Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis

Epigenetic association studies have demonstrated differential promoter methylation in the core circadian genes in breast cancer cases relative to cancer-free controls. The current pilot study aims to investigate whether epigenetic changes affecting breast cancer risk could be caused by circadian disruption through exposure to light at night. Archived DNA samples extracted from whole blood of 117 female subjects from a prospective cohort conducted in Denmark were included in this study. A polymerase chain reaction (PCR)-based method was used for detection of gene-promoter methylation, whereas genome-wide methylation analysis was performed using the Illumina Infinium Methylation Chip. Long-term shiftwork resulted in the same promoter hypomethylation of CLOCK and hypermethylation of CRY2, as was previously observed in breast cancer case-control studies. Genome-wide methylation analysis further discovered widespread methylation alterations in shiftworkers, including changes in many methylation- and cancer-relevant genes. Pathway analysis of the genes with altered methylation patterns revealed several cancer-related pathways. One of the top three networks generated was designated as "DNA replication, recombination, and repair, gene expression, behavior" with ESR1 (estrogen receptor α) featured most prominently in the network, underscoring the potential breast cancer relevance of the genes differentially methylated in long-term shiftworkers. These results, although exploratory, demonstrate the first evidence of the cancer-relevant epigenetic effects of night shiftwork, which warrant further investigation. Considering there are millions of shiftworkers worldwide, understanding the effects of this exposure may lead to novel strategies for cancer prevention and new policies regulating shiftwork.     

EPIGENETIC EFFECTS OF SHIFTWORK ON BLOOD DNA METHYLATION

In the present study, the authors investigated the effects of shiftwork exposure on DNA methylation using peripheral blood DNA from subjects working in two chemical plants in Northern Italy. The investigation was designed to evaluate (a) DNA methylation changes in Alu and long interspersed nuclear element-1 (LINE-1) repetitive elements as a surrogate of global methylation and (b) promoter methylation of glucocorticoid receptor (GCR), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ). One hundred and fifty white male workers (mean?±?SD: 41.0?±?9 yrs of age) were examined: 100 3?×?8 rotating shiftworkers (40.4?±?8.7 yrs of age) and 50 day workers (42.2?±?9.4 yrs of age). The authors used bisulfite-pyrosequencing to estimate repetitive elements and gene-specific methylation. Multiple regression analysis, adjusted for age, body mass index (BMI), and job seniority, did not show any significant association between the five DNA methylation markers and shiftwork. However, job seniority, in all subjects, was significantly associated with Alu (β?=??0.019, p?=?.033) and IFN-γ (β?=??0.224, p?<?.001) methylation, whereas TNF-α methylation was inversely correlated with age (β?=??0.093, p?<?.001). Considering only shiftworkers, multiple regression analysis, adjusted for age, BMI, and job seniority, showed a significant difference between morning and evening types in TNF-α methylation (mean morning type [MT] 11.425 %5mC versus evening type [ET] 12.975 %5mC; β?=?1.33, p?=?.022). No difference was observed between good and poor tolerance to shiftwork. Increasing job seniority (<5, 5–15, >15 yrs) was associated with significantly lower Alu (β?=??0.86, p?=?.006) and IFN-γ methylation (β?=??6.50, p?=?.007) after adjustment for age, BMI, and morningness/eveningness. In addition, GCR significantly increased with length of shiftwork (β?=?3.33, p?=?.05). The data showed alterations in blood DNA methylation in a group of shiftworkers, including changes in Alu repetitive elements methylation and gene-specific methylation of IFN-γ and TNF-α promoters. Further studies are required to determine the role of such alterations in mediating the effects of shiftwork on human health. (Author correspondence: giovanni.costa@unimi.it)     

Mood,Alertness, and Performance in Response to Sleep Deprivation and Recovery Sleep in Experienced Shiftworkers Versus Non-Shiftworkers

Previous studies have shown increased sleepiness and mood changes in shiftworkers, which may be due to sleep deprivation or circadian disruption. Few studies, however, have compared responses of experienced shiftworkers and non-shiftworkers to sleep deprivation in an identical laboratory setting. The aim of this laboratory study, therefore, was to compare long-term shiftworkers and non-shiftworkers and to investigate the effects of one night of total sleep deprivation (30.5?h of continuous wakefulness) and recovery sleep on psychomotor vigilance, self-rated alertness, and mood. Eleven experienced male shiftworkers (shiftwork ≥5 yrs) were matched with 14 non-shiftworkers for age (mean?±?SD: 35.7?±?7.2 and 32.5?±?6.2 yrs, respectively) and body mass index (BMI) (28.7?±?3.8 and 26.6?±?3.4?kg/m², respectively). After keeping a 7-d self-selected sleep/wake cycle (7.5/8?h nocturnal sleep), both groups entered a laboratory session consisting of a night of adaptation sleep and a baseline sleep (each 7.5/8?h), a sleep deprivation night, and recovery sleep (4-h nap plus 7.5/8?h nighttime sleep). Subjective alertness and mood were assessed with the Karolinska Sleepiness Scale (KSS) and 9-digit rating scales, and vigilance was measured by the visual psychomotor vigilance test (PVT). A mixed-model regression analysis was carried out on data collected every hour during the sleep deprivation night and on all days (except for the adaptation day), at .25, 4.25, 5.25, 11.5, 12.5, and 13.5?h after habitual wake-up time. Despite similar circadian phase (melatonin onset), demographics, food intake, body posture, and environmental light, shiftworkers felt significantly more alert, more cheerful, more elated, and calmer than non-shiftworkers throughout the laboratory study. In addition, shiftworkers showed a faster median reaction time (RT) compared to non-shiftworkers, although four other PVT parameters did not differ between the groups. As expected, both groups showed a decrease in subjective alertness and PVT performance during and following the sleep deprivation night. Subjective sleepiness and most aspects of PVT performance returned to baseline levels after a nap and recovery sleep. The mechanisms underlying the observed differences between shiftworkers and non-shiftworkers require further study, but may be related to the absence of shiftwork the week prior to and during the laboratory study as well as selection into and out of shiftwork. (Author correspondence: sophie.wehrens@surrey.ac.uk)     

Shiftwork and Metabolic Dysfunction

Many of the health problems that are more prevalent among shiftworkers are thought to be linked to their heightened susceptibility to metabolic syndrome, i.e., the association of even moderate degrees of visceral obesity, dyslipidemia, abnormal blood pressure, and serum glucose levels in the same individual. Although previous studies have identified associations between shiftwork and metabolic syndrome, there is relatively little evidence to date of how the risk of developing it varies as a function of exposure to shiftwork. The current study seeks to confirm earlier findings of an association between shiftwork exposure and metabolic dysfunction, and to examine the impact of exposure duration, while adjusting for a number of covariates in the analyses. The analyses were based on data from VISAT, a study involving the measurement of physiological, behavioral, and subjective outcomes from 1757 participants, 989 being current or former shiftworkers. The sample comprised employed and retired wage earners, male and female, who were 32, 42, 52, and 62 yrs old. The first analysis sought to confirm previous findings of an association between exposure to shiftwork and the risk of developing metabolic syndrome. It indicated that participants who were or who had previously been shiftworkers (i.e., working schedules that involved rotating shifts; not being able to go to bed before midnight; having to get up before 05:00?h; or being prevented from sleeping during the night) were more likely to exhibit symptoms of metabolic syndrome, after adjusting for age, sex, socioeconomic status, smoking, alcohol intake, perceived stress, and sleep difficulty (odds ratio [OR] 1.78; 95% confidence interval [CI] 1.03–3.08). The results suggest the association between shiftwork and metabolic syndrome cannot be fully accounted for by either higher levels of strain or increased sleep difficulty among shiftworkers, although it remains a possibility that either one or both of these factors may have played a contributing role. The second analysis addressed the issue of duration of exposure to shiftwork. Participants with >10 yrs' experience of working rotating shifts were more likely to exhibit symptoms of metabolic syndrome than participants without exposure to shiftwork, i.e., dayworkers, even after adjusting for age and sex (OR 1.96; 95% CI 1.03–3.75). Thus, the current study confirms the association between shiftwork exposure and metabolic syndrome. It also provides new information regarding the time course of the development of the illness as function of exposure duration, although this was only examined in relation to rotating shiftwork. It is concluded that those responsible for monitoring workers' health should pay particular attention to indices of metabolic dysfunction in workers who have been exposed to shiftwork for >10 yrs. (Author correspondence: p.t.tucker@swansea.ac.uk)     

Measurements of Light at Night (LAN) for a Sample of Female School Teachers

Epidemiological studies have shown an association between rotating shiftwork and breast cancer (BC) risk. Recently, light at night (LAN) measured by satellite photometry and by self-reports of bedroom brightness has been shown to be associated with BC risk, irrespective of shiftwork history. Importance has been placed on these associations because retinal light exposures at night can suppress the hormone melatonin and/or disrupt circadian entrainment to the local 24-h light-dark cycle. The present study examined whether it was valid to use satellite photometry and self-reports of brightness to characterize light, as it might stimulate the circadian system and thereby affect BC incidence. Calibrated photometric measurements were made at the bedroom windows and in the bedrooms of a sample of female school teachers, who worked regular dayshifts and lived in a variety of satellite-measured sky brightness categories. The light levels at both locations were usually very low and were independent of the amount of satellite-measured light. Calibrated photometric measurements were also obtained at the corneas of these female school teachers together with calibrated accelerometer measurements for seven consecutive days and evenings. Based upon these personal light exposure and activity measurements, the female teachers who participated in this study did not have disrupted light-dark cycles like those associated with rotating shiftworkers who do exhibit a higher risk for BC. Rather, this sample of female school teachers had 24-h light-dark and activity-rest patterns very much like those experienced by dayshift nurses examined in an earlier study who are not at an elevated risk of BC. No relationship was found between the amount of satellite-measured light levels and the 24-h light-dark patterns these women experienced. It was concluded from the present study that satellite photometry is unrelated to personal light exposures as they might affect melatonin suppression and/or circadian disruption. More generally, photometric devices calibrated in terms of the operational characteristics of the human circadian system must be used to meaningfully link LAN and BC incidence. (Authors correspondence: E-mail: ream@rpi.edu)     

LIGHT INTENSITY EXPOSURE,SLEEP DURATION,PHYSICAL ACTIVITY,AND BIOMARKERS OF MELATONIN AMONG ROTATING SHIFT NURSES

Long-term, night shiftwork has been identified as a potential carcinogenic risk factor. It is hypothesized that increased light at night exposure during shiftwork reduces melatonin production, which is associated with increased cancer risk. Sleep duration has been hypothesized to influence both melatonin levels and cancer risk, and it has been suggested that sleep duration could be used as a proxy for melatonin production. Finally, physical activity has been shown to reduce cancer risk, and laboratory studies indicate it may influence melatonin levels. A cross-sectional study of light exposure, sleep duration, physical activity, and melatonin levels was conducted among 61 female rotating shift nurses (work schedule: two 12?h days, two 12?h nights, five days off). Light intensity was measured using a light-intensity data logger, and sleep duration and physical activity were self-reported in a study diary and questionnaire. Melatonin concentrations were measured from urine and saliva samples. The characteristics of nurses working day and night shifts were similar. Light intensity was significantly higher during sleep for those working at night (p<?0.0001), while urinary melatonin levels following sleep were significantly higher among those working days (p?=?0.0003). Mean sleep duration for nurses working during the day (8.27?h) was significantly longer than for those working at night (4.78?h, p<?0.0001). An inverse association (p?=?0.002) between light exposure and urinary melatonin levels was observed; however, this was not significant when stratified by shift group. There was no significant correlation between sleep duration and melatonin, and no consistent relationship between physical activity and melatonin. Analysis of salivary melatonin levels indicated that the circadian rhythms of night workers were not altered, meaning peak melatonin production occurred at night. This study indicates that two nights of rotating shift work may not change the timing of melatonin production to the day among those working at night. Additionally, in this study, sleep duration was not correlated with urinary melatonin levels, suggesting it may not be a good proxy for melatonin production. (Author correspondence: anne.grundy@queensu.ca)     

20th International Symposium on Shiftwork and Working Time: Biological Mechanisms,Recovery, and Risk Management in the 24-h Society

This dedicated issue of Chronobiology International is devoted to the selected proceedings of the 20th International Symposium on Shift Work and Working Time held in Stockholm, Sweden, 28 June to 1 July 2011. It constitutes the fifth such issue of the journal since 2004 dedicated to the selected proceedings to the meetings of the Working Time Society. The key theme of the 20th Symposium was “Biological Mechanisms, Recovery, and Risk Management in the 24-h Society.” The collection of papers of this dedicated issue represents the best of contemporary research on the effects of night and rotating shift schedules on worker health and safety. The contents cover such topics as sleep restriction, injuries, health, and performance of night work and rotating shiftwork, plus light treatment as a countermeasure against the circadian disruption of shiftwork. The majority of the papers are observational field studies, including some of large sample size, and three studies are well-designed laboratory experiments. (Author correspondence: goran.kecklund@stress.su.se)     

GENE-GENE INTERACTION BETWEEN SEROTONIN TRANSPORTER (SLC6A4) AND CLOCK MODULATES THE RISK OF METABOLIC SYNDROME IN ROTATING SHIFTWORKERS

Serotonergic neurotransmission and the master circadian CLOCK gene are physiological modulators of the circadian system. In addition, both are involved in the physiopathology of metabolic syndrome (MS). The authors sought to examine the potential effect of the gene-gene interaction between the functional 44-bp insertion/deletion polymorphism in the promoter region (serotonin-transporter-linked promoter region polymorphism or 5-HTTLPR) of the serotonin transporter gene (SLC6A4) and common variants of the gene CLOCK on the genetic risk underlying MS of shift-workers. To test this hypothesis, 856 men were studied; 518 dayworkers were compared with 338 rotating shiftworkers. Medical history, health examination including anthropometric and arterial blood pressure measurements, a questionnaire on health-related behaviors, and biochemical determinations were obtained from every participant. 5-HTTLPR genotypes were determined using polymerase chain reaction followed by gel electrophoresis. Six tag single-nucleotide polymorphisms (SNPs) in the CLOCK gene with a minor allele frequency >10 % (rs1554483 C/G, rs11932595 A/G, rs4580704 C/G, rs6843722 A/C, rs6850524 C/G, and rs4864548 A/G), encompassing 117 kb of chromosome 4 and representing 115 polymorphic sites (r²?>?.8), were genotyped. A significant interaction between the 5-HTTLPR variant and the haplotype rs1554483–rs4864548 of the CLOCK gene was detected for diastolic (p?=?.0058) and systolic blood pressure (p?=?.0014), arterial hypertension (p?=?.033), plasma triglycerides levels (p?=?.033), and number of MS components (p?=?.01). In all these cases, the higher values were observed in rotating shiftworkers homozygous for the SLC6A4 S allele and carrying the haplotype composed by the CLOCK rs1554483 G and rs4864548 A variants. In conclusion, these data suggest a potential interaction (epistatic effect) of serotonin transporter and CLOCK gene variation on the genetic susceptibility to develop MS by rotating shiftworkers. (Author correspondence: cpirola@ciudad.com.ar or pirola.carlos@lanari.fmed.uba.ar)     

Does the Modern Urbanized Sleeping Habitat Pose a Breast Cancer Risk?

Due to its disruptive effects on circadian rhythms and sleep deprivation at night, shiftworking is currently recognized as a risk factor for breast cancer (BC). As revealed by the present analysis based on a comparative case-control study of 1679 women, exposure to light-at-night (LAN) in the “sleeping habitat” is significantly associated with BC risk (odds ratio [OR]?=?1.220, 95% confidence interval [CI]?=?1.118–1.311; p?<?.001), controlling for education, ethnicity, fertility, and alcohol consumption. The novelty of the present research is that, to the best of the authors' knowledge, it is the first study to have identified an unequivocal positive association between bedroom-light intensity and BC risk. Thus, according to the results of the present study, not only should artificial light exposure in the working environment be considered as a potential risk factor for BC, but also LAN in the “sleeping habitat.” (Author correspondence: ahaim@research.haifa.ac.il)     

Factors Associated With Shift Work Disorder in Nurses Working With Rapid-Rotation Schedules in Japan: The Nurses’ Sleep Health Project

Workers who meet the criteria for shift work disorder (SWD) have elevated levels of risk for various health and behavioral problems. However, the impact of having SWD on shiftworkers engaged in rapid-rotation schedules is unknown. Moreover, the risk factors for the occurrence of SWD remain unclear. To clarify these issues, we conducted a questionnaire-based, cross-sectional survey on a sample of shiftworking nurses. Responses were obtained from 1202 nurses working at university hospitals in Tokyo, Japan, including 727 two-shift workers and 315 three-shift workers. The questionnaire included items relevant to age, gender, family structure, work environment, health-related quality of life (QOL), diurnal type, depressive symptoms, and SWD. Participants who reported insomnia and/or excessive sleepiness for at least 1 mo that was subjectively relevant to their shiftwork schedules were categorized as having SWD. The prevalence of SWD in the sampled shiftworking nurses was 24.4%; shiftworking nurses with SWD showed lower health-related QOL and more severe depressive symptoms, with greater rates of both actual accidents/errors and near misses, than those without SWD. The results of logistic regression analyses showed that more time spent working at night, frequent missing of nap opportunities during night work, and having an eveningness-oriented chronotype were significantly associated with SWD. The present study indicated that SWD might be associated with reduced health-related QOL and decreased work performance in shiftworking nurses on rapid-rotation schedules. The results also suggested that missing napping opportunities during night work, long nighttime working hours, and the delay of circadian rhythms are associated with the occurrence of SWD among shiftworking nurses on rapid-rotation schedules. (Author correspondence: yuinoue@tokyo-med.ac.jp)     

Unaltered Instrumental Learning and Attenuated Body-Weight Gain in Rats During Non-rotating Simulated Shiftwork

Exposure to shiftwork has been associated with multiple health disorders and cognitive impairments in humans. We tested if we could replicate metabolic and cognitive consequences of shiftwork, as reported in humans, in a rat model comparable to 5 wks of non-rotating night shifts. The following hypotheses were addressed: (i) shiftwork enhances body-weight gain, which would indicate metabolic effects; and (ii) shiftwork negatively affects learning of a simple goal-directed behavior, i.e., the association of lever pressing with food reward (instrumental learning), which would indicate cognitive effects. We used a novel method of forced locomotion to model work during the animals' normal resting period. We first show that Wistar rats, indeed, are active throughout a shiftwork protocol. In contrast with previous findings, the shiftwork protocol attenuated the normal weight gain to 76?±?8?g in 5 wks as compared to 123?±?15?g in the control group. The discrepancy with previous work may be explained by the concurrent observation that with our shiftwork protocol rats did not adjust their between-work circadian activity pattern. They maintained a normal level of activity during the “off-work” periods. In the control experiment, rats were kept active during the dark period, normally dominated by activity. This demonstrated that forced activity, per se, did not affect body-weight gain (mean±SEM: 85?±?11?g over 5 wks as compared to 84?±?11?g in the control group). Rats were trained on an instrumental learning paradigm during the fifth week of the protocol. All groups showed equivalent increases in lever pressing from the first (3.8?±?.7) to the sixth (21.3?±?2.4) session, and needed a similar amount of sessions (5.1?±?.3) to reach a learning criterion (≥27 out of 30 lever presses). These results suggest that while on prolonged non-rotating shiftwork, not fully reversing the circadian rhythm might actually be beneficial to prevent body-weight gain and cognitive impairments. (Author correspondence: C.Leenaars@nin.knaw.nl)     

An Integrated Approach to Uncover Driver Genes in Breast Cancer Methylation Genomes

Background

Cancer cells typically exhibit large-scale aberrant methylation of gene promoters. Some of the genes with promoter methylation alterations play “driver” roles in tumorigenesis, whereas others are only “passengers”.

Results

Based on the assumption that promoter methylation alteration of a driver gene may lead to expression alternation of a set of genes associated with cancer pathways, we developed a computational framework for integrating promoter methylation and gene expression data to identify driver methylation aberrations of cancer. Applying this approach to breast cancer data, we identified many novel cancer driver genes and found that some of the identified driver genes were subtype-specific for basal-like, luminal-A and HER2+ subtypes of breast cancer.

Conclusion

The proposed framework proved effective in identifying cancer driver genes from genome-wide gene methylation and expression data of cancer. These results may provide new molecular targets for potential targeted and selective epigenetic therapy.     

Impact of Nightshift Work on Overweight and Abdominal Obesity Among Workers of a Poultry Processing Plant in Southern Brazil

The authors examined the associations of shiftwork with overweight and abdominal obesity through a cross-sectional study of 1206 employees 18 to 50 yrs of age who were working on a production line in a poultry processing plant. Night-shift workers (n?=?800) were considered exposed, whereas day shiftworkers (n?=?406) were considered nonexposed. Overweight was defined as a body mass index ≥25?kg/m² and abdominal obesity as a waist circumference ≥88?cm in women and ≥102?cm in men. The mean age of the workers was 30.5 yrs (standard deviation?=?8.7 yrs), and 65.2% were women. Nightshift workers compared to dayshift workers showed higher prevalences of overweight (42.2% vs. 34.3%; p?=?.020) and abdominal obesity (24.9% vs. 19.5%; p?=?.037). After adjusting for sociodemographics, parental overweight status, behavioral characteristics, and sleep characteristics, including hours of sleep, the prevalence ratios for overweight and abdominal obesity were 1.27 (95% confidence interval [ CI]: 1.00–1.61) and 1.45 (95% CI: 1.10–1.92), respectively, for the nightshift workers compared to the dayshift workers. A consistent finding in our study was the independent contribution of night shiftwork to overweight and abdominal obesity among Brazilian workers. Further studies are needed to understand the biological mechanisms involved and the complex behavioral and social adaptations experienced by night-shift workers. (Author correspondence: mtolinto@gmail.com)     

THE CIRCADIAN CLOCK GENE PER1 SUPPRESSES CANCER CELL PROLIFERATION AND TUMOR GROWTH AT SPECIFIC TIMES OF DAY

Cell cycle progression is tightly regulated. The expressions of cell cycle regulators, the products of which either promote or inhibit cell proliferation, oscillate during each cell cycle. Cellular proliferation and the expression of cell cycle regulators are also controlled by the circadian clock. Disruption of the circadian clock may thereby lead to deregulated cell proliferation. Mammalian Per2 is a core clock gene, the product of which suppresses cancer cell proliferation and tumor growth in vivo and in vitro. Because Per1, another key clock gene, is mutated in human breast cancers, and because its clock functions are similar and complementary to those of Per2, we have studied its role in modulating breast cancer cell proliferation and tumor growth. We find that breast cancer growth rate is gated by the circadian clock with two daily peaks and troughs, and that they are coupled to the daily expression patterns of clock-controlled genes that regulate cell proliferation. Down-regulation of the expression of tumor Per1 increases cancer cell growth in vitro and tumor growth in vivo by enhancing the circadian amplitude of the two daily tumor growth peaks. The data of the study suggest Per1 has tumor-suppressor function that diminishes cancer proliferation and tumor growth, but only at specific times of day. (Author correspondence: williamhrushesky@gmail.com).     

Working against our endogenous circadian clock: Breast cancer and electric lighting in the modern world

Breast cancer incidence increases rapidly as societies industrialize. Many changes occur during the industrialization process, one of which is a dramatic alteration in the lighted environment from a sun-based system to an electricity-based system. Increasingly, the natural dark period at night is being seriously eroded for the bulk of humanity. Based on the fact that light during the night can suppress melatonin, and also disrupt the circadian rhythm, it was proposed in 1987 that increasing use of electricity to light the night accounts in part for the rising risk of breast cancer globally. Predictions from the theory include: non-day shift work increases risk, blindness lowers risk, long sleep duration lowers risk, and population level community nighttime light level co-distributes with breast cancer incidence. Thus far, studies of these predictions are consistent in support of the theory. A new avenue of research has been on function of circadian genes and whether these are related to breast cancer risk. In particular, a length variant of Per3 (5-VNTR) has been associated with increased risk in young women, and this same 5-VNTR variant has also been found to predict morning diurnal type and shorter sleep duration compared to the 4-VNTR variant. An important question is how an effect of light-at-night (LAN) exposure on breast cancer risk might be modified by polymorphisms and/or epigenetic alterations in the circadian genes, and conversely whether light-at-night exposure (e.g., shift work) can induce deleterious epigenetic changes in these genes.     

A different methylation profile of circadian genes promoter in breast cancer patients according to clinicopathological features

ABSTRACT

One of the supposed mechanisms that may lead to breast cancer (BC) is an alteration of circadian gene expression and DNA methylation. We undertook an integrated approach to identify methylation pattern of core circadian promoter regions in BC patients with regard to clinical features. We performed a quantitative methylation-specific real-time PCR analysis of a promoter methylation profile in 107 breast tumor and matched non-tumor tissues. A panel of circadian genes CLOCK, BMAL1, PERIOD (PER1, 2, 3), CRYPTOCHROME (CRY1, 2) and TIMELESS as well as their association with clinicopathological characteristics were included in the analysis. Three out of the eight analyzed genes exhibited marked hypermethylation (PER1, 2, 3), whereas CLOCK, BMAL1, CRY2 showed significantly lower promoter CpG methylation in the BC tissues when compared to the non-tumor tissues. Among variously methylated genes we found an association between the elevated methylation level of PERs promoter region and molecular subtypes, histological subtypes and tumor grading of BC. Methylation status may be associated with a gene expression level of circadian genes in BC patients. An aberrant methylation pattern in circadian genes in BC may provide information that could be used as novel biomarkers in clinics and molecular epidemiology as well as play an important role in BC etiology.     

CLOCK,PER2 and BMAL1 DNA Methylation: Association with Obesity and Metabolic Syndrome Characteristics and Monounsaturated Fat Intake

The circadian clock system instructs 24-h rhythmicity on gene expression in essentially all cells, including adipocytes, and epigenetic mechanisms may participate in this regulation. The aim of this research was to investigate the influence of obesity and metabolic syndrome (MetS) features in clock gene methylation and the involvement of these epigenetic modifications in the outcomes. Sixty normal-weight, overweight and obese women followed a 16-weeks weight reduction program. DNA methylation levels at different CpG sites of CLOCK, BMAL1 and PER2 genes were analyzed by Sequenom's MassARRAY in white blood cells obtained before the treatment. Statistical differences between normal-weight and overweight?+?obese subjects were found in the methylation status of different CpG sites of CLOCK (CpGs 1, 5-6, 8 and 11-14) and, with lower statistical significance, in BMAL1 (CpGs 6-7, 8, 15 and 16-17). The methylation pattern of different CpG sites of the three genes showed significant associations with anthropometric parameters such as body mass index and adiposity, and with a MetS score. Moreover, the baseline methylation levels of CLOCK CpG 1 and PER2 CpGs 2-3 and 25 correlated with the magnitude of weight loss. Interestingly, the percentage of methylation of CLOCK CpGs 1 and 8 showed associations with the intake of monounsaturated and polyunsaturated fatty acids. This study demonstrates for the first time an association between methylation status of CpG sites located in clock genes (CLOCK, BMAL1 and PER2) with obesity, MetS and weight loss. Moreover, the methylation status of different CpG sites in CLOCK and PER2 could be used as biomarkers of weight-loss success, particularly CLOCK CPGs 5-6. (Author correspondence: garaulet@um.es)     

Assessment of a New Dynamic Light Regimen in a Nuclear Power Control Room Without Windows on Quickly Rotating Shiftworkers—Effects on Health,Wakefulness, and Circadian Alignment: A Pilot Study

The aim of the study was to test whether a new dynamic light regime would improve alertness, sleep, and adaptation to rotating shiftwork. The illumination level in a control room without windows at a nuclear power station was ～200 lux (straight-forward horizontal gaze) using a weak yellow light of 200 lux, 3000 K (Philips Master TLD 36 W 830). New lighting equipment was installed in one area of the control room above the positions of the reactor operators. The new lights were shielded from the control group by a distance of >6?m, and the other operators worked at desks turned away from the new light. The new lights were designed to give three different light exposures: (i) white/blue strong light of 745 lux, 6000 K; (ii) weak yellow light of 650 lux, 4000 K; and (iii) yellow moderate light of 700 lux, 4000 K. In a crossover design, the normal and new light exposures were given during a sequence of three night shifts, two free days, two morning shifts, and one afternoon shift (NNN?+?MMA), with 7 wks between sessions. The operators consisted of two groups; seven reactor operators from seven work teams were at one time exposed to the new equipment and 16 other operators were used as controls. The study was conducted during winter with reduced opportunities of daylight exposure during work, after night work, or before morning work. Operators wore actigraphs, filled in a sleep/wake diary, including ratings of sleepiness on the Karolinska Sleepiness Scale (KSS) every 2?h, and provided saliva samples for analysis of melatonin at work (every 2nd h during one night shift and first 3?h during one morning shift). Results from the wake/sleep diary showed the new light treatment increased alertness during the 2nd night shift (interaction group?×?light?×?time, p < .01). Time of waking was delayed in the light condition after the 3rd night shift (group?×?light, p < .05), but the amount of wake time during the sleep span increased after the 2nd night shift (p < .05), also showing a tendency to affect sleep efficiency (p < .10). Effects on circadian phase were difficult to establish given the small sample size and infrequent sampling of saliva melatonin. Nonetheless, it seems that appropriate dynamic light in rooms without windows during the dark Nordic season may promote alertness, sleep, and better adaptation to quickly rotating shiftwork. (Author correspondence: arne.lowden@stress.su.se)     

SLEEP AND SLEEPINESS: IMPACT OF ENTERING OR LEAVING SHIFTWORK—A PROSPECTIVE STUDY

Very little is known about the effects on sleep and sleepiness of entering or exiting shiftwork. The present study used a longitudinal database (n?=?3637). Participants completed a questionnaire on work hours, sleep, and work environment at the start and end of a 5-yr period. Changes in shift/day work status were related to change in a number of subjective sleep variables using logistic regression analysis. The analyses were adjusted for age, sex, and differences in socioeconomic status, work demands, work control, physical workload, marriage status, and number of children. In comparison with constant day work, entering shiftwork (with or without night shifts) from day work increased the risk of difficulties in falling asleep, and leaving shiftwork reduced this risk (odds ratio [OR]?=?2.8 [confidence interval, CI?=?1.8–4.5]). Also falling asleep at work showed a consistent pattern; an increased risk of falling asleep for those with shiftwork on both occasions, and for those with night work on both occasions. Also entering night work was associated with a strongly increased risk of falling asleep at work (OR?=?2.9 [CI?=?1.3–6.7]). These results suggest that entering and leaving shiftwork has a considerable impact on sleep and alertness. However, there is a need for large and more extended longitudinal studies to support our findings. (Author correspondence: Torbjorn.akerstedt@ki.se)