Diurnal relationship between core clock gene BMAL1, antioxidant SOD1 and oxidative RNA/DNA damage in young and older healthy women.

Abstract

BMAL1 is a core clock gene that positively regulates circadian rhythms. In animals, BMAL1 further acts as transcription factor for the SOD1 gene which encodes the major antioxidant enzyme superoxide dismutase. SOD1 protects against oxidative damage that is a major factor for human aging. Mice lacking mBmal1 exhibit premature aging phenotypes which might be related to a reduced expression of SOD1. The purpose of this study was to explore the circadian relationship between BMAL1, SOD1, oxidative RNA/DNA damage and aging in healthy humans. In a cross-sectional study design, buccal mucosa cells and saliva samples were obtained from 21 young (23.7±2.3yrs) and 21 older women (66.8±5.7yrs) within 24h at 4-h intervals (08:00, 12:00, 16:00, 20:00, 24:00 and 04:00h). Transcript levels of BMAL1 and SOD1 were measured by real-time quantitative PCR. Protein levels of SOD1 were determined by immunoblotting and densitometry. Levels of oxidative RNA/DNA damage and melatonin were quantified by enzyme immunosorbent assays. Transcript levels of BMAL1 and SOD1 mRNAs as well as protein levels of SOD1 and melatonin exhibited significant 24-h variation in each age group (P<0.010, Friedman tests). The mRNA expression patterns of BMAL1 and SOD1 showed similar 24-h rhythmicity and positive relationships were found. Strongest relationships occurred in young women at 12:00h (rs=0.81, P=0.005) and in older women at 08:00h (r=0.84, P<0.001). Maximum levels of SOD1 mRNA appeared within 24h in both age groups at 24:00h. In both age groups, the timing of maximum level for SOD1 protein was delayed relative to the timing of maximum level for SOD1 mRNA. This delay was larger in older women (8h) compared to young women (4h). Older women showed higher oxidative RNA/DNA damage at all time-points compared to young women (P<0.020). The oxidative RNA/DNA damage decreased continuously from 08:00 to 20:00h (P<0.001) in both age groups. Although oxidative damage and SOD1 protein levels declined simultaneously, only weak and non-significant relationships were noted. This study demonstrates that transcript levels of SOD1 and protein level of SOD1 follow a circadian pattern of expression in healthy young and older women. The time-shift found between the 24-h maximum levels of SOD1 mRNA and SOD1 protein could be explained by the time needed for translation of SOD1 protein. The positive relationship found between expression levels of SOD1 mRNA and BMAL1 mRNA is in line with animal studies showing that BMAL1 acts as transcription factor and regulates the circadian synthesis of SOD1 mRNA. Differences between young and older women found in the timing of 24-h maxima as well as differences observed in 24-h relationships between characters might be due to age-related alterations in the circadian system. Absence of relationships between levels of SOD1 and oxidative RNA/DNA damage argue for a more complex interaction between the antioxidant system and the circadian system.