinds of DNA damage, which is an abnormal chemical structure in DNA and includes oxidative damage, depurination, depyrimidination, and pathologic EDSBs [10, 14]. Alu methylation levels in white blood cells were found to inversely correlate with 8-hydroxy-2′-deoxyguanosine (8-OHdG) oxidative damage and apurinic/apyrimidinic sites (AP sites) [37]. Transfection of cells with Alu small interfering RNA (Alu siRNA) increased Alu methylation and reduced endogenous 8-OHdG and AP sites [37]. Interestingly, Alu siRNA also increased cell division and resistance to DNA damage-causing agents [37]. This evidence indirectly suggests that Alu methylation stabilizes the human genome. DNA methylation also prevents pathologic EDSBs. The chromosomal rearrangements and deletions of DNA commonly found in cancer cells treated with DNA demethylating agents and DNA methyltransferase (DNMT) knockout mice and naturally occurring mutations in the cytosine DNA methyltransferase DNMT3B suggest that pathologic EDSBs are the intermediate products of hypomethylation that drive genomic instability [10, 48–50].

#### **2.5 DNA lesions as a molecular pathogenesis mechanism of the aging process and NCDs**

A number of studies support the idea that accumulation of DNA damage drives the aging process. First, congenital defects in DNA repair accelerate aging. For example, progeroid syndrome patients with ERCC4 mutations have premature aging of many organs. ERCC4 is a protein designated as the DNA repair endonuclease XPF that is critical for many DNA repair pathways, including NER [51]. Second, genotoxic agents accelerate the aging process in cancer survivor patients. For example, 50-year-old survivors of childhood cancer have an increased incidence of age-related diseases compared to their siblings [52]. Third, there is evidence of DNA damage accumulation when cells age. Pathologic EDSBs are accumulated in chronological aging yeast [17]. Many kinds of DNA damage from base modifications to γH2AX foci, representing pathologic EDSBs, have been reported in several organs

*Epigenetics*

**2. Genome-wide hypomethylation**

genomic instability in the elderly.

**2.1 Interspersed repetitive sequence methylation**

**2.2 Alu hypomethylation in aging and NCDs**

**2.3 Mechanism causing global hypomethylation**

Genome-wide hypomethylation reduces the DNA methylation level of the whole genome. DNA methylation possesses two basic roles, gene regulation and the prevention of genomic instability, which we emphasize here [20]. The majority of DNA methylation in the human genome is on interspersed repetitive sequences (IRSs). Genome-wide hypomethylation or global hypomethylation mostly reflects a decrease in the DNA methylation of IRSs [11, 21]. Here, I will describe how IRS methylation occurs, how hypomethylation occurs, and how hypomethylation drives

To evaluate the global methylation level, most recent studies have used PCR techniques to measure the DNA methylation level of each IRS, including Alu elements (Alu), long interspersed element-1s (LINE-1s), and several types of human endogenous retroviruses (HERVs). A reduction in Alu element methylation represents a genome-wide hypomethylation, driving genomic instability more than that of LINE-1 s and HERVs [11]. Throughout the human genome, there are over 1 million copies of Alu elements [22]. Although there is also a vast number of LINE-1 s, only approximately 3000 copies of LINE-1s contain a 5' UTR where LINE-1 methylation was usually measured [23, 24]. Because there are several classes of HERVs, each PCR measured DNA methylation of one class and as a result, covered a smaller percentage of the genome [25]. Furthermore, methylation of LINE-1 and HERV was reported to possess gene regulation functions [24, 26]. The tissue-specific methylation level of LINE-1 is locus dependent [27, 28]. In contrast, the global hypomethylation occurs as a generalized process [11, 28]. Therefore, methylation of LINE-1 and HERV repre-

sents global methylation in a lesser proportion than that of Alu elements.

Although global hypomethylation has been reported in the elderly, not all IRSs are hypomethylated. We investigated Alu, LINE-1, and HERV-K and found Alu and HERV-K hypomethylation in aging but not LINE-1 [11]. Therefore, methylation of LINE-1 and Alu may possess different roles. Global hypomethylation is also associated with the aging phenotype. First, lower global DNA methylation is associated with higher cardiovascular risk in postmenopausal women [29]. Second, Alu hypomethylation was observed in individuals with lower bone mass, osteopenia, osteoporosis, and a high body mass index [12]. Finally, Alu hypomethylation was reported in diabetes mellitus patients and was directly correlated with high fasting blood sugar, HbA1C, and blood pressure [13]. Interestingly, the Alu methylation level was also high in catch-up growth in a 20-year-old offspring [30]. These studies indicated the positive role of Alu methylation in the human growth process and the role of Alu hypomethylation as an epigenetic cause of the human aging process.

The direct correlation between IRS methylation levels suggests that the mechanisms causing global hypomethylation in both aging cells and cancer are a generalizing process [11, 28]. The actual mechanism causing global hypomethylation in aging remains to be explored. Nevertheless, exposure to oxidative stress, benzene, air pollution, UV light, radiation, smoke, and folate deficiency facilitates genome-wide hypomethylation processes [31–37]. Therefore, the accumulation of

**68**

of animals and humans [53–56]. Finally, a reduction in DNA repair efficiency was reported in aging cells of many organisms [57–59]. In NCDs, the accumulation of oxidative DNA damage has been reported in patients with cardiovascular disease, diabetes and metabolic syndrome, chronic obstructive pulmonary disease, osteoporosis, and neurological degeneration, including Alzheimer's disease and Parkinson's disease [5]. DNA damage triggers DDR. To facilitate DNA repair and prevent mutation accumulation, DDR arrests cell cycle progression until repair is complete. While DDR can prevent cancer development, DDR leads to many unwanted effects, including inflammation, metabolic rewiring, senescence, apoptosis, and aging [60–62]. The DDR signaling pathway consists of signal sensors, transducers, and effectors. The sensors of this pathway are proteins that recognize DNA damage. The main transducers are ATM and ATR and their downstream kinases. The effectors of this pathway are substrates of ATM and ATR and their downstream kinases. These effectors of DDR involve many proteins, including P53, BRCA1, and CDC25s [60–63].

## **2.6 DNA methylation possesses a long-range effect in stabilizing the human genome in cis**

A direct association between loss of DNA methylation and rearrangements in the pericentromeric heterochromatin was demonstrated in ICF syndrome (immunodeficiency, chromosomal instability, and facial anomalies) and loss-of-function mutations in DNMT3B [50, 64]. Therefore, hypomethylation could lead to spontaneous mutations in cis, which are epigenetic and genetic events occurring in the same chromosome. Notably, Alu siRNA increased Alu methylation levels in HEK293 cells from 60 to 70% [14]. Because there are approximately 1 million copies of Alu, by rough estimation, Alu siRNA methylates 10% of Alu elements or approximately 100,000 Alu elements in 3000 Mb of the human genome. In other words, Alu siRNA transfection methylated one locus of every 30 kb of human genome on average. Furthermore, Alu siRNA reduced 75% of endogenous 8-OHdG [14]. Therefore, even if Alu siRNA increases methylation in a limited location, the transfection stabilized the genome far beyond the methylated Alu elements (**Figure 2**).

## **2.7 Hypotheses: DNA methylation prevents genomic instability mechanisms**

There are at least three possible mechanisms by which Alu methylation reduces endogenous DNA damage and increases resistance to DNA damage-causing agents. The extension of genomic stability from methylated Alu loci supports my first hypothesis that DNA methylation stabilizes the genome by homing Youth-DNA-GAPs, Phy-RIND-EDSBs, and that the gaps extended the stabilizing effect to the entire genome [14]. Another reason that supports the Phy-RIND-EDSBs mediating the DNA methylation role in stabilizing the genome is that Phy-RIND-EDSBs are localized in hypermethylated DNA [15]. Moreover, Phy-RIND-EDSBs possess a redundant topoisomerase which relieve tension of double-helix spin and torsion from any DNA activity [17]. The second hypothesis would be the spreading of DNA methylation and consequently heterochromatin [65]. However, this mechanism is unlikely because the spreading would need to extend to cover the whole genome and would interfere with cellular function. A reduction in cell viability by Alu siRNA was not observed. The last and unlikely hypothesis was that DNA methylation somehow enhanced DNA repair activity [66], although this mechanism is also unlikely because most DNA repair machinery starts with specific sensors to recognize DNA lesions.

**71**

**Figure 2.**

*and white circles are unmethylated DNA.*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

**3. Phy-RIND-EDSBs represent epigenetic marks as youth-DNA-GAPs**

*DNA methylation possesses a long-range effect in stabilizing the human genome in cis. This diagram represents a fraction of the human genome before and after Alu siRNA transfection. While Alu siRNA methylated only 10% of Alu loci, Alu siRNA reduced 75% of the 8-OHdG in the entire genome [14]. Therefore, DNA methylation possesses a long-range effect in stabilizing the human genome. Blue circles are DNA methylation* 

and reduced in chronological aging yeast [15, 17, 19]. A reduction in Phy-RIND-EDSBs decreased cell viability and augmented pathologic EDSB production [19]. Phy-RIND-EDSBs are devoid of DDR and are repaired by the error-free repair pathway [16]. Therefore, Phy-RIND-EDSBs are Youth-DNA-GAPs epigenetic marks

**3.1 IRS-EDSB ligation-mediated PCR (IRS-EDSB-LMPCR) to measure EDSBs**

Ligation-mediated PCR (LMPCR) is the method that we used for EDSB detection [15]. Previously, this PCR technique was used to characterize the signal end and coding end of EDSBs occurring during the V(D)J recombination process [67]. For V(D)J recombination, the signal end and coding end of EDSBs occur at the T-cell receptor or antibody genes in lymphoblasts. To detect the signal end and coding end, DNA from lymphoblasts was ligated to a linker, and PCR was performed using linker primer and oligonucleotide sequences of T-cell receptor or antibody genes. Generalized EDSBs can occur anywhere in the genome. Therefore, we replaced IRS as a primer instead of T-cell receptor or antibody genes [67, 68]. As a result, IRS-EDSB-LMPCR yields two types of amplicons, IRS-EDSB and IRS-IRS sequences, and we detected linker sequences that represent EDSB amplicons. In brief, IRS-EDSB-LMPCR was performed as follows. First, the oligonucleotide linker, EDSB linker, was ligated to high-molecular-weight DNA (HMWDNA) or nucleus. Second, real-time quantitative PCR was performed using two PCR primers. The first was homologous to IRS, and the other had the same sequence as the 5′ end of the ligation linker. The number of EDSBs could be measured by Taqman probe homology to the 3′ end of the ligation linker sequence. The HMWDNA or

that prevent genomic instability in eukaryotic genomes.

Phy-RIND-EDSBs are found in all eukaryotic cells, produced by certain proteins,

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

**Figure 2.**

*Epigenetics*

[60–63].

**genome in cis**

of animals and humans [53–56]. Finally, a reduction in DNA repair efficiency was reported in aging cells of many organisms [57–59]. In NCDs, the accumulation of oxidative DNA damage has been reported in patients with cardiovascular disease, diabetes and metabolic syndrome, chronic obstructive pulmonary disease, osteoporosis, and neurological degeneration, including Alzheimer's disease and Parkinson's disease [5]. DNA damage triggers DDR. To facilitate DNA repair and prevent mutation accumulation, DDR arrests cell cycle progression until repair is complete. While DDR can prevent cancer development, DDR leads to many unwanted effects, including inflammation, metabolic rewiring, senescence, apoptosis, and aging [60–62]. The DDR signaling pathway consists of signal sensors, transducers, and effectors. The sensors of this pathway are proteins that recognize DNA damage. The main transducers are ATM and ATR and their downstream kinases. The effectors of this pathway are substrates of ATM and ATR and their downstream kinases. These effectors of DDR involve many proteins, including P53, BRCA1, and CDC25s

**2.6 DNA methylation possesses a long-range effect in stabilizing the human** 

**2.7 Hypotheses: DNA methylation prevents genomic instability mechanisms**

There are at least three possible mechanisms by which Alu methylation reduces endogenous DNA damage and increases resistance to DNA damage-causing agents. The extension of genomic stability from methylated Alu loci supports my first hypothesis that DNA methylation stabilizes the genome by homing Youth-DNA-GAPs, Phy-RIND-EDSBs, and that the gaps extended the stabilizing effect to the entire genome [14]. Another reason that supports the Phy-RIND-EDSBs mediating the DNA methylation role in stabilizing the genome is that Phy-RIND-EDSBs are localized in hypermethylated DNA [15]. Moreover, Phy-RIND-EDSBs possess a redundant topoisomerase which relieve tension of double-helix spin and torsion from any DNA activity [17]. The second hypothesis would be the spreading of DNA methylation and consequently heterochromatin [65]. However, this mechanism is unlikely because the spreading would need to extend to cover the whole genome and would interfere with cellular function. A reduction in cell viability by Alu siRNA was not observed. The last and unlikely hypothesis was that DNA methylation somehow enhanced DNA repair activity [66], although this mechanism is also unlikely because most DNA repair machinery starts with specific sensors to recognize DNA lesions.

A direct association between loss of DNA methylation and rearrangements in the pericentromeric heterochromatin was demonstrated in ICF syndrome (immunodeficiency, chromosomal instability, and facial anomalies) and loss-of-function mutations in DNMT3B [50, 64]. Therefore, hypomethylation could lead to spontaneous mutations in cis, which are epigenetic and genetic events occurring in the same chromosome. Notably, Alu siRNA increased Alu methylation levels in HEK293 cells from 60 to 70% [14]. Because there are approximately 1 million copies of Alu, by rough estimation, Alu siRNA methylates 10% of Alu elements or approximately 100,000 Alu elements in 3000 Mb of the human genome. In other words, Alu siRNA transfection methylated one locus of every 30 kb of human genome on average. Furthermore, Alu siRNA reduced 75% of endogenous 8-OHdG [14]. Therefore, even if Alu siRNA increases methylation in a limited location, the transfection stabilized the genome far beyond the methylated Alu elements (**Figure 2**).

**70**

*DNA methylation possesses a long-range effect in stabilizing the human genome in cis. This diagram represents a fraction of the human genome before and after Alu siRNA transfection. While Alu siRNA methylated only 10% of Alu loci, Alu siRNA reduced 75% of the 8-OHdG in the entire genome [14]. Therefore, DNA methylation possesses a long-range effect in stabilizing the human genome. Blue circles are DNA methylation and white circles are unmethylated DNA.*

## **3. Phy-RIND-EDSBs represent epigenetic marks as youth-DNA-GAPs**

Phy-RIND-EDSBs are found in all eukaryotic cells, produced by certain proteins, and reduced in chronological aging yeast [15, 17, 19]. A reduction in Phy-RIND-EDSBs decreased cell viability and augmented pathologic EDSB production [19]. Phy-RIND-EDSBs are devoid of DDR and are repaired by the error-free repair pathway [16]. Therefore, Phy-RIND-EDSBs are Youth-DNA-GAPs epigenetic marks that prevent genomic instability in eukaryotic genomes.

#### **3.1 IRS-EDSB ligation-mediated PCR (IRS-EDSB-LMPCR) to measure EDSBs**

Ligation-mediated PCR (LMPCR) is the method that we used for EDSB detection [15]. Previously, this PCR technique was used to characterize the signal end and coding end of EDSBs occurring during the V(D)J recombination process [67]. For V(D)J recombination, the signal end and coding end of EDSBs occur at the T-cell receptor or antibody genes in lymphoblasts. To detect the signal end and coding end, DNA from lymphoblasts was ligated to a linker, and PCR was performed using linker primer and oligonucleotide sequences of T-cell receptor or antibody genes. Generalized EDSBs can occur anywhere in the genome. Therefore, we replaced IRS as a primer instead of T-cell receptor or antibody genes [67, 68]. As a result, IRS-EDSB-LMPCR yields two types of amplicons, IRS-EDSB and IRS-IRS sequences, and we detected linker sequences that represent EDSB amplicons. In brief, IRS-EDSB-LMPCR was performed as follows. First, the oligonucleotide linker, EDSB linker, was ligated to high-molecular-weight DNA (HMWDNA) or nucleus. Second, real-time quantitative PCR was performed using two PCR primers. The first was homologous to IRS, and the other had the same sequence as the 5′ end of the ligation linker. The number of EDSBs could be measured by Taqman probe homology to the 3′ end of the ligation linker sequence. The HMWDNA or

nucleus served as a source of EDSBs, and the EDSB linker detected and ligated EDSBs. The first PCR cycle polymerized DNA from genome-wide distributed IRSs. The polymerization through EDSBs generated an EDSB-LMPCR linker template. The IRS-EDSB-linker sequences were generated, detected, and quantitated by the Taqman probe during PCR cycle (**Figure 3**) [15].

Common criticism of IRS-EDSB-LMPCR is the possibility of DNA shearing from HMWDNA preparation. However, the characteristics of the DSBs generated by DNA preparation are different from RIND-EDSBs. In humans, the sequence around RIND-EDSBs is always hypermethylated, whereas methylation levels of DSBs from mechanical shearing possess less methylation than RIND-EDSBs [15]. To prove that the RIND-EDSBs are real, we compared EDSBs from linker ligated to HMWDNA and nucleus and found that RIND-EDSBs analyzed directly from in situ ligation displayed the same pattern as IRS-EDSB-LMPCR from HMWDNA [17]. Therefore, DSBs detected by IRS-EDSB-LMPCR were endogenous in origin.

## **3.2 Phy-RIND-EDSBs are evolutionarily conserved epigenetic marks**

Nature has conserved all epigenetic marks by conserving the genes that produce epigenetic marks [7]. Epigenetic marks have a specific biological role, whether it is gene expression, genomic stability, or interacting with DNA. Therefore, the genome distribution of epigenetic markers will not be random. Finally, epigenetic marks are usually crucial for cell survival and therefore should be ubiquitously present in all cells. To search for genes that produce or maintain Phy-RIND-EDSBs, we evaluated RIND-EDSB levels in yeast strains that lack functional mutation genes encoding various DNA repair regulators, chromatin formation, endonucleases, topoisomerase, and chromatin-condensing proteins [17]. We found low levels of RIND-EDSBs in cells lacking high-mobility group box (HMGB) proteins and Sir2. Thus, HMGB proteins and Sir2 play roles in producing and maintaining Phy-RIND-EDSBs [17]. Phy-RIND-EDSBs are distributed in the genome nonrandomly [18]. In humans, Phy-RIND-EDSBs are localized within hypermethylated DNA [15]. In yeast, DNA sequences 5′ end to RIND-EDSBs were not random; certain four-nucleotide sequences were more likely to be present immediately prior to the breaks. Moreover, RIND-EDSBs were prevented from occurring or were never observed following certain four-base combinations [18]. RIND-EDSBs were found in yeast and in the human genome, and therefore, Phy-RIND-EDSBs are conserved in eukaryotic organisms [15, 17]. In humans, RIND-EDSBs were detectable in all cell types and found within the hypermethylated genome in all phases of the cell cycle [15]. In yeast, we found a very strong direct correlation between cell viability and Phy-RIND-EDSB levels (r = 0.94, p < 0.0001) [19]. In other words, the more Phy-RIND-EDSBs a cell possesses, the better the cell survives [19]. When Phy-RIND-EDSB levels were reduced by homothallic switching (HO) endonuclease induction or NHP6A gene deletion, cell viability decreased [19]. In conclusion, Phy-RIND-EDSBs are epigenetic markers that are important in all eukaryotic cells [19].

Most of the RIND-EDSBs under normal physiologic conditions are not DNA damage, signals of the DDR, or precursors of mutations [16]. While sequences around human RIND-EDSBs are hypermethylated, γH2AX-binding DNA is hypomethylated. Therefore, most RIND-EDSBs are devoid of γH2AX [16]. γH2AX is a H2AX molecule that is phosphorylated at serine 139 by the signaling cascade of DDR of pathologic DSBs [69]. Most RIND-EDSBs are repaired by a more precise ATM-dependent pathway, and therefore, most RIND-EDSBs under normal physiologic conditions are Phy-RIND-EDSBs [16].

**73**

**Figure 3.**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

*IRS-EDSB-LMPCR diagram demonstrating IRS-EDSB-LMPCR. LMPCR linker ligates to EDSB. The 5*′ *end of the LMPCR linker is the same sequence as the PCR primer. The 3*′ *end of the LMPCR linker is homologous to the Taqman probe. The Taqman probe is used for quantitation of EDSBs by real-time PCR. The IRS primer is* 

*a PCR primer with IRS sequences to polymerize numerous locations of the genome [15].*

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

**Figure 3.**

*Epigenetics*

endogenous in origin.

nucleus served as a source of EDSBs, and the EDSB linker detected and ligated EDSBs. The first PCR cycle polymerized DNA from genome-wide distributed IRSs. The polymerization through EDSBs generated an EDSB-LMPCR linker template. The IRS-EDSB-linker sequences were generated, detected, and quantitated by the

Common criticism of IRS-EDSB-LMPCR is the possibility of DNA shearing from HMWDNA preparation. However, the characteristics of the DSBs generated by DNA preparation are different from RIND-EDSBs. In humans, the sequence around RIND-EDSBs is always hypermethylated, whereas methylation levels of DSBs from mechanical shearing possess less methylation than RIND-EDSBs [15]. To prove that the RIND-EDSBs are real, we compared EDSBs from linker ligated to HMWDNA and nucleus and found that RIND-EDSBs analyzed directly from in situ ligation displayed the same pattern as IRS-EDSB-LMPCR from HMWDNA [17]. Therefore, DSBs detected by IRS-EDSB-LMPCR were

**3.2 Phy-RIND-EDSBs are evolutionarily conserved epigenetic marks**

Nature has conserved all epigenetic marks by conserving the genes that produce epigenetic marks [7]. Epigenetic marks have a specific biological role, whether it is gene expression, genomic stability, or interacting with DNA. Therefore, the genome distribution of epigenetic markers will not be random. Finally, epigenetic marks are usually crucial for cell survival and therefore should be ubiquitously present in all cells. To search for genes that produce or maintain Phy-RIND-EDSBs, we evaluated RIND-EDSB levels in yeast strains that lack functional mutation genes encoding various DNA repair regulators, chromatin formation, endonucleases, topoisomerase, and chromatin-condensing proteins [17]. We found low levels of RIND-EDSBs in cells lacking high-mobility group box (HMGB) proteins and Sir2. Thus, HMGB proteins and Sir2 play roles in producing and maintaining Phy-RIND-EDSBs [17]. Phy-RIND-EDSBs are distributed in the genome nonrandomly [18]. In humans, Phy-RIND-EDSBs are localized within hypermethylated DNA [15]. In yeast, DNA sequences 5′ end to RIND-EDSBs were not random; certain four-nucleotide sequences were more likely to be present immediately prior to the breaks. Moreover, RIND-EDSBs were prevented from occurring or were never observed following certain four-base combinations [18]. RIND-EDSBs were found in yeast and in the human genome, and therefore, Phy-RIND-EDSBs are conserved in eukaryotic organisms [15, 17]. In humans, RIND-EDSBs were detectable in all cell types and found within the hypermethylated genome in all phases of the cell cycle [15]. In yeast, we found a very strong direct correlation between cell viability and Phy-RIND-EDSB levels (r = 0.94, p < 0.0001) [19]. In other words, the more Phy-RIND-EDSBs a cell possesses, the better the cell survives [19]. When Phy-RIND-EDSB levels were reduced by homothallic switching (HO) endonuclease induction or NHP6A gene deletion, cell viability decreased [19]. In conclusion, Phy-RIND-EDSBs are epigenetic markers

Most of the RIND-EDSBs under normal physiologic conditions are not DNA damage, signals of the DDR, or precursors of mutations [16]. While sequences around human RIND-EDSBs are hypermethylated, γH2AX-binding DNA is hypomethylated. Therefore, most RIND-EDSBs are devoid of γH2AX [16]. γH2AX is a H2AX molecule that is phosphorylated at serine 139 by the signaling cascade of DDR of pathologic DSBs [69]. Most RIND-EDSBs are repaired by a more precise ATM-dependent pathway, and therefore, most RIND-EDSBs under normal physi-

Taqman probe during PCR cycle (**Figure 3**) [15].

that are important in all eukaryotic cells [19].

ologic conditions are Phy-RIND-EDSBs [16].

**72**

*IRS-EDSB-LMPCR diagram demonstrating IRS-EDSB-LMPCR. LMPCR linker ligates to EDSB. The 5*′ *end of the LMPCR linker is the same sequence as the PCR primer. The 3*′ *end of the LMPCR linker is homologous to the Taqman probe. The Taqman probe is used for quantitation of EDSBs by real-time PCR. The IRS primer is a PCR primer with IRS sequences to polymerize numerous locations of the genome [15].*

## **3.3 Phy-RIND-EDSB or youth-DNA-GAP complex**

Human Phy-RIND-EDSBs are localized in hypermethylated DNA regions and deacetylated histones [15, 16]. Phy-RIND-EDSBs are reduced in cells lacking HMGB proteins and Sir2 and NAD-dependent deacetylase [17, 70]. The human Sir2 homolog, sirtuin 1 (SIRT1), binds to the HMGB1 protein and deacetylates DNMT1 [71, 72]. Furthermore, HMGB1 possesses deoxyribophosphate lyase activity [73]. Therefore, we propose a hypothesis here that HMGB1 cuts DNA to produce Phy-RIND-EDSBs. SIRT1-bound HMGB1 deacetylates histones, keeping Phy-RIND-EDSB ends within the heterochromatin to shield them from the DDR signal. Finally, the interaction between SIRT1 and DNMT1 or deacetylated histone and DNA methylation may be the reason why sequences around human Phy-RIND-EDSBs are hypermethylated (**Figure 4**).

Interestingly, both HMGB1 and Sir2 have other functions that can be related to Phy-RIND-EDSBs. HMGB1 is a protein in another physiologic EDSB complex, the signal end and coding end of V(D)J recombination [74]. Moreover, yeast lacking the NHP6A protein, a type of yeast HMGB, shows increased endogenous DNA damage and sensitivity to UV light [75]. Finally, HMGB1 has the ability to bend DNA [76]. To prevent DNA torsion, it is reasonable to create Phy-RIND-EDSBs while bending DNA. Sir2 can deacetylate the histone, while Phy-RIND-EDSBs are localized in the deacetylated histone. Interestingly, Sir2 and SIRT1 are known to prevent the aging process [77, 78].

### **3.4 Spontaneous pathologic RIND-EDSBs and modified ends with insertion at the breaks**

Independent of DNA replication, EDSB-LMPCR could detect pathologic RIND-EDSBs (Path-RIND-EDSBs) as excess EDSBs when DSB repair was inhibited by chemical inhibition or DSB repair gene mutation [19]. When we treated G0 yeast cells with caffeine, a DSB repair inhibitor, we observed a spontaneous increase in RIND-EDSBs [19]. These excess RIND-EDSBs did not possess the same 5′ end-sequence-four-base combinations as Phy-RIND-EDSBs odds ratio (OR) > 1 breaks. Notably, we called four-base combinations that are unlikely to be found in Phy-RIND-EDSBs as OR ≤ 1 breaks [18]. Moreover, we also observed that the 5′ end sequence downstream of the break did not match with the genomic sequence from the first base as reads with modified ends with insertion at the breaks (MIBs) [57]. We found that caffeine treatment increased the proportion

#### **Figure 4.**

*Phy-RIND-EDSB or youth-DNA-GAP complex: I hypothesize that the HMGB group initiates Phy-RIND-EDSB and that HMGB interacts with SIR2 or SIRT1. SIRT1 deacetylates histones and DNMT1, and DNMT1 methylates DNA. The role of Phy-RIND-EDSB or youth-DNA-GAP is to prevent DNA damage anywhere along the same chromosome.*

**75**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

RIND-EDSBs are immediately repaired, while Phy-RIND-EDSBs are

of MIBs [57]. Therefore, MIBs might be a mechanism that compensates for repair defects, such as alternate repair of the DSB pathway, or prevents EDSB ends from stimulating DDR. Seven repair defect yeast strains, *mec1*Δ, *mre11*Δ, *nej1*Δ, *rad51*Δ, *tel1*Δ, *yku70*Δ, and *yku80*Δ, were studied. Except for *nej1*Δ, the percentages of OR ≤ 1 breaks and MIBs were significantly increased in all samples when compared to the wild type. We also examined whether there was an association between MIBs and types of breaks (OR > 1 breaks and OR ≤ 1 breaks) and found that in the wild type, MIBs occurred at OR ≤ 1 breaks. In contrast, in *mec1*Δ, *mre11*Δ, *rad51*Δ, *tel1*Δ, *yku70*Δ, MIBs occurred at both OR > 1 breaks and OR ≤ 1 breaks [57]. Therefore, both Phy-RIND-EDSBs and Path-RIND-EDSBs are produced in the genome independent of DNA replication. However, most Path-

**3.5 Variation in RIND-EDSB level and reduction in Phy-RIND-EDSBs in aging** 

Path-RIND-EDSBs are spontaneously produced and immediately repaired, while

Phy-RIND-EDSBs are produced and retained by the Phy-RIND-EDSB complex formation process. We observed an increase in RIND-EDSB levels in yeast lacking a DSB repair gene, topoisomerase and endonuclease. Analysis of EDSB sequences suggested that DSB repair inhibition causes retention of both Phy-RIND-EDSBs and Path-RIND-EDSBs. For topoisomerase and endonuclease mutants, we postulated that Phy-RIND-EDSBs may have redundant roles with topoisomerase and endonuclease in stabilizing the genome. Nevertheless, sequence analysis is needed to prove this hypothesis. As mentioned earlier, one yeast strain lacking the *HMGB* gene or *SIR2* possessed a low level of RIND-EDSBs. Therefore, we hypothesized that HMGB and Sir2 play roles in PHY-RIND-EDSB complex formation and retention. We observed that three chemicals can alter RIND-EDSB levels. Whereas caffeine and vanillin, DSB repair inhibitors, increased RIND-EDSB levels, trichostatin A, a histone deacetylase inhibitor, decreased the EDSBs. The reduction in RIND-EDSBs by trichostatin A suggested that Phy-RIND-EDSBs are retained within facultative heterochromatin. This result is similar to the low level of RIND-EDSBs in yeast

DDR signals to repair Path-RIND-EDSBs can repair and consequently reduce Phy-RIND-EDSBs [19]. The retention of Phy-RIND-EDSBs and the immediate repair of Path-RIND-EDSBs led to the finding that the majority of RIND-EDSBs under normal physiologic conditions are Phy-RIND-EDSBs and that a reduction in RIND-EDSBs in any condition is a reduction in Phy-RIND-EDSBs. In addition to gene mutation and histone acetylation, we could reduce RIND-EDSB levels in yeast by inducing a Path-RIND-EDSB by HO endonuclease induction [19]. HO endonuclease is a site-specific endonuclease that cleaves a site in the MAT locus on chromosome III [79]. After induction in nondividing yeast, we observed a sustained reduction in RIND-EDSBs for up to 4 days. However, when we induced HO in yeast lacking *MEC1*, a DSB repair protein, the reduction was not observed [19]. These experiments suggested that Path-RIND-EDSB production can ignite the global DSB repair process, and consequently, the retained Phy-RIND-EDSBs are repaired [19]. This mechanism is one possible explanation for the reduction in RIND-EDSBs in

Phy-RIND-EDSB levels in the elderly should be low. We found low levels of RIND-EDSBs in chronologically aging yeast and in the human cancer cells, HeLa and SW480, which are cervical cancer and colon cancer cell lines, respectively [15, 19]. Phy-RIND-EDSBs are localized in hypermethylated genomic regions [15].

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

retained [57].

lacking *SIR2*.

chronologically aging yeast.

**and hypomethylated cells**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

of MIBs [57]. Therefore, MIBs might be a mechanism that compensates for repair defects, such as alternate repair of the DSB pathway, or prevents EDSB ends from stimulating DDR. Seven repair defect yeast strains, *mec1*Δ, *mre11*Δ, *nej1*Δ, *rad51*Δ, *tel1*Δ, *yku70*Δ, and *yku80*Δ, were studied. Except for *nej1*Δ, the percentages of OR ≤ 1 breaks and MIBs were significantly increased in all samples when compared to the wild type. We also examined whether there was an association between MIBs and types of breaks (OR > 1 breaks and OR ≤ 1 breaks) and found that in the wild type, MIBs occurred at OR ≤ 1 breaks. In contrast, in *mec1*Δ, *mre11*Δ, *rad51*Δ, *tel1*Δ, *yku70*Δ, MIBs occurred at both OR > 1 breaks and OR ≤ 1 breaks [57]. Therefore, both Phy-RIND-EDSBs and Path-RIND-EDSBs are produced in the genome independent of DNA replication. However, most Path-RIND-EDSBs are immediately repaired, while Phy-RIND-EDSBs are retained [57].

### **3.5 Variation in RIND-EDSB level and reduction in Phy-RIND-EDSBs in aging and hypomethylated cells**

Path-RIND-EDSBs are spontaneously produced and immediately repaired, while Phy-RIND-EDSBs are produced and retained by the Phy-RIND-EDSB complex formation process. We observed an increase in RIND-EDSB levels in yeast lacking a DSB repair gene, topoisomerase and endonuclease. Analysis of EDSB sequences suggested that DSB repair inhibition causes retention of both Phy-RIND-EDSBs and Path-RIND-EDSBs. For topoisomerase and endonuclease mutants, we postulated that Phy-RIND-EDSBs may have redundant roles with topoisomerase and endonuclease in stabilizing the genome. Nevertheless, sequence analysis is needed to prove this hypothesis. As mentioned earlier, one yeast strain lacking the *HMGB* gene or *SIR2* possessed a low level of RIND-EDSBs. Therefore, we hypothesized that HMGB and Sir2 play roles in PHY-RIND-EDSB complex formation and retention. We observed that three chemicals can alter RIND-EDSB levels. Whereas caffeine and vanillin, DSB repair inhibitors, increased RIND-EDSB levels, trichostatin A, a histone deacetylase inhibitor, decreased the EDSBs. The reduction in RIND-EDSBs by trichostatin A suggested that Phy-RIND-EDSBs are retained within facultative heterochromatin. This result is similar to the low level of RIND-EDSBs in yeast lacking *SIR2*.

DDR signals to repair Path-RIND-EDSBs can repair and consequently reduce Phy-RIND-EDSBs [19]. The retention of Phy-RIND-EDSBs and the immediate repair of Path-RIND-EDSBs led to the finding that the majority of RIND-EDSBs under normal physiologic conditions are Phy-RIND-EDSBs and that a reduction in RIND-EDSBs in any condition is a reduction in Phy-RIND-EDSBs. In addition to gene mutation and histone acetylation, we could reduce RIND-EDSB levels in yeast by inducing a Path-RIND-EDSB by HO endonuclease induction [19]. HO endonuclease is a site-specific endonuclease that cleaves a site in the MAT locus on chromosome III [79]. After induction in nondividing yeast, we observed a sustained reduction in RIND-EDSBs for up to 4 days. However, when we induced HO in yeast lacking *MEC1*, a DSB repair protein, the reduction was not observed [19]. These experiments suggested that Path-RIND-EDSB production can ignite the global DSB repair process, and consequently, the retained Phy-RIND-EDSBs are repaired [19]. This mechanism is one possible explanation for the reduction in RIND-EDSBs in chronologically aging yeast.

Phy-RIND-EDSB levels in the elderly should be low. We found low levels of RIND-EDSBs in chronologically aging yeast and in the human cancer cells, HeLa and SW480, which are cervical cancer and colon cancer cell lines, respectively [15, 19]. Phy-RIND-EDSBs are localized in hypermethylated genomic regions [15].

*Epigenetics*

**3.3 Phy-RIND-EDSB or youth-DNA-GAP complex**

hypermethylated (**Figure 4**).

process [77, 78].

**the breaks**

Human Phy-RIND-EDSBs are localized in hypermethylated DNA regions and deacetylated histones [15, 16]. Phy-RIND-EDSBs are reduced in cells lacking HMGB proteins and Sir2 and NAD-dependent deacetylase [17, 70]. The human Sir2 homolog, sirtuin 1 (SIRT1), binds to the HMGB1 protein and deacetylates DNMT1 [71, 72]. Furthermore, HMGB1 possesses deoxyribophosphate lyase activity [73]. Therefore, we propose a hypothesis here that HMGB1 cuts DNA to produce Phy-RIND-EDSBs. SIRT1-bound HMGB1 deacetylates histones, keeping Phy-RIND-EDSB ends within the heterochromatin to shield them from the DDR signal. Finally, the interaction between SIRT1 and DNMT1 or deacetylated histone and DNA methylation may be the reason why sequences around human Phy-RIND-EDSBs are

Interestingly, both HMGB1 and Sir2 have other functions that can be related to Phy-RIND-EDSBs. HMGB1 is a protein in another physiologic EDSB complex, the signal end and coding end of V(D)J recombination [74]. Moreover, yeast lacking the NHP6A protein, a type of yeast HMGB, shows increased endogenous DNA damage and sensitivity to UV light [75]. Finally, HMGB1 has the ability to bend DNA [76]. To prevent DNA torsion, it is reasonable to create Phy-RIND-EDSBs while bending DNA. Sir2 can deacetylate the histone, while Phy-RIND-EDSBs are localized in the deacetylated histone. Interestingly, Sir2 and SIRT1 are known to prevent the aging

**3.4 Spontaneous pathologic RIND-EDSBs and modified ends with insertion at** 

Independent of DNA replication, EDSB-LMPCR could detect pathologic RIND-EDSBs (Path-RIND-EDSBs) as excess EDSBs when DSB repair was inhibited by chemical inhibition or DSB repair gene mutation [19]. When we treated G0 yeast cells with caffeine, a DSB repair inhibitor, we observed a spontaneous increase in RIND-EDSBs [19]. These excess RIND-EDSBs did not possess the same 5′ end-sequence-four-base combinations as Phy-RIND-EDSBs odds ratio (OR) > 1 breaks. Notably, we called four-base combinations that are unlikely to be found in Phy-RIND-EDSBs as OR ≤ 1 breaks [18]. Moreover, we also observed that the 5′ end sequence downstream of the break did not match with the genomic sequence from the first base as reads with modified ends with insertion at the breaks (MIBs) [57]. We found that caffeine treatment increased the proportion

*Phy-RIND-EDSB or youth-DNA-GAP complex: I hypothesize that the HMGB group initiates Phy-RIND-EDSB and that HMGB interacts with SIR2 or SIRT1. SIRT1 deacetylates histones and DNMT1, and DNMT1 methylates DNA. The role of Phy-RIND-EDSB or youth-DNA-GAP is to prevent DNA damage anywhere* 

**74**

**Figure 4.**

*along the same chromosome.*

Therefore, cancer genome hypomethylation may explain why RIND-EDSB levels in cancer cells were low [15, 27]. We have not reported RIND-EDSB levels in the elderly. However, our unpublished data demonstrated results similar to those in chronologically aging yeast and in cancer cells.

## **3.6 Reduction in Phy-RIND-EDSBs augments pathologic EDSB production**

To define the molecular mechanism by which the reduction in Phy-RIND-EDSBs in chronological aging in yeast reduced cell viability and to evaluate the consequences of Phy-RIND-EDSB reduction, we analyzed yeast cells with low levels of Phy-RIND-EDSBs, including HO endonuclease and *nhp6a*∆, a highmobility group box protein mutant [19]. Very high levels of Path-RIND-EDSBs were observed in both strains possessing low levels of Phy-RIND-EDSBs after treatment with caffeine, a DSB repair inhibitor. The new Path-RIND-EDSBs were not in the same location as Phy-RIND-EDSBs. Therefore, similar to DNA methylation, Phy-RIND-EDSB stabilizes the genome far beyond the Phy-RIND-EDSB complex (**Figure 4**). These experiments led to my conclusion that the role of Phy-RIND-EDSBs is similar to that of EDSBs induced by topoisomerase, which is DNA torsion prevention and DNA tension reduction from DNA spinning due to any DNA activity, including transcription, replication, and repair. The role of Phy-RIND-EDSBs can be imagined as gaps in a railroad track that prevent track torsion from track expansion by heat. Phy-RIND-EDSB levels decreased in chronologically aging yeast, and the reduction was directly correlated with reduced cell viability. Therefore, Phy-RIND-EDSBs play a Youth-DNA-GAPs role in preventing Path-RIND-EDSBs and DNA damage lesions [19]. Moreover, the *nhp6a* gene is known to prevent other types of DNA lesions, such as pyrimidine dimers [75]. Therefore, it is reasonable to hypothesize that the scatter distribution of the Phy-RIND-EDSB complex prevents all kinds of DNA damage along the length of the whole genome (**Figure 4**).

## **3.7 DNA repair activity may be compromised in aging cells by a reduction in Phy-RIND-EDSBs**

A reduction in Phy-RIND-EDSBs during chronological aging may be a cause of DNA repair defects in the elderly. DNA repair machinery is known to be compromised and error-prone with age [59, 75]. Numerous studies have found a significant decline in all commonly known repair pathway activities with aging, including double-strand break repair activities [53–56]. We demonstrated that the reduction in the Phy-RIND-EDSB complex will increase the production of DNA damage [19]. Therefore, aging cells have to repair DNA damage more often than younger cells. As a result, more DNA repair machinery is required for older cells. Consequently, DNA repair substrates are consumed more quickly than they are produced, resulting in DNA repair defects in the elderly.

## **4. Conclusion**

All evidences described in this chapter suggest that genomic instability in the elderly is a vicious cycle of interactive networks among DNA damage, DNA repair, DNA demethylation, and reduction in Youth-DNA-GAPs (**Figure 5**). DNA damage occurs spontaneously. Then, the DNA repair process, in addition to repairing DNA damage, has consequences of reducing epigenetic marks. While NER demethylates DNA, the DSB repair pathway will repair Phy-RIND-EDSBs. DNA demethylation

**77**

**Figure 5.**

*and DDR arrests and ages cells.*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

results in global hypomethylation and consequently reduces the homing of Phy-RIND-EDSBs. The depletion of the Phy-RIND-EDSB complex will then augment DNA damage production. Cells need to use many DNA repair substrates to eliminate DNA damage faster than these substrates are produced and eventually lose the capability of DNA repair. As a result, aging cells continue to accumulate DNA damage and send DDR signals, halting the cell cycle, causing metabolic rewiring,

*Destructive network of aging DNA. DNA damage can occur spontaneously. The base modification repair consequence is DNA demethylation, and DSB repair for pathologic DSB will also globally repair Phy-RIND-EDSBs. Continuous DNA demethylation results in genome-wide hypomethylation, which, together with global Phy-RIND-EDSB repair, reduces the Phy-RIND-EDSB complex. A reduction in the Phy-RIND-EDSB complex augments DNA damage, and a large amount of DNA damage requires extensive DDR. Cells extensively use DDR until the DNA repair machinery is exhausted and defective, at which point, DNA damage accumulates* 

and eventually driving cells to enter senescence.

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

#### **Figure 5.**

*Epigenetics*

(**Figure 4**).

**Phy-RIND-EDSBs**

DNA repair defects in the elderly.

**4. Conclusion**

Therefore, cancer genome hypomethylation may explain why RIND-EDSB levels in cancer cells were low [15, 27]. We have not reported RIND-EDSB levels in the elderly. However, our unpublished data demonstrated results similar to those in

**3.6 Reduction in Phy-RIND-EDSBs augments pathologic EDSB production**

To define the molecular mechanism by which the reduction in Phy-RIND-EDSBs in chronological aging in yeast reduced cell viability and to evaluate the consequences of Phy-RIND-EDSB reduction, we analyzed yeast cells with low levels of Phy-RIND-EDSBs, including HO endonuclease and *nhp6a*∆, a highmobility group box protein mutant [19]. Very high levels of Path-RIND-EDSBs were observed in both strains possessing low levels of Phy-RIND-EDSBs after treatment with caffeine, a DSB repair inhibitor. The new Path-RIND-EDSBs were not in the same location as Phy-RIND-EDSBs. Therefore, similar to DNA methylation, Phy-RIND-EDSB stabilizes the genome far beyond the Phy-RIND-EDSB complex (**Figure 4**). These experiments led to my conclusion that the role of Phy-RIND-EDSBs is similar to that of EDSBs induced by topoisomerase, which is DNA torsion prevention and DNA tension reduction from DNA spinning due to any DNA activity, including transcription, replication, and repair. The role of Phy-RIND-EDSBs can be imagined as gaps in a railroad track that prevent track torsion from track expansion by heat. Phy-RIND-EDSB levels decreased in chronologically aging yeast, and the reduction was directly correlated with reduced cell viability. Therefore, Phy-RIND-EDSBs play a Youth-DNA-GAPs role in preventing Path-RIND-EDSBs and DNA damage lesions [19]. Moreover, the *nhp6a* gene is known to prevent other types of DNA lesions, such as pyrimidine dimers [75]. Therefore, it is reasonable to hypothesize that the scatter distribution of the Phy-RIND-EDSB complex prevents all kinds of DNA damage along the length of the whole genome

**3.7 DNA repair activity may be compromised in aging cells by a reduction in** 

A reduction in Phy-RIND-EDSBs during chronological aging may be a cause of DNA repair defects in the elderly. DNA repair machinery is known to be compromised and error-prone with age [59, 75]. Numerous studies have found a significant decline in all commonly known repair pathway activities with aging, including double-strand break repair activities [53–56]. We demonstrated that the reduction in the Phy-RIND-EDSB complex will increase the production of DNA damage [19]. Therefore, aging cells have to repair DNA damage more often than younger cells. As a result, more DNA repair machinery is required for older cells. Consequently, DNA repair substrates are consumed more quickly than they are produced, resulting in

All evidences described in this chapter suggest that genomic instability in the elderly is a vicious cycle of interactive networks among DNA damage, DNA repair, DNA demethylation, and reduction in Youth-DNA-GAPs (**Figure 5**). DNA damage occurs spontaneously. Then, the DNA repair process, in addition to repairing DNA damage, has consequences of reducing epigenetic marks. While NER demethylates DNA, the DSB repair pathway will repair Phy-RIND-EDSBs. DNA demethylation

chronologically aging yeast and in cancer cells.

**76**

*Destructive network of aging DNA. DNA damage can occur spontaneously. The base modification repair consequence is DNA demethylation, and DSB repair for pathologic DSB will also globally repair Phy-RIND-EDSBs. Continuous DNA demethylation results in genome-wide hypomethylation, which, together with global Phy-RIND-EDSB repair, reduces the Phy-RIND-EDSB complex. A reduction in the Phy-RIND-EDSB complex augments DNA damage, and a large amount of DNA damage requires extensive DDR. Cells extensively use DDR until the DNA repair machinery is exhausted and defective, at which point, DNA damage accumulates and DDR arrests and ages cells.*

results in global hypomethylation and consequently reduces the homing of Phy-RIND-EDSBs. The depletion of the Phy-RIND-EDSB complex will then augment DNA damage production. Cells need to use many DNA repair substrates to eliminate DNA damage faster than these substrates are produced and eventually lose the capability of DNA repair. As a result, aging cells continue to accumulate DNA damage and send DDR signals, halting the cell cycle, causing metabolic rewiring, and eventually driving cells to enter senescence.

## **Acknowledgements**

All studies in Thailand were supported by the National Science and Technology Development Agency, Thailand Research Fund, and Chulalongkorn University, Thailand. I thank Dr. Maturada Patchsung, Ms Papitchaya Watcharanurak, and Ms. Sirapat Settayanon for illustration design.

## **Conflict of interest**

The author declares no conflict of interest.

## **Abbreviation list**


**79**

**Author details**

Apiwat Mutirangura

Bangkok, Thailand

provided the original work is properly cited.

and apiwat.mutirangura@gmail.com

\*Address all correspondence to: mapiwat@chula.ac.th

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Anatomy, Faculty of Medicine, Institution(s), Center for Excellence in Molecular Genetics of Cancer and Human Diseases, Chulalongkorn University,

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

## **Author details**

*Epigenetics*

**Acknowledgements**

**Conflict of interest**

**Abbreviation list**

Sirapat Settayanon for illustration design.

The author declares no conflict of interest.

NCDs noncommunicable disease DDR DNA damage repair signal

Phy-RIND-EDSBs physiologic RIND-EDSBs

Alu Alu elements

BER base excision repair NER nucleotide excision repair

MMR mismatch repair

IRSs interspersed repetitive sequences

LINE-1s long interspersed element-1s HERVs human endogenous retroviruses

8-OHdG 8-hydroxy-2′-deoxyguanosine AP sites apurinic/apyrimidinic sites DNMT DNA methyltransferase Alu siRNA Alu small interfering RNA LMPCR ligation-mediated PCR HMW DNA high-molecular-weight DNA IRS-EDSB-LMPCR IRS-EDSB ligation-mediated PCR

HMGB high-mobility group box

Path-RIND-EDSBs pathologic RIND-EDSBs HO homothallic switching

MIBs modified ends with insertion at the breaks

SIRT1 sirtuin 1

OR odds ratio

EDSB endogenous DNA double-strand break RIND-EDSB replication-independent endogenous DNA double-strand break

Youth-DNA-GAPs youth-associated genomic-stabilizing DNA gaps

All studies in Thailand were supported by the National Science and Technology Development Agency, Thailand Research Fund, and Chulalongkorn University, Thailand. I thank Dr. Maturada Patchsung, Ms Papitchaya Watcharanurak, and Ms.

**78**

Apiwat Mutirangura Department of Anatomy, Faculty of Medicine, Institution(s), Center for Excellence in Molecular Genetics of Cancer and Human Diseases, Chulalongkorn University, Bangkok, Thailand

\*Address all correspondence to: mapiwat@chula.ac.th and apiwat.mutirangura@gmail.com

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Lopez-Otin C et al. The hallmarks of aging. Cell. 2013;**153**(6):1194-1217

[2] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;**144**(5):646-674

[3] Schumacher B, Garinis GA, Hoeijmakers JH. Age to survive: DNA damage and aging. Trends in Genetics. 2008;**24**(2):77-85

[4] Olivieri F et al. DNA damage response (DDR) and senescence: Shuttled inflamma-miRNAs on the stage of inflamm-aging. Oncotarget. 2015;**6**(34):35509-35521

[5] Milic M et al. DNA damage in noncommunicable diseases: A clinical and epidemiological perspective. Mutation Research. 2015;**776**:118-127

[6] Huang B, Jiang C, Zhang R. Epigenetics: The language of the cell? Epigenomics. 2014;**6**(1):73-88

[7] Mazzio EA, Soliman KF. Basic concepts of epigenetics: Impact of environmental signals on gene expression. Epigenetics. 2012;**7**(2):119-130

[8] De Bont R, van Larebeke N. Endogenous DNA damage in humans: A review of quantitative data. Mutagenesis. 2004;**19**(3):169-185

[9] Unnikrishnan A et al. Revisiting the genomic hypomethylation hypothesis of aging. Annals of the New York Academy of Sciences. 2018;**1418**(1):69-79

[10] Chen RZ et al. DNA hypomethylation leads to elevated mutation rates. Nature. 1998;**395**(6697):89-93

[11] Jintaridth P, Mutirangura A. Distinctive patterns of agedependent hypomethylation in interspersed repetitive sequences. Physiological Genomics. 2010;**41**(2):194-200

[12] Jintaridth P et al. Hypomethylation of Alu elements in post-menopausal women with osteoporosis. PLoS One. 2013;**8**(8):e70386

[13] Thongsroy J, Patchsung M, Mutirangura A. The association between Alu hypomethylation and severity of type 2 diabetes mellitus. Clinical Epigenetics. 2017;**9**:93

[14] Patchsung M et al. Alu siRNA to increase Alu element methylation and prevent DNA damage. Epigenomics. 2018;**10**(2):175-185

[15] Pornthanakasem W et al. LINE-1 methylation status of endogenous DNA double-strand breaks. Nucleic Acids Research. 2008;**36**(11):3667-3675

[16] Kongruttanachok N et al. Replication independent DNA doublestrand break retention may prevent genomic instability. Molecular Cancer. 2010;**9**:70

[17] Thongsroy J et al. Replicationindependent endogenous DNA doublestrand breaks in *Saccharomyces cerevisiae* model. PLoS One. 2013;**8**(8):e72706

[18] Pongpanich M et al. Characteristics of replication-independent endogenous double-strand breaks in *Saccharomyces cerevisiae*. BMC Genomics. 2014;**15**:750

[19] Thongsroy J et al. Reduction in replication-independent endogenous DNA double-strand breaks promotes genomic instability during chronological aging in yeast. The FASEB Journal. 2018:fj201800218RR

[20] Tirado-Magallanes R et al. Whole genome DNA methylation: Beyond genes silencing. Oncotarget. 2017;**8**(3):5629-5637

**81**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

[31] Bollati V et al. Changes in DNA methylation patterns in subjects exposed to low-dose benzene. Cancer

[32] Mittal A et al. Exceptionally high protection of photocarcinogenesis

(−-)-epigallocatechin-3-gallate in hydrophilic cream in SKH-1 hairless mouse model: Relationship to inhibition of UVB-induced global DNA hypomethylation. Neoplasia.

[33] Koturbash I et al. Radiation-

[34] Puttipanyalears C et al. Alu hypomethylation in smoke-exposed epithelia and oral squamous carcinoma.

Asian Pacific Journal of Cancer Prevention. 2013;**14**(9):5495-5501

[35] Wangsri S et al. Patterns and possible roles of LINE-1 methylation changes in smoke-exposed epithelia.

[36] Crider KS et al. Folate and DNA methylation: A review of molecular mechanisms and the evidence for folate's role. Advances in Nutrition.

[38] Grin I, Ishchenko AA. An interplay

of the base excision repair and mismatch repair pathways in active DNA demethylation. Nucleic Acids Research. 2016;**44**(8):3713-3727

[39] Wu SC, Zhang Y. Active DNA demethylation: Many roads lead to Rome. Nature Reviews. Molecular Cell

Biology. 2010;**11**(9):607-620

PLoS One. 2012;**7**(9):e45292

[37] Patchsung M et al. Long interspersed nuclear element-1 hypomethylation and oxidative stress: Correlation and bladder cancer diagnostic potential. PLoS One.

2012;**3**(1):21-38

2012;**7**(5):e37009

induced changes in DNA methylation of repetitive elements in the mouse heart. Mutation Research. 2016;**787**:43-53

Research. 2007;**67**(3):876-880

by topical application of

2003;**5**(6):555-565

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

[21] Zheng Y et al. Prediction of genome-wide DNA methylation in repetitive elements. Nucleic Acids Research. 2017;**45**(15):8697-8711

[22] International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004;**431**(7011):931-945

[23] Penzkofer T et al. L1Base 2: More retrotransposition-active LINE-1s, more mammalian genomes. Nucleic Acids Research. 2017;**45**(D1):D68-D73

[25] Gifford RJ et al. Nomenclature for endogenous retrovirus (ERV) loci. Retrovirology. 2018;**15**(1):59

Enrichment analysis of specific human endogenous retrovirus patterns and their neighboring genes. PLoS One.

[27] Chalitchagorn K et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene.

methylation patterns of different loci in normal and cancerous cells. Nucleic Acids Research. 2008;**36**(17):5704-5712

[26] Tongyoo P et al. EnHERV:

[24] Aporntewan C et al. Hypomethylation of intragenic LINE-1 represses transcription in cancer cells through AGO2. PLoS One.

2011;**6**(3):e17934

2017;**12**(5):e0177119

2004;**23**(54):8841-8846

Genetics. 2016;**17**(1):71

2015;**10**(3):e0120032

[28] Phokaew C et al. LINE-1

[29] Ramos RB et al. Association between global leukocyte DNA methylation and cardiovascular risk in postmenopausal women. BMC Medical

[30] Rerkasem K et al. Higher Alu methylation levels in catch-up growth in twenty-year-old offsprings. PLoS One.

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

[21] Zheng Y et al. Prediction of genome-wide DNA methylation in repetitive elements. Nucleic Acids Research. 2017;**45**(15):8697-8711

[22] International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature. 2004;**431**(7011):931-945

[23] Penzkofer T et al. L1Base 2: More retrotransposition-active LINE-1s, more mammalian genomes. Nucleic Acids Research. 2017;**45**(D1):D68-D73

[24] Aporntewan C et al. Hypomethylation of intragenic LINE-1 represses transcription in cancer cells through AGO2. PLoS One. 2011;**6**(3):e17934

[25] Gifford RJ et al. Nomenclature for endogenous retrovirus (ERV) loci. Retrovirology. 2018;**15**(1):59

[26] Tongyoo P et al. EnHERV: Enrichment analysis of specific human endogenous retrovirus patterns and their neighboring genes. PLoS One. 2017;**12**(5):e0177119

[27] Chalitchagorn K et al. Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene. 2004;**23**(54):8841-8846

[28] Phokaew C et al. LINE-1 methylation patterns of different loci in normal and cancerous cells. Nucleic Acids Research. 2008;**36**(17):5704-5712

[29] Ramos RB et al. Association between global leukocyte DNA methylation and cardiovascular risk in postmenopausal women. BMC Medical Genetics. 2016;**17**(1):71

[30] Rerkasem K et al. Higher Alu methylation levels in catch-up growth in twenty-year-old offsprings. PLoS One. 2015;**10**(3):e0120032

[31] Bollati V et al. Changes in DNA methylation patterns in subjects exposed to low-dose benzene. Cancer Research. 2007;**67**(3):876-880

[32] Mittal A et al. Exceptionally high protection of photocarcinogenesis by topical application of (−-)-epigallocatechin-3-gallate in hydrophilic cream in SKH-1 hairless mouse model: Relationship to inhibition of UVB-induced global DNA hypomethylation. Neoplasia. 2003;**5**(6):555-565

[33] Koturbash I et al. Radiationinduced changes in DNA methylation of repetitive elements in the mouse heart. Mutation Research. 2016;**787**:43-53

[34] Puttipanyalears C et al. Alu hypomethylation in smoke-exposed epithelia and oral squamous carcinoma. Asian Pacific Journal of Cancer Prevention. 2013;**14**(9):5495-5501

[35] Wangsri S et al. Patterns and possible roles of LINE-1 methylation changes in smoke-exposed epithelia. PLoS One. 2012;**7**(9):e45292

[36] Crider KS et al. Folate and DNA methylation: A review of molecular mechanisms and the evidence for folate's role. Advances in Nutrition. 2012;**3**(1):21-38

[37] Patchsung M et al. Long interspersed nuclear element-1 hypomethylation and oxidative stress: Correlation and bladder cancer diagnostic potential. PLoS One. 2012;**7**(5):e37009

[38] Grin I, Ishchenko AA. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation. Nucleic Acids Research. 2016;**44**(8):3713-3727

[39] Wu SC, Zhang Y. Active DNA demethylation: Many roads lead to Rome. Nature Reviews. Molecular Cell Biology. 2010;**11**(9):607-620

**80**

*Epigenetics*

**References**

2011;**144**(5):646-674

2008;**24**(2):77-85

[3] Schumacher B, Garinis GA, Hoeijmakers JH. Age to survive: DNA damage and aging. Trends in Genetics.

[4] Olivieri F et al. DNA damage response (DDR) and senescence: Shuttled inflamma-miRNAs on the stage of inflamm-aging. Oncotarget.

[5] Milic M et al. DNA damage in noncommunicable diseases: A clinical and epidemiological perspective. Mutation

R. Epigenetics: The language of the cell?

2015;**6**(34):35509-35521

Research. 2015;**776**:118-127

[6] Huang B, Jiang C, Zhang

Epigenomics. 2014;**6**(1):73-88

2012;**7**(2):119-130

[7] Mazzio EA, Soliman KF. Basic concepts of epigenetics: Impact of environmental signals on gene expression. Epigenetics.

[8] De Bont R, van Larebeke N. Endogenous DNA damage in humans:

A review of quantitative data. Mutagenesis. 2004;**19**(3):169-185

of Sciences. 2018;**1418**(1):69-79

[10] Chen RZ et al. DNA hypomethylation leads to elevated mutation rates. Nature.

1998;**395**(6697):89-93

[11] Jintaridth P, Mutirangura A. Distinctive patterns of agedependent hypomethylation in

[9] Unnikrishnan A et al. Revisiting the genomic hypomethylation hypothesis of aging. Annals of the New York Academy

[1] Lopez-Otin C et al. The hallmarks of aging. Cell. 2013;**153**(6):1194-1217

interspersed repetitive sequences.

[13] Thongsroy J, Patchsung M, Mutirangura A. The association between Alu hypomethylation and severity of type 2 diabetes mellitus. Clinical Epigenetics. 2017;**9**:93

[14] Patchsung M et al. Alu siRNA to increase Alu element methylation and prevent DNA damage. Epigenomics.

[15] Pornthanakasem W et al. LINE-1 methylation status of endogenous DNA double-strand breaks. Nucleic Acids Research. 2008;**36**(11):3667-3675

Replication independent DNA doublestrand break retention may prevent genomic instability. Molecular Cancer.

[17] Thongsroy J et al. Replicationindependent endogenous DNA doublestrand breaks in *Saccharomyces cerevisiae* model. PLoS One. 2013;**8**(8):e72706

[18] Pongpanich M et al. Characteristics of replication-independent endogenous double-strand breaks in *Saccharomyces cerevisiae*. BMC Genomics. 2014;**15**:750

[19] Thongsroy J et al. Reduction in replication-independent endogenous

promotes genomic instability during chronological aging in yeast. The FASEB

DNA double-strand breaks

Journal. 2018:fj201800218RR

[20] Tirado-Magallanes R et al. Whole genome DNA methylation: Beyond genes silencing. Oncotarget.

2017;**8**(3):5629-5637

[16] Kongruttanachok N et al.

[12] Jintaridth P et al. Hypomethylation of Alu elements in post-menopausal women with osteoporosis. PLoS One.

Physiological Genomics. 2010;**41**(2):194-200

2013;**8**(8):e70386

2018;**10**(2):175-185

2010;**9**:70

[2] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell.

[40] Globisch D et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One. 2010;**5**(12):e15367

[41] Guo JU et al. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell. 2011;**145**(3):423-434

[42] Pfaffeneder T et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angewandte Chemie (International Ed. in English). 2011;**50**(31):7008-7012

[43] He YF et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science. 2011;**333**(6047):1303-1307

[44] Ito S et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;**333**(6047):1300-1303

[45] Hitchler MJ, Domann FE. An epigenetic perspective on the free radical theory of development. Free Radical Biology & Medicine. 2007;**43**(7):1023-1036

[46] Miller JW et al. Folate-deficiencyinduced homocysteinaemia in rats: Disruption of S-adenosylmethionine's coordinate regulation of homocysteine metabolism. The Biochemical Journal. 1994;**298**(Pt 2):415-419

[47] Miller JW et al. Folate, DNA methylation, and mouse models of breast tumorigenesis. Nutrition Reviews. 2008;**66**(Suppl 1):S59-S64

[48] Eden A et al. Chromosomal instability and tumors promoted by DNA hypomethylation. Science. 2003;**300**(5618):455

[49] Lengauer C, Kinzler KW, Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells. Proceedings of the National Academy

of Sciences of the United States of America. 1997;**94**(6):2545-2550

[50] Xu GL et al. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature. 1999;**402**(6758):187-191

[51] Niedernhofer LJ et al. A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis. Nature. 2006;**444**(7122):1038-1043

[52] Armstrong GT et al. Aging and risk of severe, disabling, life-threatening, and fatal events in the childhood cancer survivor study. Journal of Clinical Oncology. 2014;**32**(12):1218-1227

[53] Siddiqui MS et al. Persistent gammaH2AX: A promising molecular marker of DNA damage and aging. Mutation Research, Reviews in Mutation Research. 2015;**766**:1-19

[54] Voss P, Siems W. Clinical oxidation parameters of aging. Free Radical Research. 2006;**40**(12):1339-1349

[55] Poljsak B, Dahmane R. Free radicals and extrinsic skin aging. Dermatology Research and Practice. 2012;**2012**:135206

[56] Al-Mashhadi S et al. Oxidative glial cell damage associated with white matter lesions in the aging human brain. Brain Pathology. 2015;**25**(5):565-574

[57] Pongpanich M, Patchsung M, Mutirangura A. Pathologic replicationindependent endogenous DNA double-Strand breaks repair defect in chronological aging yeast. Frontiers in Genetics. 2018;**9**:501

[58] Gorbunova V et al. Changes in DNA repair during aging. Nucleic Acids Research. 2007;**35**(22):7466-7474

[59] Li W, Vijg J. Measuring genome instability in aging—A mini-review. Gerontology. 2012;**58**(2):129-138

**83**

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide…*

silencing and more. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(26):14030-14032

[71] Hwang JS et al. Deacetylationmediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia. Scientific Reports.

[72] Peng L et al. SIRT1 deacetylates the DNA methyltransferase 1 (DNMT1) protein and alters its activities. Molecular and Cellular Biology.

[73] Prasad R et al. HMGB1 is a cofactor in mammalian base excision repair. Molecular Cell. 2007;**27**(5):829-841

[74] Ru H et al. Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures. Cell.

[75] Giavara S et al. Yeast Nhp6A/B and mammalian Hmgb1 facilitate the maintenance of genome stability. Current Biology. 2005;**15**(1):68-72

[76] Stros M. HMGB proteins:

Biochimica et Biophysica Acta.

[77] Poulose N, Raju R. Sirtuin regulation in aging and injury. Biochimica et Biophysica Acta. 2015;**1852**(11):2442-2455

[78] Wierman MB, Smith JS. Yeast sirtuins and the regulation of aging. FEMS Yeast Research. 2014;**14**(1):73-88

[79] Nasmyth K. Regulating the HO endonuclease in yeast. Current Opinion in Genetics & Development.

1993;**3**(2):286-294

2010;**1799**(1-2):101-113

Interactions with DNA and chromatin.

2015;**5**:15971

2011;**31**(23):4720-4734

2015;**163**(5):1138-1152

*DOI: http://dx.doi.org/10.5772/intechopen.83372*

[60] Ciccia A, Elledge SJ. The DNA damage response: Making it safe to play with knives. Molecular Cell.

[61] Harper JW, Elledge SJ. The DNA damage response: Ten years after. Molecular Cell. 2007;**28**(5):739-745

Poulogiannis G. DNA damage, repair, and cancer metabolism. Frontiers in

[63] Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harbor Perspectives in

hypomethylation in Wilms tumors of DNA in chromosomes 1 and 16*.* Cancer Genetics and Cytogenetics, 1999.

[65] Meng H et al. DNA methylation, its mediators and genome integrity. International Journal of Biological Sciences. 2015;**11**(5):604-617

[66] Putiri EL, Robertson KD. Epigenetic mechanisms and genome stability. Clinical Epigenetics. 2011;**2**(2):299-314

[67] Steen SB et al. Initiation of V(D) J recombination in vivo: Role of recombination signal sequences in formation of single and paired doublestrand breaks. The EMBO Journal.

1997;**16**(10):2656-2664

1998;**18**(4):2029-2037

[68] Schlissel MS. Structure of

Molecular and Cellular Biology.

[70] Shore D. The Sir2 protein family: A novel deacetylase for gene

nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination.

[69] Kuo LJ, Yang LX. Gamma-H2AX—A novel biomarker for DNA double-strand breaks. In Vivo. 2008;**22**(3):305-309

[62] Turgeon MO, Perry NJS,

2010;**40**(2):179-204

Oncology. 2018;**8**:15

Biology. 2013:**5**(9)

**109**(1): p. 34-39

[64] Qu GZ et al. Frequent

*A Hypothesis to Explain How the DNA of Elderly People Is Prone to Damage: Genome-Wide… DOI: http://dx.doi.org/10.5772/intechopen.83372*

[60] Ciccia A, Elledge SJ. The DNA damage response: Making it safe to play with knives. Molecular Cell. 2010;**40**(2):179-204

*Epigenetics*

[40] Globisch D et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates.

of Sciences of the United States of America. 1997;**94**(6):2545-2550

[50] Xu GL et al. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature.

[51] Niedernhofer LJ et al. A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis. Nature. 2006;**444**(7122):1038-1043

[53] Siddiqui MS et al. Persistent gammaH2AX: A promising molecular marker of DNA damage and aging. Mutation Research, Reviews in Mutation

[55] Poljsak B, Dahmane R. Free radicals and extrinsic skin aging. Dermatology Research and Practice.

[56] Al-Mashhadi S et al. Oxidative glial cell damage associated with white matter lesions in the aging human brain. Brain Pathology. 2015;**25**(5):565-574

[57] Pongpanich M, Patchsung M, Mutirangura A. Pathologic replication-

[58] Gorbunova V et al. Changes in DNA repair during aging. Nucleic Acids Research. 2007;**35**(22):7466-7474

[59] Li W, Vijg J. Measuring genome instability in aging—A mini-review. Gerontology. 2012;**58**(2):129-138

independent endogenous DNA double-Strand breaks repair defect in chronological aging yeast. Frontiers in

Genetics. 2018;**9**:501

[54] Voss P, Siems W. Clinical oxidation parameters of aging. Free Radical Research. 2006;**40**(12):1339-1349

Research. 2015;**766**:1-19

2012;**2012**:135206

[52] Armstrong GT et al. Aging and risk of severe, disabling, life-threatening, and fatal events in the childhood cancer survivor study. Journal of Clinical Oncology. 2014;**32**(12):1218-1227

1999;**402**(6758):187-191

PLoS One. 2010;**5**(12):e15367

2011;**50**(31):7008-7012

2011;**333**(6047):1303-1307

[41] Guo JU et al. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell. 2011;**145**(3):423-434

[42] Pfaffeneder T et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angewandte Chemie (International Ed. in English).

[43] He YF et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science.

[44] Ito S et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;**333**(6047):1300-1303

[45] Hitchler MJ, Domann FE. An epigenetic perspective on the free radical theory of development. Free Radical Biology & Medicine.

[46] Miller JW et al. Folate-deficiencyinduced homocysteinaemia in rats: Disruption of S-adenosylmethionine's coordinate regulation of homocysteine metabolism. The Biochemical Journal.

2007;**43**(7):1023-1036

1994;**298**(Pt 2):415-419

2003;**300**(5618):455

[47] Miller JW et al. Folate, DNA methylation, and mouse models of breast tumorigenesis. Nutrition Reviews. 2008;**66**(Suppl 1):S59-S64

[48] Eden A et al. Chromosomal instability and tumors promoted by DNA hypomethylation. Science.

B. DNA methylation and genetic instability in colorectal cancer cells. Proceedings of the National Academy

[49] Lengauer C, Kinzler KW, Vogelstein

**82**

[61] Harper JW, Elledge SJ. The DNA damage response: Ten years after. Molecular Cell. 2007;**28**(5):739-745

[62] Turgeon MO, Perry NJS, Poulogiannis G. DNA damage, repair, and cancer metabolism. Frontiers in Oncology. 2018;**8**:15

[63] Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harbor Perspectives in Biology. 2013:**5**(9)

[64] Qu GZ et al. Frequent hypomethylation in Wilms tumors of DNA in chromosomes 1 and 16*.* Cancer Genetics and Cytogenetics, 1999. **109**(1): p. 34-39

[65] Meng H et al. DNA methylation, its mediators and genome integrity. International Journal of Biological Sciences. 2015;**11**(5):604-617

[66] Putiri EL, Robertson KD. Epigenetic mechanisms and genome stability. Clinical Epigenetics. 2011;**2**(2):299-314

[67] Steen SB et al. Initiation of V(D) J recombination in vivo: Role of recombination signal sequences in formation of single and paired doublestrand breaks. The EMBO Journal. 1997;**16**(10):2656-2664

[68] Schlissel MS. Structure of nonhairpin coding-end DNA breaks in cells undergoing V(D)J recombination. Molecular and Cellular Biology. 1998;**18**(4):2029-2037

[69] Kuo LJ, Yang LX. Gamma-H2AX—A novel biomarker for DNA double-strand breaks. In Vivo. 2008;**22**(3):305-309

[70] Shore D. The Sir2 protein family: A novel deacetylase for gene silencing and more. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(26):14030-14032

[71] Hwang JS et al. Deacetylationmediated interaction of SIRT1-HMGB1 improves survival in a mouse model of endotoxemia. Scientific Reports. 2015;**5**:15971

[72] Peng L et al. SIRT1 deacetylates the DNA methyltransferase 1 (DNMT1) protein and alters its activities. Molecular and Cellular Biology. 2011;**31**(23):4720-4734

[73] Prasad R et al. HMGB1 is a cofactor in mammalian base excision repair. Molecular Cell. 2007;**27**(5):829-841

[74] Ru H et al. Molecular mechanism of V(D)J recombination from synaptic RAG1-RAG2 complex structures. Cell. 2015;**163**(5):1138-1152

[75] Giavara S et al. Yeast Nhp6A/B and mammalian Hmgb1 facilitate the maintenance of genome stability. Current Biology. 2005;**15**(1):68-72

[76] Stros M. HMGB proteins: Interactions with DNA and chromatin. Biochimica et Biophysica Acta. 2010;**1799**(1-2):101-113

[77] Poulose N, Raju R. Sirtuin regulation in aging and injury. Biochimica et Biophysica Acta. 2015;**1852**(11):2442-2455

[78] Wierman MB, Smith JS. Yeast sirtuins and the regulation of aging. FEMS Yeast Research. 2014;**14**(1):73-88

[79] Nasmyth K. Regulating the HO endonuclease in yeast. Current Opinion in Genetics & Development. 1993;**3**(2):286-294

## *Edited by Rosaria Meccariello*

Epigenetic changes are heritable and reversible modifications that significantly affect gene expression without any change in DNA sequence. The epigenetic signature is remodelled during the lifespan as a direct consequence of both environment and lifestyle. Therefore, health or disease status strongly depends on epigenetic marks. This book summarizes the current knowledge in the field and includes chapters on epigenetics in plants and epigenetics in health and disease. It is written for a wide audience of basic and clinical scientists, teachers and students interested in gaining a better understanding of epigenetics.

Published in London, UK © 2019 IntechOpen © kirstypargeter / iStock

Epigenetics

Epigenetics

*Edited by Rosaria Meccariello*