Night shift schedule causes circadian dysregulation of DNA repair genes and elevated DNA damage in humans

Abstract

Circadian disruption has been identified as a risk factor for health disorders such as obesity, cardiovascular disease, and cancer. Although epidemiological studies suggest an increased risk of various cancers associated with circadian misalignment due to night shift work, the underlying mechanisms have yet to be elucidated. We sought to investigate the potential mechanistic role that circadian disruption of cancer hallmark pathway genes may play in the increased cancer risk in shift workers. In a controlled laboratory study, we investigated the circadian transcriptome of cancer hallmark pathway genes and associated biological pathways in circulating leukocytes obtained from healthy young adults during a 24-hour constant routine protocol following 3 days of simulated day shift or night shift. The simulated night shift schedule significantly altered the normal circadian rhythmicity of genes involved in cancer hallmark pathways. A DNA repair pathway showed significant enrichment of rhythmic genes following the simulated day shift schedule, but not following the simulated night shift schedule. In functional assessments, we demonstrated that there was an increased sensitivity to both endogenous and exogenous sources of DNA damage after exposure to simulated night shift. Our results suggest that circadian dysregulation of DNA repair may increase DNA damage and potentiate elevated cancer risk in night shift workers.

Keywords: circadian misalignment; genomic stability; hallmarks of cancer; shift work.

FIGURE 1

Design of the in-laboratory, simulated shift work study in humans. Left and right panels represent the simulated day and night shift conditions, respectively. After three days on the simulated day or night shift schedule, subjects underwent a 24-hour constant routine protocol during which blood samples were collected at 3-hour intervals.

FIGURE 2

Circadian analysis of cancer hallmark gene expression as observed under 24-hour constant routine after exposure to simulated day or night shift schedule. (A) Heatmap of significantly (p<0.05, cosinor analysis t-test) rhythmic expression profiles of cancer hallmark genes during 24-hour constant routine following the three-day simulated day shift (left) and night shift (right) conditions. Genes are ordered in the heatmap by time of peak expression (acrophase) for genes significantly rhythmic after day shift only, after night shift only, and after both – with genes displaying a significant timing difference between conditions set apart in the bottom rows. (B) Enrichment for rhythmic genes in cancer hallmark pathways after day shift only, after night shift only, or after both (*p<0.05, Fisher’s exact test). (C) Heatmap of significantly rhythmic (p<0.05, cosinor analysis t-test) transcripts in the DNA repair pathway during 24-hour constant routine following the simulated day shift (left) and night shift (right) conditions. Genes are ordered in the heatmap as in panel (A). (D) Circadian rhythms in the expression of selected genetic markers of DNA repair during 24-hour constant routine after simulated day versus night shift.

FIGURE 3

DNA damage under 24-hour constant routine after exposure to simulated day or night shift schedule. (A) Representative DNA migration (head to tail) in leukocytes after simulated day shift (left) versus night shift (right) as assessed with alkaline comet assay. (B) Percentage (mean ± SE) of tail genomic DNA across the 24-hour constant routine, after simulated day shift or night shift (F_7,70=4.55, p<0.001, mixed-effects ANOVA condition by time interaction; *p<0.05, **p<0.01, pairwise contrasts). (C) Immunofluorescence quantification of leukocytes with BRCA1 (top) and γH2AX (bottom) foci averaged over the 24-hour constant routine after simulated day or night shift (**p<0.01, ***p<0.001, two-sample t-test). The y-axis indicates the average percentage ± SE. (D) Immunofluorescence quantification of BRCA1 (top) and γH2AX (bottom) foci in leukocytes upon IR treatment of samples collected at 07:30 (AM) or 19:30 (PM) during the 24-hour constant routine after simulated day or night shift (*p<0.05, **p<0.01, two-sample t-test). The y-axis indicates log₂ fold change ± SE of the number of foci in IR treated cells versus untreated controls. (E) Representative immunoblot of the DNA damage response upon IR exposure of samples collected at 07:30 (AM) or 19:30 (PM) during the 24-hour constant routine after a simulated day or night shift. M denotes “mock” (i.e., sample not exposed to IR).

FIGURE 4

Schematic drawing showing how a night shift schedule may elevate cancer risk through circadian dysregulation of cell cycle, apoptosis and DNA repair mechanisms leading to increased DNA damage.