Circulating cell-free DNA in health and disease - the relationship to health behaviours, ageing phenotypes and metabolomics.

Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17-82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.

The burden of overweight: Higher body mass index, but not vital exhaustion, is associated with higher DNA damage and lower DNA repair capacity.

BACKGROUND/OBJECTIVES: DNA damage and the capacity to repair damaged DNA have been associated with the pathogenesis of several diseases such as cancer. While it is well known that external mutagenic agents can induce DNA damage, less is known about endogenous contributors to genomic instability. The aim of this study was to investigate whether excess body weight as a physiological factor and vital exhaustion as a psychological factor would be associated with basal levels of DNA damage as well as DNA repair capacity. SUBJECTS/METHODS: In a cross-sectional between-subject design we recruited 53 apparently healthy men within the normal to non-obese overweight range (mean BMI: 25.2 ± 0.5) who were either vitally exhausted (VE) (VE-score ≥ 10) or non-exhausted (VE-score ≤ 3). Vital exhaustion was assessed using the Maastricht Vital Exhaustion Questionnaire. We assessed DNA damage and repair in terms of strand breaks in PBMCs by means of the automated Fluorimetric Detection of Alkaline Unwinding (FADU) assay. DNA repair capacity was assessed by repeatedly measuring the amount of intact DNA up to 90 min after standardized X-irradiation of the cells. RESULTS: General linear models revealed that elevated levels of basal DNA damage (ß=-0.34, p=0.013, f=0.33) as well as impaired capacity to repair damaged DNA (F(1/50)=5.40, p=0.024, f=0.33) with increasing BMI, but not with vital exhaustion (p's ≥ 0.63). CONCLUSION: Our findings point to DNA integrity impairments with increasing BMI, already in the overweight range, and suggest impaired DNA repair as a potential underlying molecular mechanism. In contrast, the psychological factor vital exhaustion was not associated with DNA damage or DNA repair capacity.

Procaine-The Controversial Geroprotector Candidate: New Insights Regarding Its Molecular and Cellular Effects.

Since its discovery in 1905 and its employment in everyday medical practice as a local anesthetic, to its highly controversial endorsement as an "anti-aging" molecule in the sixties and seventies, procaine is part of the history of medicine and gerontoprophylaxis. Procaine can be considered a "veteran" drug due to its long-time use in clinical practice, but is also a molecule which continues to incite interest, revealing new biological and pharmacological effects within novel experimental approaches. Therefore, this review is aimed at exploring and systematizing recent data on the biochemical, cellular, and molecular mechanisms involved in the antioxidant and potential geroprotective effects of procaine, focusing on the following aspects: (1) the research state-of-the-art, through an objective examination of scientific literature within the last 30 years, describing the positive, as well as the negative reports; (2) the experimental data supporting the beneficial effects of procaine in preventing or alleviating age-related pathology; and (3) the multifactorial pathways procaine impacts oxidative stress, inflammation, atherogenesis, cerebral age-related pathology, DNA damage, and methylation. According to reviewed data, procaine displayed antioxidant and cytoprotective actions in experimental models of myocardial ischemia/reperfusion injury, lipoprotein oxidation, endothelial-dependent vasorelaxation, inflammation, sepsis, intoxication, ionizing irradiation, cancer, and neurodegeneration. This analysis painted a complex pharmacological profile of procaine: a molecule that has not yet fully expressed its therapeutic potential in the treatment and prevention of aging-associated diseases. The numerous recent reports found demonstrate the rising interest in researching the multiple actions of procaine regulating key processes involved in cellular senescence. Its beneficial effects on cell/tissue functions and metabolism could designate procaine as a valuable candidate for the well-established Geroprotectors database.

Age-related activity of Poly (ADP-Ribose) Polymerase (PARP) in men with localized prostate cancer.

Mutations in DNA repair genes have been connected with familial prostate cancer and sensitivity to targeted drugs like PARP-inhibitors. Clinical use of this information is limited by the small fraction of prostate cancer risk gene carriers, variants of unknown pathogenicity and the focus on monogenic disease mechanisms. Functional assays capturing mono- and polygenic defects were shown to detect breast and ovarian cancer risk in blood-derived cells. Here, we comparatively analyzed lymphocytes from prostate cancer patients and controls applying a sensitive DNA double-strand break (DSB) repair assay and a flow cytometrybased assay measuring the activity of Poly(ADP-Ribose)-Polymerase, a target in treatment of metastatic prostate cancer. Contrary to breast and ovarian cancer patients, error-prone DNA double-strand break repair was not activated in prostate cancer patients. Yet, the activity of PARP discriminated between prostate cancer cases and controls. PARylation also correlated with the age of male probands, suggesting male-specific links between mutation-based and aging-associated DNA damage accumulation and PARP. Our work identifies prostate cancer-specific DNA repair phenotypes characterized by increased PARP activities and carboplatin-sensitivities, detected by functional testing of lymphocytes. This provides new insights for further investigation of PARP and carboplatin sensitivity as biomarkers in peripheral cells of men and prostate cancer patients.

Medication Intake Is Associated with Lower Plasma Carotenoids and Higher Fat-Soluble Vitamins in the Cross-Sectional MARK-AGE Study in Older Individuals.

The regular use of medication may interfere with micronutrient metabolism on several levels, such as absorption, turnover rate, and tissue distribution, and this might be amplified during aging. This study evaluates the impact of self-reported medication intake on plasma micronutrients in the MARK-AGE Project, a cross-sectional observational study in 2217 subjects (age- and sex-stratified) aged 35-75 years from six European countries that were grouped according to age. Polypharmacy as possible determinant of micronutrient concentrations was assessed using multiple linear regression models adjusted for age-group, dietary fruit, vegetables, and juice intake, and other confounders. Younger participants reported taking fewer drugs than older participants. Inverse associations between medication intake and lutein (-3.31% difference per increase in medication group), ß-carotene (-11.44%), α-carotene (-8.50%) and positive associations with retinol (+2.26%), α-tocopherol/cholesterol (+2.89%) and γ-tocopherol/cholesterol (+1.36%) occurred in multiple adjusted regression models. Combined usage of a higher number of medical drugs was associated with poorer status of carotenoids on the one hand and higher plasma concentrations of retinol, α- and γ-tocopherol on the other hand. Our results raise concerns regarding the safety of drug combinations via the significant and surprisingly multifaceted disturbance of the concentrations of relevant micronutrients.

Single-Cell RNA-Sequencing Identifies Activation of TP53 and STAT1 Pathways in Human T Lymphocyte Subpopulations in Response to Ex Vivo Radiation Exposure.

Detrimental health consequences from exposure to space radiation are a major concern for long-duration human exploration missions to the Moon or Mars. Cellular responses to radiation are expected to be heterogeneous for space radiation exposure, where only high-energy protons and other particles traverse a fraction of the cells. Therefore, assessing DNA damage and DNA damage response in individual cells is crucial in understanding the mechanisms by which cells respond to different particle types and energies in space. In this project, we identified a cell-specific signature for radiation response by using single-cell transcriptomics of human lymphocyte subpopulations. We investigated gene expression in individual human T lymphocytes 3 h after ex vivo exposure to 2-Gy gamma rays while using the single-cell sequencing technique (10X Genomics). In the process, RNA was isolated from ~700 irradiated and ~700 non-irradiated control cells, and then sequenced with ~50 k reads/cell. RNA in each of the cells was distinctively barcoded prior to extraction to allow for quantification for individual cells. Principal component and clustering analysis of the unique molecular identifier (UMI) counts classified the cells into three groups or sub-types, which correspond to CD4+, naïve, and CD8+/NK cells. Gene expression changes after radiation exposure were evaluated using negative binomial regression. On average, BBC3, PCNA, and other TP53 related genes that are known to respond to radiation in human T cells showed increased activation. While most of the TP53 responsive genes were upregulated in all groups of cells, the expressions of IRF1, STAT1, and BATF were only upregulated in the CD4+ and naïve groups, but were unchanged in the CD8+/NK group, which suggests that the interferon-gamma pathway does not respond to radiation in CD8+/NK cells. Thus, single-cell RNA sequencing technique was useful for simultaneously identifying the expression of a set of genes in individual cells and T lymphocyte subpopulation after gamma radiation exposure. The degree of dependence of UMI counts between pairs of upregulated genes was also evaluated to construct a similarity matrix for cluster analysis. The cluster analysis identified a group of TP53-responsive genes and a group of genes that are involved in the interferon gamma pathway, which demonstrate the potential of this method for identifying previously unknown groups of genes with similar expression patterns.

Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells.

The implementation of rotating-wall vessels (RWVs) for studying the effect of lack of gravity has attracted attention, especially in the fields of stem cells, tissue regeneration, and cancer research. Immune cells incubated in RWVs exhibit several features of immunosuppression including impaired leukocyte proliferation, cytokine responses, and antibody production. Interestingly, stress hormones influence cellular immune pathways affected by microgravity, such as cell proliferation, apoptosis, DNA repair, and T cell activation. These pathways are crucial defense mechanisms that protect the cell from toxins, pathogens, and radiation. Despite the importance of the adrenergic receptor in regulating the immune system, the effect of microgravity on the adrenergic system has been poorly studied. Thus, we elected to investigate the synergistic effects of isoproterenol (a sympathomimetic drug), radiation, and microgravity in nonstimulated immune cells. Peripheral blood mononuclear cells were treated with the sympathomimetic drug isoproterenol, exposed to 0.8 or 2 Gy γ-radiation, and incubated in RWVs. Mixed model regression analyses showed significant synergistic effects on the expression of the ß2-adrenergic receptor gene (ADRB2). Radiation alone increased ADRB2 expression, and cells incubated in microgravity had more DNA strand breaks than cells incubated in normal gravity. We observed radiation-induced cytokine production only in microgravity. Prior treatment with isoproterenol clearly prevents most of the microgravity-mediated effects. RWVs may be a useful tool to provide insight into novel regulatory pathways, providing benefit not only to astronauts but also to patients suffering from immune disorders or undergoing radiotherapy.

The degree of radiation-induced DNA strand breaks is altered by acute sleep deprivation and psychological stress and is associated with cognitive performance in humans.

Study Objectives: Sleep deprivation is associated with impaired immune responses, cancer, and morbidity and mortality, and can degrade cognitive performance, although individual differences exist in such responses. Sleep deprivation induces DNA strand breaks and DNA base oxidation in animals, and psychological stress is associated with increased DNA damage in humans. It remains unknown whether sleep deprivation or psychological stress in humans affects DNA damage response from environmental stressors, and whether these responses predict cognitive performance during sleep deprivation. Methods: Sixteen healthy adults (ages 29-52 years; mean age ± SD, 36.4 ± 7.1 years; seven women) participated in a 5-day experiment involving two 8 hr time-in-bed (TIB) baseline nights, followed by 39 hr total sleep deprivation (TSD), and two 8-10 hr TIB recovery nights. A modified Trier Social Stress Test was conducted on the day after TSD. The Psychomotor Vigilance Test measured behavioral attention. DNA damage was assessed in blood cells collected at 5 time points, and blood cells were irradiated ex vivo. Results: TSD, alone or in combination with psychological stress, did not induce significant increases in DNA damage. By contrast, radiation-induced DNA damage decreased significantly in response to TSD, but increased back to baseline when combined with psychological stress. Cognitively vulnerable individuals had more radiation-induced DNA strand breaks before TSD, indicating their greater sensitivity to DNA damage from environmental stressors. Conclusions: Our results provide novel insights into the molecular consequences of sleep deprivation, psychological stress, and performance vulnerability. They are important for fields involving sleep loss, radiation exposure, and cognitive deficits, including cancer therapy, environmental toxicology, and space medicine.

Impaired PARP activity in response to the ß-adrenergic receptor agonist isoproterenol.

Psychological stress has been associated with DNA damage, thus increasing the risk of numerous diseases including cancer. Here, we investigate the effect of acute and chronic stress on poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage and DNA repair initiator. In order to mimic the chronic release of epinephrine, human peripheral blood mononuclear cells (PBMCs) were treated repeatedly with the sympathomimetic drug isoproterenol. We found significant induction of DNA strand breaks that remained unrepaired 24 h after ex vivo incubation. Isoproterenol-induced DNA strand breaks could be partially prevented by pre-treatment with the ß-adrenergic receptor antagonist propranolol. Furthermore, the level of PARP-1 protein and PARP activity decreased and the levels of the PARP substrate nicotinamide adenine dinucleotide (NAD+) and of adenosine triphosphate (ATP), necessary to replenish NAD+ pools, were lowered by isoproterenol treatment. In conclusion our data provide novel insights into the mechanisms of isoproterenol-induced genotoxicity linking ß-adrenergic stimulation and PARP-1.

DNA Hydroxymethylation Levels Are Altered in Blood Cells From Down Syndrome Persons Enrolled in the MARK-AGE Project.

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the aging process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work, we investigated the levels of 5-hydroxymethylcytosine and of the Ten-eleven translocation dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in aging. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease in 5-hydroxymethylcytosine, TET1, and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS.

Zinc-Induced Metallothionein in Centenarian Offspring From a Large European Population: The MARK-AGE Project.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35-75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan.

Apoptosis and expression of apoptosis-related genes in mouse intestinal tissue after whole-body proton exposure.

Energetic protons are the most abundant particle type in space and can pose serious health risks to astronauts during long-duration missions. The health effects of proton exposure are also a concern for cancer patients undergoing radiation treatment with accelerated protons. To investigate the damage induced by energetic protons in vivo to radiosensitive organs, 6-week-old BALB/c male mice were subjected to 250 MeV proton radiation at whole-body doses of 0.1, 1, and 2 Gy. The gastrointestinal (GI) tract of each exposed animal was dissected 4 h post-irradiation, and the isolated small intestinal tissue was analyzed for histopathological and gene expression changes. Histopathologic observation of the tissue using standard hematoxylin and eosin (H&E) staining methods to screen for morphologic changes showed a marked increase in apoptotic lesions for even the lowest dose of 0.1 Gy, similar to X- or γ rays. The percentage of apoptotic cells increased dose-dependently, but the dose response appeared supralinear, indicating hypersensitivity at low doses. A significant decrease in surviving crypts and mucosal surface area, as well as in cell proliferation, was also observed in irradiated mice. Gene expression analysis of 84 genes involved in the apoptotic process showed that most of the genes affected by protons were common between the low (0.1 Gy) and high (1 and 2 Gy) doses. However, the genes that were distinctively responsive to the low or high doses suggest that high doses of protons may cause apoptosis in the small intestine by direct damage to the DNA, whereas low doses of protons may trigger apoptosis through a different stress response mechanism.

Increased single-strand annealing rather than non-homologous end-joining predicts hereditary ovarian carcinoma.

Mutations in genes encoding DNA double-strand break (DSB) repair components, especially homologous recombination (HR) proteins, were found to predispose to breast and ovarian cancer. Beyond high penetrance risk gene mutations underlying monogenic defects, low risk gene mutations generate polygenic defects, enlarging the fraction of individuals with a predisposing phenotype. DSB repair dysfunction opens new options for targeted therapies; poly (ADP-ribose) polymerase (PARP) inhibitors have been approved for BRCA-mutated and platinum-responsive ovarian cancers. In this work, we performed functional analyses in peripheral blood lymphocytes (PBLs) using a case-control design. We examined 38 women with familial history of breast and/or ovarian cancer, 40 women with primary ovarian cancer and 34 healthy controls. Using a GFP-based test we analyzed error-prone DSB repair mechanisms which are known to compensate for HR defects and to generate chromosomal instabilities. While non-homologous end-joining (NHEJ) did not discriminate between cases and controls, we found increases of single-strand annealing (SSA) in women with familial risk vs. controls (P=0.016) and patients with ovarian cancer vs. controls (P=0.002). Consistent with compromised HR we also detected increased sensitivities to carboplatin in PBLs from high-risk individuals (P<0.0001) as well as patients (P=0.0011) compared to controls. Conversely, neither PARP inhibitor responses nor PARP activities were altered in PBLs from the case groups, but PARP activities increased with age in high-risk individuals, providing novel clues for differential drug mode-of-action. Our findings indicate the great potential of detecting SSA activities to deliver an estimate of ovarian cancer susceptibility and therapeutic responsiveness beyond the limitations of genotyping.

Combined Effects of Simulated Microgravity and Radiation Exposure on Osteoclast Cell Fusion.

The loss of bone mass and alteration in bone physiology during space flight are one of the major health risks for astronauts. Although the lack of weight bearing in microgravity is considered a risk factor for bone loss and possible osteoporosis, organisms living in space are also exposed to cosmic radiation and other environmental stress factors. As such, it is still unclear as to whether and by how much radiation exposure contributes to bone loss during space travel, and whether the effects of microgravity and radiation exposure are additive or synergistic. Bone is continuously renewed through the resorption of old bone by osteoclast cells and the formation of new bone by osteoblast cells. In this study, we investigated the combined effects of microgravity and radiation by evaluating the maturation of a hematopoietic cell line to mature osteoclasts. RAW 264.7 monocyte/macrophage cells were cultured in rotating wall vessels that simulate microgravity on the ground. Cells under static 1g or simulated microgravity were exposed to γ rays of varying doses, and then cultured in receptor activator of nuclear factor-κB ligand (RANKL) for the formation of osteoclast giant multinucleated cells (GMCs) and for gene expression analysis. Results of the study showed that radiation alone at doses as low as 0.1 Gy may stimulate osteoclast cell fusion as assessed by GMCs and the expression of signature genes such as tartrate resistant acid phosphatase (Trap) and dendritic cell-specific transmembrane protein (Dcstamp). However, osteoclast cell fusion decreased for doses greater than 0.5 Gy. In comparison to radiation exposure, simulated microgravity induced higher levels of cell fusion, and the effects of these two environmental factors appeared additive. Interestingly, the microgravity effect on osteoclast stimulatory transmembrane protein (Ocstamp) and Dcstamp expressions was significantly higher than the radiation effect, suggesting that radiation may not increase the synthesis of adhesion molecules as much as microgravity.

Associations between Specific Redox Biomarkers and Age in a Large European Cohort: The MARK-AGE Project.

Oxidative stress and antioxidants play a role in age-related diseases and in the aging process. We here present data on protein carbonyls, 3-nitrotyrosine, malondialdehyde, and cellular and plasma antioxidants (glutathione, cysteine, ascorbic acid, uric acid, α-tocopherol, and lycopene) and their relation with age in the European multicenter study MARK-AGE. To avoid confounding, only data from countries which recruited subjects from all three study groups (five of eight centers) and only participants aged ≥55 years were selected resulting in data from 1559 participants. These included subjects from (1) the general population, (2) members from long-living families, and (3) their spouses. In addition, 683 middle-aged reference participants (35-54 years) served as a control. After adjustment for age, BMI, smoking status, gender, and country, there were differences in protein carbonyls, malondialdehyde, 3-nitrotyrosine, α-tocopherol, cysteine, and glutathione between the 3 study groups. Protein carbonyls and 3-nitrotyrosine as well as cysteine, uric acid, and lycopene were identified as independent biomarkers with the highest correlation with age. Interestingly, from all antioxidants measured, only lycopene was lower in all aged groups and from the oxidative stress biomarkers, only 3-nitrotyrosine was increased in the descendants from long-living families compared to the middle-aged control group. We conclude that both lifestyle and genetics may be important contributors to redox biomarkers in an aging population.

Interplay of space radiation and microgravity in DNA damage and DNA damage response.

In space, multiple unique environmental factors, particularly microgravity and space radiation, pose constant threat to the DNA integrity of living organisms. Specifically, space radiation can cause damage to DNA directly, through the interaction of charged particles with the DNA molecules themselves, or indirectly through the production of free radicals. Although organisms have evolved strategies on Earth to confront such damage, space environmental conditions, especially microgravity, can impact DNA repair resulting in accumulation of severe DNA lesions. Ultimately these lesions, namely double strand breaks, chromosome aberrations, micronucleus formation, or mutations, can increase the risk for adverse health effects, such as cancer. How spaceflight factors affect DNA damage and the DNA damage response has been investigated since the early days of the human space program. Over the years, these experiments have been conducted either in space or using ground-based analogs. This review summarizes the evidence for DNA damage induction by space radiation and/or microgravity as well as spaceflight-related impacts on the DNA damage response. The review also discusses the conflicting results from studies aimed at addressing the question of potential synergies between microgravity and radiation with regard to DNA damage and cellular repair processes. We conclude that further experiments need to be performed in the true space environment in order to address this critical question.

Plasma Carotenoids, Tocopherols, and Retinol in the Age-Stratified (35-74 Years) General Population: A Cross-Sectional Study in Six European Countries.

Blood micronutrient status may change with age. We analyzed plasma carotenoids, α-/γ-tocopherol, and retinol and their associations with age, demographic characteristics, and dietary habits (assessed by a short food frequency questionnaire) in a cross-sectional study of 2118 women and men (age-stratified from 35 to 74 years) of the general population from six European countries. Higher age was associated with lower lycopene and α-/ß-carotene and higher ß-cryptoxanthin, lutein, zeaxanthin, α-/γ-tocopherol, and retinol levels. Significant correlations with age were observed for lycopene (r = -0.248), α-tocopherol (r = 0.208), α-carotene (r = -0.112), and ß-cryptoxanthin (r = 0.125; all p < 0.001). Age was inversely associated with lycopene (-6.5% per five-year age increase) and this association remained in the multiple regression model with the significant predictors (covariables) being country, season, cholesterol, gender, smoking status, body mass index (BMI (kg/m²)), and dietary habits. The positive association of α-tocopherol with age remained when all covariates including cholesterol and use of vitamin supplements were included (1.7% vs. 2.4% per five-year age increase). The association of higher ß-cryptoxanthin with higher age was no longer statistically significant after adjustment for fruit consumption, whereas the inverse association of α-carotene with age remained in the fully adjusted multivariable model (-4.8% vs. -3.8% per five-year age increase). We conclude from our study that age is an independent predictor of plasma lycopene, α-tocopherol, and α-carotene.

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study.

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

Stress Hormone-Mediated DNA Damage Response--Implications for Cellular Senescence and Tumour Progression.

When DNA damage occurs, cells stop the cell cycle and DNA repair can take place. However, if DNA damage exceeds DNA repair capacities, cells undergo either apoptosis or senescence. These mechanisms preclude the proliferation of cells with heavily damaged DNA, thus protecting the organism against tumour development. When individuals are exposed to stress, the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic- adrenal-medullary (SAM) system can be activated leading to secretion of corticosteroids and catecholamines, respectively. The influences of these stress-related hormones have been proposed to promote cellular senescence. But paradoxically, chronic stimulation of the HPA axis is associated with higher risk of developing cancer. Focusing on the DNA damage response pathway, this review discusses whether stress hormones induce senescence or tumour progression or both and presents historical and recent data that might help resolve some of these controversies.

Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients.

AIMS: Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics and DNA damage, in peripheral blood mononuclear cells (PBMCs) of AD and control participants, for biomarker discovery. METHODS: PBMCs were isolated from 53 patients with AD of mild to moderate degree and 30 age-matched healthy controls. Tests were performed on the PBMCs from as many of these participants as possible. We measured glycolysis and mitochondrial respiration fluxes using the Seahorse Bioscience flux analyzer, mitochondrial ROS production using flow cytometry, dNTP levels by way of a DNA polymerization assay, DNA strand breaks using the Fluorometric detection of Alkaline DNA Unwinding (FADU) assay, and APE1 incision activity (in cell lysates) on a DNA substrate containing an AP site (to estimate DNA repair efficiency). RESULTS: In the PBMCs of AD patients, we found reduced basal mitochondrial oxygen consumption, reduced proton leak, higher dATP level, and lower AP endonuclease 1 activity, depending on adjustments for gender and/or age. CONCLUSIONS: This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD.