Biomarkers for circadian rhythm disruption independent of time of day

Frequent shift work causes disruption of the circadian rhythm and might on the long-term result in increased health risk. Current biomarkers evaluating the presence of circadian rhythm disturbance (CRD), including melatonin, cortisol and body temperature, require 24-hr ("around the clock") measurements, which is tedious. Therefore, these markers are not eligible to be used in large-scale (human) studies.

Transcription-coupled and global genome repair differentially influence UV-B-induced acute skin effects and syste

Exposure to UV-B radiation impairs immune responses in mammals by inhibiting especially Th1-mediated contact hypersensitivity and delayed-type hypersensitivity. Immunomodulation is not restricted to the exposed skin, but is also observed at distant sites, indicating the existence of mediating factors such as products from exposed skin cells or photoactivated factors present in the superficial layers. DNA damage appears to play a key role, because enhanced nucleotide excision repair (NER) strongly counteracts immunosuppression. To determine the effects of the type and genomic location of UV-induced DNA damage on immunosuppression and acute skin reactions (edema and erythema) four congenic mouse strains carrying different defects in NER were compared: CSB and XPC mice lacking transcription-coupled or global genome NER, respectively, as well as XPA and TTD/XPD mice carrying complete or partial defects in both NER subpathways, respectively. The major conclusions are that 1) transcription-coupled DNA repair is the dominant determinant in protection against acute skin effects; 2) systemic immunomodulation is only affected when both NER subpathways are compromised; and 3) sunburn is not related to UV-B-induced immunosuppression

High Preservation of CpG Cytosine Methylation Patterns at Imprinted Gene Loci in Liver and Brain of Aged Mice

A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the

Preoperative fasting protects against renal ischemia-reperfusion injury in aged and overweight mice

Ischemia-reperfusion injury (IRI) is inevitable during kidney transplantation leading to oxidative stress and inflammation. We previously reported that preoperative fasting in young-lean male mice protects against IRI. Since patients are generally of older age with morbidities possibly leading to a different response to fasting, we investigated the effects of preoperative fasting on renal IRI in aged-overweight male and female mice. Male and female F1-FVB/C57BL6-hybrid mice, average age 73 weeks weighing 47.2 grams, were randomized to preoperative ad libitum feeding or 3 days fasting, followed by renal IRI. Body weight, kidney function and survival of the animals were monitored until day 28 postoperatively. Kidney histopathology was scored for all animals and gene expression profiles after fasting were analyzed in kidneys of young and aged male mice. Preoperative fasting significantly improved survival after renal IRI in both sexes compared with normal fed mice. Fasted groups had a better kidney function shown by lower serum urea levels after renal IRI. Histopathology showed less acute tubular necrosis and more regeneration in kidneys from fasted mice. A mRNA analysis indicated the involvement of metabolic processes including fatty acid oxidation and retinol metabolism, and the NRF2-mediated stress response. Similar to young-lean, healthy male mice, preoperative fasting protects against renal IRI in aged-overweight mice of both genders. These findings suggest a general protective response of fasting against renal IRI regardless of age, gender, body weight and genetic background. Therefore, fasting could be a non-invasive intervention inducing increased oxidative stress resistance in older and overweight patients as well

Mouse model for the DNA repair/basal transcription disorder Trichothiodystrophy reveals cancer predisposition.

Patients with the nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) are highly predisposed to develop sunlight-induced skin cancer, in remarkable contrast to photosensitive NER-deficient trichothiodystrophy (TTD) patients carrying mutations in the same XPD gene. XPD encodes a helicase subunit of the dually functional DNA repair/basal transcription complex TFIIH. The pleiotropic disease phenotype is hypothesized to be, in part, derived from a repair defect causing UV sensitivity and, in part, from a subtle, viable basal transcription deficiency accounting for the cutaneous, developmental, and the typical brittle hair features of TTD. To understand the relationship between deficient NER and tumor susceptibility, we used a mouse model for TTD that mimics an XPD point mutation of a TTD patient in the mouse germline. Like the fibroblasts from the patient, mouse cells exhibit a partial NER defect, evident from the reduced UV-induced DNA repair synthesis (residual repair capacity approximately 25%), limited recovery of RNA synthesis after UV exposure, and a relatively mild hypersensitivity to cell killing by UV or 7,12-dimethylbenz[a]anthracene. In accordance with the cellular studies, TTD mice exhibit a modestly increased sensitivity to UV-induced inflammation and hyperplasia of the skin. In striking contrast to the human syndrome, TTD mice manifest a dear susceptibility to UV- and 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis, albeit not as pronounced as the totally NER-deficient XPA mice. These findings open up the possibility that TTD is associated with a so far unnoticed cancer predisposition and support the notion that a NER deficiency enhances cancer susceptibility. These findings have important implications for the etiology of the human disorder and for the impact of NER on carcinogenesis

A signature of renal stress resistance induced by short-Term dietary restriction, fasting, and protein restriction

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-Term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate-or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-Analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-Term DR

The progeroid phenotype of Ku80 deficiency Is dominant over DNA-PK CS deficiency

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80-/- mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs -/- mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80-/-, dna-pkcs -/- and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver a

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.</p

TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts

Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[. a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[. a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C. &gt;. T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers' lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.</p

The transcriptomic response to irinotecan in colon carcinoma bearing mice preconditioned by fasting

Background: Irinotecan use is limited due to severe toxicity. Preconditioning by fasting (PBF) protects against side effects of irinotecan while preserving its antitumor activity. The mechanisms underlying the effects of PBF still need to be elucidated. Here, we investigated the transcriptional responses of PBF on irinotecan in both tumor and healthy liver tissue. Experimental approach: Male BALB/c mice were subcutaneously injected with C26 colon carcinoma cells. Twelve days after tumor inoculation, two groups were fasted for three days and two groups were allowed food ad libitum (AL). Subsequently, both groups received one dose of irinotecan. Twelve hours after administration mice were sacrificed and blood, tumor and liver tissue were harvested. Blood samples were analyzed to determine liver, kidney and bone marrow function, tissues were used for transcriptome analyses. Key results: The AL irinotecan group showed worsened organ function and decreased leukocyte numbers. These effects were abated in PBF animals. PBF led to an altered transcriptional response in the liver of irinotecan-treated mice, including decreased cellular injury and increased stress resistance. Hepatic metabolism of irinotecan was also significantly changed due to PBF. The transcriptional response of tumor tissue observed after PBF was hardly affected compared to AL fed animals. Conclusions: Transcriptional changes after PBF to irinotecan treatment showed an improved protective stress response in healthy liver but not in tumor tissue, including changes in irinotecan metabolism. These data help to unravel the mechanisms underlying the effects of fasting on irinotecan and help to improve outcome of chemotherapeutic treatment in cancer patients