Evaluation of circadian gene expression changes in human peripheral blood cells as biomarkers of circadian disruption in shift workers: application to studies of breast and prostate cancer chemoprevention

Abstract

Epidemiological studies indicate that disruption of circadian rhythm by shift work is associated with increased risk of breast and prostate cancers. Rat studies demonstrated that carcinogens also disrupt the rhythmic expression of circadian and circadian controlled genes (CCG). More importantly, chemopreventive methylselenocysteine (MSC) resets the expression levels and phases of circadian and CCG genes, including estrogen receptor β (ER β ) (Fang, Zhang & Zarbl Cancer Prev. Res. 3: 640–652, 2010). Unpublished findings indicate that MSC resets rhythmic oscillations in histone and Bmal1 acetylation on the Per2 gene promoter, suggesting a mechanism for MSC- mediated resetting of circadian rhythm. The goal of the present study was to determine if changes in circadian and CCG expression in white blood cells can serve as biomarkers of circadian disruption in shift workers, and to assess the ability of MSC to reset the circadian rhythm in peripheral cells. Phase I: Fifteen hospital interns and residents (age 21–34) were recruited to a cross-over biomarker study. Blood samples were drawn at 8 AM, noon and 8 PM after completing at least one week on the day shift, and at 8 AM and 8 PM, after completing one week of service on the night shift. Serum melatonin and expression of circadian genes (Per2, NR1D1) and CCG (ER β ) were measured in peripheral blood samples. Descriptive statistics and mixed linear models evaluated changes in gene expression. Phase II: Results of this study were used to design a blinded randomized placebo-controlled intervention trial to explore the ability of a dietary supplement of MSC to reset normal circadian and circadian controlled gene expression. One hundred healthy volunteers, who work predominantly at night, were randomized to a placebo or MSC interventions groups (50 subjects per group). Blood samples were collected prior to the intervention to determine baseline levels of biomarkers. Subjects were asked to take a daily supplement containing MSC (3 ppm Se) or a placebo for a period of 30 days, while keeping a sleep log. Biomarkers are reassessed after termination of the intervention. Results showed that when working the day-shift, the average Per2 mRNA levels were lower at night vs morning; however, when working the night-shift, per2 mRNA was significantly higher at night vs morning ( p = 0.01), indicating a change in Per2 mRNA expression with a change from day-to night-shift. We also observed a significant increase in Per2 mRNA expression ( p = 0.034) during night- vs day-shift based on the samples collected in the evening. However, NR1D1 and ER β mRNA expression, very low at these time points, were not significantly affected by schedule changes. These findings formed the basis for a MSC interventional trial in 100 shift workers. The interventional study has been completed and the circadian biomarker analysis is in progress. These results indicate that night- vs day-shift has a significant impact on Per2 gene expression, suggesting that it can be used as a biomarker to indicate disrupted circadian rhythm in shift workers. Per2 expression shows promise as a useful in longitudinal studies of MSC chemoprevention of cancer in shift workers. These studies are supported by a Translational Research Grant from the V Foundation for Cancer Research, and NIH grants U19ES011387 (H. Zarbl, P.I.) and P30ES005022 (H. Zarbl, P.I.) from the National Institute for Environmental Health Sciences. We thank Kathy Kelly McNeil, Michelle Robertson, Susan Goodin and Benjamin Crabtree for contributions to this study.