Understanding the association between sleep, shift work and COVID-19 vaccine immune response efficacy: Protocol of the S-CORE study.

This protocol describes an innovative study to investigate the relationship between sleep, shift work and the immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2; coronavirus disease 2019 [COVID-19]) vaccination. As the COVID-19 pandemic is a global crisis with devastating health, social and economic impacts, there is a pressing need for effective vaccination programmes. Previous influenza and hepatitis vaccination studies suggest that lack of sleep can negatively alter immune responsiveness, while circadian misalignment most likely may also play an important role in the immune response to vaccination. Our present study will be the first to address this question in actual shift workers and in relation to COVID-19 vaccination. We hypothesise that the occurrence of recent night shifts and diminished sleep will negatively alter the immune response to vaccination in shift workers compared to dayworkers. We aim to recruit 50 shift workers and 50 dayworkers. Participants will receive an mRNA-based vaccination, through the Dutch vaccination programme. To assess immune responsiveness, blood will be drawn at baseline (before first vaccination), 10 days after first vaccination, the day prior to the second vaccination; and 28 days, 6 and 12 months after the second vaccination. Actigraphy and daily sleep e-diaries will be implemented for 7 days around each vaccination to assess sleep. The Pittsburgh Sleep Quality Index will be used to monitor sleep in the long term. Optimising the efficacy of the COVID-19 vaccines is of outmost importance and results of this study could provide insights to develop sleep and circadian-based interventions to enhance vaccination immunity, and thereby improve global health.

The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

Genome-wide DNA methylation patterns associated with sleep and mental health in children: a population-based study.

BACKGROUND: DNA methylation (DNAm) has been implicated in the biology of sleep. Yet, how DNAm patterns across the genome relate to different sleep outcomes, and whether these associations overlap with mental health is currently unknown. Here, we investigated associations of DNAm with sleep and mental health in a pediatric population. METHODS: This cross-sectional study included 465 10-year-old children (51.3% female) from the Generation R Study. Genome-wide DNAm levels were measured using the Illumina 450K array (peripheral blood). Sleep problems were assessed from self-report and mental health outcomes from maternal questionnaires. Wrist actigraphy was used in 188 11-year-old children to calculate sleep duration and midpoint sleep. Weighted gene co-expression network analysis was used to identify highly comethylated DNAm 'modules', which were tested for associations with sleep and mental health outcomes. RESULTS: We identified 64 DNAm modules, one of which associated with sleep duration after covariate and multiple testing adjustment. This module included CpG sites spanning 9 genes on chromosome 17, including MAPT - a key regulator of Tau proteins in the brain involved in neuronal function - as well as genes previously implicated in sleep duration. Follow-up analyses suggested that DNAm variation in this region is under considerable genetic control and shows strong blood-brain concordance. DNAm modules associated with sleep did not overlap with those associated with mental health. CONCLUSIONS: We identified one DNAm region associated with sleep duration, including genes previously reported by recent GWAS studies. Further research is warranted to examine the functional role of this region and its longitudinal association with sleep.

Differential roles for cryptochromes in the mammalian retinal clock.

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.

CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock.

Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein-protein interactions revealed that CRY-mediated periodic binding of CK2ß to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1-CK2ß binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1-P-BMAL1 loop is an integral part of the core clock oscillator.

Clinical chronobiology: a timely consideration in critical care medicine.

A fundamental aspect of human physiology is its cyclical nature over a 24-h period, a feature conserved across most life on Earth. Organisms compartmentalise processes with respect to time in order to promote survival, in a manner that mirrors the rotation of the planet and accompanying diurnal cycles of light and darkness. The influence of circadian rhythms can no longer be overlooked in clinical settings; this review provides intensivists with an up-to-date understanding of the burgeoning field of chronobiology, and suggests ways to incorporate these concepts into daily practice to improve patient outcomes. We outline the function of molecular clocks in remote tissues, which adjust cellular and global physiological function according to the time of day, and the potential clinical advantages to keeping in time with them. We highlight the consequences of "chronopathology", when this harmony is lost, and the risk factors for this condition in critically ill patients. We introduce the concept of "chronofitness" as a new target in the treatment of critical illness: preserving the internal synchronisation of clocks in different tissues, as well as external synchronisation with the environment. We describe methods for monitoring circadian rhythms in a clinical setting, and how this technology may be used for identifying optimal time windows for interventions, or to alert the physician to a critical deterioration of circadian rhythmicity. We suggest a chronobiological approach to critical illness, involving multicomponent strategies to promote chronofitness (chronobundles), and further investment in the development of personalised, time-based treatment for critically ill patients.

Flexible phase adjustment of circadian albumin D site-binding protein (DBP) gene expression by CRYPTOCHROME1.

The albumin D site-binding protein (DBP) governs circadian transcription of a number of hepatic detoxification and metabolic enzymes prior to the activity phase and subsequent food intake of mice. However, the behavior of mice is drastically affected by the photoperiod. Therefore, continuous adjustment of the phase of circadian Dbp expression is required in the liver. Here we describe a direct impact of CRYPTOCHROME1 (CRY1) on the phase of Dbp expression. Dbp and the nuclear receptor Rev-Erbalpha are circadian target genes of BMAL1 and CLOCK. Surprisingly, dynamic CRY1 binding to the Dbp promoter region delayed BMAL1 and CLOCK-mediated transcription of Dbp compared with Rev-Erbalpha. Extended presence of CRY1 in the nucleus enabled continuous uncoupling of the phase of Dbp from Rev-Erbalpha expression upon change from short to longer photoperiods. CRY1 thus maintained the peak of DBP accumulation close to the activity phase. In contrast, Rev-Erbalpha expression was phase-locked to the circadian oscillator and shaped by accumulation of its own gene product. Our data indicate that fine-tuning of circadian transcription in the liver is even more sophisticated than expected.

Mutations in Cockayne Syndrome-Associated Genes (Csa and Csb) Predispose to Cisplatin-Induced Hearing Loss in Mice.

UNLABELLED: Cisplatin is a common and effective chemotherapeutic agent, yet it often causes permanent hearing loss as a result of sensory hair cell death. The causes of sensitivity to DNA-damaging agents in nondividing cell populations, such as cochlear hair and supporting cells, are poorly understood, as are the specific DNA repair pathways that protect these cells. Nucleotide excision repair (NER) is a conserved and versatile DNA repair pathway for many DNA-distorting lesions, including cisplatin-DNA adducts. Progressive sensorineural hearing loss is observed in a subset of NER-associated DNA repair disorders including Cockayne syndrome and some forms of xeroderma pigmentosum. We investigated whether either of the two overlapping branches that encompass NER, transcription-coupled repair or global genome repair, which are implicated in Cockayne syndrome and xeroderma pigmentosum group C, respectively, modulates cisplatin-induced hearing loss and cell death in the organ of Corti, the auditory sensory epithelium of mammals. We report that cochlear hair cells and supporting cells in transcription-coupled repair-deficient Cockayne syndrome group A (Csa(-/-)) and group B (Csb(-/-)) mice are hypersensitive to cisplatin, in contrast to global genome repair-deficient Xpc(-/-) mice, both in vitro and in vivo We show that sensory hair cells in Csa(-/-) and Csb(-/-) mice fail to remove cisplatin-DNA adducts efficiently in vitro; and unlike Xpc(-/-) mice, Csa(-/-) and Csb(-/-) mice lose hearing and manifest outer hair cell degeneration after systemic cisplatin treatment. Our results demonstrate that Csa and Csb deficiencies predispose to cisplatin-induced hearing loss and hair/supporting cell damage in the mammalian organ of Corti, and emphasize the importance of transcription-coupled DNA repair in the protection against cisplatin ototoxicity. SIGNIFICANCE STATEMENT: The utility of cisplatin in chemotherapy remains limited due to serious side effects, including sensorineural hearing loss. We show that mouse models of Cockayne syndrome, a progeroid disorder resulting from a defect in the transcription-coupled DNA repair (TCR) branch of nucleotide excision repair, are hypersensitive to cisplatin-induced hearing loss and sensory hair cell death in the organ of Corti, the mammalian auditory sensory epithelium. Our work indicates that Csa and Csb, two genes involved in TCR, are preferentially required to protect against cisplatin ototoxicity, relative to global genome repair-specific elements of nucleotide excision repair, and suggests that TCR is a major force maintaining DNA integrity in the cochlea. The Cockayne syndrome mice thus represent a model for testing the contribution of DNA repair mechanisms to cisplatin ototoxicity.

Critical cholangiocarcinogenesis control by cryptochrome clock genes.

A coordinated network of molecular circadian clocks in individual cells generates 24-hr rhythms in liver metabolism and proliferation. Circadian disruption through chronic jet lag or Per2 clock gene mutation was shown to accelerate hepatocarcinoma development in mice. As divergent effects were reported for clock genes Per and Cry regarding xenobiotic toxicity, we questioned the role of Cry1 and Cry2 in liver carcinogenesis. Male WT and Cry1-/- Cry2-/- mice (C57Bl/6 background) were chronically exposed to diethylnitrosamine (DEN) at ZT11. Rest-activity and body temperature rhythms were monitored using an implanted radiotransmitter. Serum aspartate and alanine aminotransferases (AST and ALT) were determined on four occasions during the progression stage. After 7 months, serum alkaline phosphatases (ALP) were determined, and livers were sampled for microscopic tumor nodule counting and histopathology. Five months after initiation of DEN treatment, we found that Cry1-/- Cry2-/- mice developed severe liver dysplasia, as evident from the increased AST, ALT and ALP levels, as compared to WT mice. DEN exposure induced primary liver cancers in nearly fivefold as many Cry1-/- Cry2-/- mice as compared to WT mice (p = 0.01). Microscopic study revealed no difference in the average number of hepatocarcinomas and a nearly eightfold increase in the average number of cholangiocarcinomas in Cry1-/- Cry2-/- mice, as compared to WT mice. This study validated the hypothesis that molecular circadian clock disruption dramatically increased chemically induced liver carcinogenesis. In addition, the pronounced shift toward cholangiocarcinoma in DEN exposed Cry1-/- Cry2-/- mice revealed a critical role of the Cry clock genes in bile duct carcinogenesis.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.

Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

Cockayne syndrome group B (Csb) and group a (Csa) deficiencies predispose to hearing loss and cochlear hair cell degeneration in mice.

Sensory hair cells in the cochlea, like most neuronal populations that are postmitotic, terminally differentiated, and non-regenerating, depend on robust mechanisms of self-renewal for lifelong survival. We report that hair cell homeostasis requires a specific sub-branch of the DNA damage nucleotide excision repair pathway, termed transcription-coupled repair (TCR). Cockayne syndrome (CS), caused by defects in TCR, is a rare DNA repair disorder with a broad clinical spectrum that includes sensorineural hearing loss. We tested hearing and analyzed the cellular integrity of the organ of Corti in two mouse models of this disease with mutations in the Csb gene (CSB(m/m) mice) and Csa gene (Csa(-/-) mice), respectively. Csb(m/m) and Csa(-/-) mice manifested progressive hearing loss, as measured by an increase in auditory brainstem response thresholds. In contrast to wild-type mice, mutant mice showed reduced or absent otoacoustic emissions, suggesting cochlear outer hair cell impairment. Hearing loss in Csb(m/m) and Csa(-/-) mice correlated with progressive hair cell loss in the base of the organ of Corti, starting between 6 and 13 weeks of age, which increased by 16 weeks of age in a basal-to-apical gradient, with outer hair cells more severely affected than inner hair cells. Our data indicate that the hearing loss observed in CS patients is reproduced in mouse models of this disease. We hypothesize that accumulating DNA damage, secondary to the loss of TCR, contributes to susceptibility to hearing loss.

Disruption of clock gene expression in human colorectal liver metastases.

The circadian timing system controls about 40 % of the transcriptome and is important in the regulation of a wide variety of biological processes including metabolic and proliferative functions. Disruption of the circadian clock could have significant effect on human health and has an important role in the development of cancer. Here, we compared the expression levels of core clock genes in primary colorectal cancer (CRC), colorectal liver metastases (CRLM), and liver tissue within the same patient. Surgical specimens of 15 untreated patients with primary CRC and metachronous CRLM were studied. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression of 10 clock genes: CLOCK, BMAL1, PER1, PER2, PER3, CRY1, CRY2, CSNK1E, TIM, TIPIN, and 2 clock-controlled genes: Cyclin-D1, and WEE1. Expression levels of 7 core clock genes were downregulated in CRLM: CLOCK (p = 0.006), BMAL1 (p = 0.003), PER1 (p = 0.003), PER2 (p = 0.002), PER3 (p < 0.001), CRY1 (p = 0.002), and CRY2 (p < 0.001). In CRC, 5 genes were downregulated: BMAL1 (p = 0.02), PER1 (p = 0.004), PER2 (p = 0.008), PER3 (p < 0.001), and CRY2 (p < 0.001). CSNK1E was upregulated in CRC (p = 0.02). Cyclin-D1 and WEE1 were both downregulated in CRLM and CRC. Related to clinicopathological factors, a significant correlation was found between low expression of CRY1 and female gender, and low PER3 expression and the number of CRLM. Our data demonstrate that the core clock is disrupted in CRLM and CRC tissue from the same patient. This disruption may be linked to altered cell-cycle dynamics and carcinogenesis.

Histone methyltransferase MLL3 contributes to genome-scale circadian transcription.

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian "output" genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of "core" clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis.

BACKGROUND: The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (ß-PV) play an important role in accelerating the early stages of skin tumorigenesis. METHODS: We investigated the effects of UV induced DNA damage in murine models of ß-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. RESULTS: Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1. CONCLUSION: We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.

Colorectal liver metastases with a disrupted circadian rhythm phase shift the peripheral clock in liver and kidney.

Circadian clock genes regulate 10-15% of the transcriptome, and might function as tumor suppressor genes. Relatively little is known about the circadian clock in tumors and its effect on surrounding healthy tissues. Therefore, we compared the 24-hr expression levels of key circadian clock genes in liver and kidney of healthy control mice with those of mice bearing C26 colorectal tumor metastases in the liver. Metastases were induced by injection of C26 colorectal carcinoma cells into the spleen. Subsequently, tumor, liver and kidney tissue was collected around the clock to compare circadian rhythmicity. Expression levels of five clock genes (Rev-Erbα, Per1, Per2, Bmal1 and Cry1) and three clock-controlled genes (CCGs; Dbp, p21 and Wee1) were determined by qRT-PCR. Liver and kidney tissue of healthy control mice showed normal 24-hr oscillations of all clock genes and CCGs, consistent with normal circadian rhythmicity. In colorectal liver metastases, however, 24-hr oscillations were completely absent for all clock genes and CCGs except Cry1. Liver and kidney tissue of tumor-bearing mice showed a shift in clock periodicity relative to control mice. In the liver we observed a phase advance, whereas in the kidney the phase was delayed. These data show that hepatic metastases of C26 colon carcinoma with a disrupted circadian rhythm phase shift liver and kidney tissue clocks, which strongly suggests a systemic effect on peripheral clocks. The possibility that tumors may modify peripheral clocks to escape from ordinary circadian rhythms and in this way contribute to fatigue and sleep disorders in cancer patients is discussed.

Circadian variation in tamoxifen pharmacokinetics in mice and breast cancer patients.

The anti-estrogen tamoxifen is characterized by a large variability in response, partly due to pharmacokinetic differences. We examined circadian variation in tamoxifen pharmacokinetics in mice and breast cancer patients. Pharmacokinetic analysis was performed in mice, dosed at six different times (24-h period). Tissue samples were used for mRNA expression analysis of drug-metabolizing enzymes. In patients, a cross-over study was performed. During three 24-h periods, after tamoxifen dosing at 8 a.m., 1 p.m., and 8 p.m., for at least 4 weeks, blood samples were collected for pharmacokinetic measurements. Differences in tamoxifen pharmacokinetics between administration times were assessed. The mRNA expression of drug-metabolizing enzymes showed circadian variation in mouse tissues. Tamoxifen exposure seemed to be highest after administration at midnight. In humans, marginal differences were observed in pharmacokinetic parameters between morning and evening administration. Tamoxifen C(max )and area under the curve (AUC)0-8 h were 20 % higher (P < 0.001), and tamoxifen t(max) was shorter (2.1 vs. 8.1 h; P = 0.001), indicating variation in absorption. Systemic exposure (AUC0-24 h) to endoxifen was 15 % higher (P < 0.001) following morning administration. The results suggest that dosing time is of marginal influence on tamoxifen pharmacokinetics. Our study was not designed to detect potential changes in clinical outcome or toxicity, based on a difference in the time of administration. Circadian rhythm may be one of the many determinants of the interpatient and intrapatient pharmacokinetic variability of tamoxifen.

A day and night difference in the response of the hepatic transcriptome to cyclophosphamide treatment.

Application of omics-based technologies is a widely used approach in research aiming to improve testing strategies for human health risk assessment. In most of these studies, however, temporal variations in gene expression caused by the circadian clock are a commonly neglected pitfall. In the present study, we investigated the impact of the circadian clock on the response of the hepatic transcriptome after exposure of mice to the chemotherapeutic agent cyclophosphamide (CP). Analysis of the data without considering clock progression revealed common responses in terms of regulated pathways between light and dark phase exposure, including DNA damage, oxidative stress, and a general immune response. The overall response, however, was stronger in mice exposed during the day. Use of time-matched controls, thereby eliminating non-CP-responsive circadian clock-controlled genes, showed that this difference in response was actually even more pronounced: CP-related responses were only identified in mice exposed during the day. Only minor differences were found in acute toxicity pathways, namely lymphocyte counts and kidney weights, indicating that gene expression is subject to time of day effects. This study is the first to highlight the impact of the circadian clock on the identification of toxic responses by omics approaches.

Optimization of irinotecan chronotherapy with P-glycoprotein inhibition.

The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F1 mice. A three-fold 24h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p<0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50mg/kg/day i.v.×4days) as a single agent or combined with P-gp inhibitor PSC833 (6.25mg/kg/day i.p.×4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40mg/kg/day×4days) and +/-PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p<0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p<0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ~60%. In tumor-bearing mice, body weight loss was ~halved in the mice on irinotecan or irinotecan-PSC833 combination at ZT15 as compared to ZT3 (p<0.001). PSC833-irinotecan at ZT15 increased tumor inhibition by ~40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan.

An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.