Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples.

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci 2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay.

Expanded usage of the Challenge-Comet assay as a DNA repair biomarker in human populations: protocols for fresh and cryopreserved blood samples, and for different challenge agents.

Deficiencies in DNA damage response and repair (DDRR) can cause serious pathological outcomes; therefore, having an ability to determine individual DDRR would enhance specificities in health risk assessment and in determining individual's response to cancer therapies. However, most methods for evaluating DDRR are not fully appropriate for population studies. The Challenge-Comet assay has gained acceptance for this purpose. The assay has traditionally used X-rays as challenge agent and isolated peripheral blood mononuclear cells (PBMC) as cell specimen. To enhance the usefulness of the assay, the objectives of this investigation were to use differently processed blood samples, to employ other challenge agents with different mechanisms of induction of DNA damage/repair, and to generate protocols for detecting different DDRR capacities. Fresh and frozen blood samples were challenged with bleomycin, methyl methanesulfonate (MMS) and ultraviolet light. Significant induction of damage after all treatments, and progressive and time-dependent DDRR were observed. No significant differences were obtained in the DDRR capacities of fresh or frozen whole blood samples as compared to PBMC, except that fresh blood samples showed higher MMS-induced DDRR capacity than PBMC. Results from this study show that the Challenge-Comet assay can be used as routine biomarker of DDRR capacity in human biomonitoring studies, and that whole blood is also a useful biomatrix for this assay. The collected data allow us to recommend different protocols for the Challenge-Comet assay which are useful for evaluating DDRR capacities in several key DNA repair pathways. Consequently, the usefulness of the Challenge-Comet assay can be greatly expanded.

Association of inflammatory mediators with frailty status in older adults: results from a systematic review and meta-analysis.

Frailty is a geriatric syndrome defined as a status of extreme vulnerability to stressors, leading to a higher risk of negative health-related outcomes. "Inflammaging", an age-related state of low-grade chronic inflammation, is characterized by an increased concentration of pro-inflammatory cytokines and acute phase proteins. Inflammaging has been postulated as an underlying mechanism of frailty, and several studies tested the relationship between frailty and concentration of inflammatory mediators. The aim of this systematic review and meta-analysis was to test whether inflammatory mediators are overproduced in frail older adults. Among the 758 articles identified in the literature search, 50 were included in the systematic review, and 39 in the three meta-analyses, i.e., C-reactive protein (CRP), interleukin 6 (IL6), and tumor necrosis factor α. To reduce heterogeneity, meta-analyses were restricted to studies identifying frailty by the Fried et al. [1] [J. Gerontol. A. Biol. Sci. Med. Sci. 56, M146-56] phenotypic criteria. Quantitative analyses measuring the association between frailty and biomarker concentrations showed significant differences when frail subjects were compared to non-frail and pre-frail subjects for CRP and IL6. This work established strong association between inflammatory biomarkers and frailty, confirming the role of age-related chronic inflammation in frailty development.

Low Vitamin D Levels and Frailty Status in Older Adults: A Systematic Review and Meta-Analysis.

Serum vitamin D deficiency is widespread among older adults and is a potential modifiable risk factor for frailty. Moreover, frailty has been suggested as an intermediate step in the association between low levels of vitamin D and mortality. Hence, we conducted a systematic review of the literature and meta-analysis to test the possible association of low concentrations of serum 25-hydroxyvitamin D (25(OH)D), a marker of vitamin D status, with frailty in later life. We reviewed cross-sectional or longitudinal studies evaluating populations of older adults and identifying frailty by a currently validated scale. Meta-analyses were restricted to cross-sectional data from studies using Fried's phenotype to identify frailty. Twenty-six studies were considered in the qualitative synthesis, and thirteen studies were included in the meta-analyses. Quantitative analyses showed significant differences in the comparisons of frail (standardized mean difference (SMD)-1.31, 95% confidence interval (CI) (-2.47, -0.15), p = 0.0271) and pre-frail (SMD-0.79, 95% CI (-1.58, -0.003), p = 0.0491) subjects vs. non-frail subjects. Sensitivity analyses reduced heterogeneity, resulting in a smaller but still highly significant between-groups difference. Results obtained indicate that lower 25(OH)D levels are significantly associated with increasing frailty severity. Future challenges include interventional studies testing the possible benefits of vitamin D supplementation in older adults to prevent/palliate frailty and its associated outcomes.

The impact of comet assay data normalization in human biomonitoring studies outcomes.

The comet assay has been extensively used in biomonitoring studies. To avoid intra-experimental variability, the incorporation of assay controls in each work session for data normalization has been suggested by some authors but has never been thoroughly analyzed. The aim of this study was to address the impact of data normalization in the results of a biomonitoring study using different normalization models. Human peripheral blood mononuclear cells (PBMC) from 140 healthy individuals were analyzed using the alkaline and FPG-modified version of the comet assay across seven different work sessions. In addition to negative standards, methyl methanesulfonate (MMS) and Ro 19-8022 plus light treated PBMC, were also included in the assay as positive standards. To verify the impact of data normalization, some demographic, lifestyle and environmental exposure-related variables were selected. Significant associations with independent study variables were observed using normalized comet endpoints, as opposed to raw data. After normalization, levels of DNA strand breaks were significantly higher among males and older individuals (>71 years), while net FPG-sensitive sites were positively related to smoking habits and environmental exposures (i.e. air pollution and bottled water consumption). This study highlights how the normalization strategies can influence the statistical results of a human biomonitoring study and lead to different data interpretations.

Serum cortisol but not oxidative stress biomarkers are related to frailty: results of a cross-sectional study in Spanish older adults.

Frailty is a multidimensional geriatric syndrome of loss of reserves and increased vulnerability to negative health outcomes. Cortisol, the major hormone of the hypothalamic pituitary adrenal (HPA) axis, and oxidative stress may be influenced by multiple endogenous and environmental factors throughout the lifespan, triggering changes in organism functioning. Association of elevated levels of cortisol and oxidative stress biomarkers with aging and several age-related diseases is well documented. However, the possible role of these factors on frailty status in older adults has not been extensively studied. Hence, the aim of this study was to conduct a cross-sectional study in 252 older adults (≥65 years old) classified according to their frailty status. Plasma cortisol and biomarkers related to oxidative stress including reactive oxygen/nitrogen species, oxidative DNA damage, and total antioxidant capacity were determined in non-frail, pre-frail, and frail subjects. Results showed significantly increasing cortisol concentrations with frailty burden, but no marked association between any oxidative stress biomarker and frailty status. In addition, dependence on activities of daily living and 10-year mortality risk were also correlated with elevated cortisol levels. Current results support the hypothesis that age-related HPA axis dysregulation is associated with frailty status, although further research is necessary to establish the role of cortisol in the pathophysiology of frailty.

Exploring Genetic Outcomes as Frailty Biomarkers.

Frailty has emerged as a reliable measure of the aging process. Because the early detection of frailty is crucial to prevent or even revert it, the use of biomarkers would allow an earlier and more objective identification of frail individuals. To improve the understanding of the biological features associated with frailty as well as to explore different biomarkers for its early identification, several genetic outcomes-mutagenicity, different types of genetic damage, and cellular repair capacity-were analyzed in a population of older adults classified into frail, prefrail, and nonfrail. Besides, influence of clinical parameters-nutritional status and cognitive status-was evaluated. No association of mutation rate or primary DNA damage with frailty was observed. However, DNA repair capacity showed a nonsignificant tendency to decrease with frailty, and persistent levels of phosphorylated H2AX, as indicative of DNA breakage, increased progressively with frailty severity. These results support the possible use of H2AX phosphorylation to provide information regarding frailty severity. Further investigation is necessary to determine the consistency of the current findings in different populations and larger sample sizes, to eventually standardize biomarkers to be used in clinics, and to fully understand the influence of cognitive impairment.

Frailty in Older Adults Is Associated With Plasma Concentrations of Inflammatory Mediators but Not With Lymphocyte Subpopulations.

Frailty denotes a multidimensional syndrome that gives rise to vulnerability to stressors and leads to an increase of the age-related decline of different physiological systems and cognitive abilities. Aging-related alterations of the immune system may compromise its competence culminating in a chronic low-grade inflammation state. Thus, it has been proposed that frailty is associated with alterations in the concentration of pro-inflammatory molecules and in different lymphocyte subpopulations. To provide further support to the validity of that hypothesis, we conducted a cross-sectional study in a population of Spanish older adults (N = 259, aged 65 and over) classified according to their frailty status. Biomarkers analyzed included percentages of several lymphocyte subsets and several inflammation mediators, namely concentrations of interleukin 6 (IL6), C-reactive protein (CRP), tumor necrosis factor α (TNFα), and 75 kDa soluble TNFα receptor II (sTNF-RII). Reference ranges for the inflammation mediators were established for the first time in robust older adults. A significant increase in the CD4+/CD8+ ratio and a significant decrease in the % CD19+ cells were observed in the frail group. Progressive increases with frailty severity were obtained in all inflammatory mediator concentrations, especially notable for IL6 and sTNF-RII. Area under the receiver-operating characteristic curve obtained for sTNF-RII (0.90, 95% CI 0.85-0.94, P < 0.001) indicates a high accuracy in the predictive value of this biomarker for frailty. Although results from the current study revealed limited strength associations between frailty and the lymphocyte subsets assessed, data obtained for the inflammatory mediators provide further support to involvement of inflammaging in frailty status in older adults.

Frailty Syndrome and Genomic Instability in Older Adults: Suitability of the Cytome Micronucleus Assay As a Diagnostic Tool.

Frailty, a condition involving increased risk of disability and mortality in older adults, has emerged as a reliable way to predict the effect of aging. Genomic instability may help to anticipate recognition of frail individuals and improving frailty outcomes. Our objective was to evaluate the potential of the micronucleus frequency, evaluated in lymphocytes and buccal cells, to anticipate frailty identification and improve diagnosis reliability. Our results, from a group of older adults over 65, showed that frail individuals had significantly higher frequencies of micronucleus in lymphocytes (19.16 ± 0.66 vs. 13.07 ± 0.78, p < .001) and of binucleated buccal cells (82.65 ± 3.42 vs. 37.16 ± 2.85, p < .001) and lower frequencies of pyknotic and condensed chromatin buccal cells, than nonfrail subjects. When cognitive status was considered, similar results were obtained. Moreover, the presence of frailty and cognitive impairment were independently related to increases in frequencies of lymphocyte micronucleus and binucleated buccal cells. Our results encourage the use of micronucleus frequency in lymphocytes as a complement to clinical parameters in frailty identification. However, these results have to be further evaluated in prefrail patients, to better understand the connection between genomic instability and frailty and to establish these parameters as actual biomarkers of frailty in clinical practice.

Clinical and genomic safety of treatment with Ginkgo biloba L. leaf extract (IDN 5933/Ginkgoselect®Plus) in elderly: a randomised placebo-controlled clinical trial [GiBiEx].

BACKGROUND: Numerous health benefits have been attributed to the Ginkgo biloba leaf extract (GBLE), one of the most extensively used phytopharmaceutical drugs worldwide. Recently, concerns of the safety of the extract have been raised after a report from US National Toxicology Program (NTP) claimed high doses of GBLE increased liver and thyroid cancer incidence in mice and rats. A safety study has been designed to assess, in a population of elderly residents in nursing homes, clinical and genomic risks associated to GBLE treatment. METHODS: GiBiEx is a multicentre randomized clinical trial, placebo controlled, double blinded, which compared subjects randomized to twice-daily doses of either 120-mg of IDN 5933 (also known as Ginkgoselect®Plus) or to placebo for a 6-months period. IDN 5933 is extracted from dried leaves and contains 24.3% flavone glycosides and 6.1% of terpene lactones (2.9% bilobalide, 1.38% ginkgolide A, 0.66% ginkgolide B, 1.12% ginkgolide C) as determined by HPLC. The study was completed by 47 subjects, 20 in the placebo group and 27 in the treatment group. Clinical (adverse clinical effect and liver injury) and genomic (micronucleus frequency, comet assay, c-myc, p53, and ctnnb1 expression profile in lymphocytes) endpoints were assessed at the start and at the end of the study. RESULTS: No adverse clinical effects or increase of liver injury markers were reported in the treatment group. The frequency of micronuclei [Mean Ratio (MR) = 1.01, 95% Confidence Intervals (95% CI) 0.86-1.18), and DNA breaks (comet assay) (MR = 0.91; 95% CI 0.58-1.43), did not differ in the two study groups. No significant difference was found in the expression profile of the three genes investigated. CONCLUSIONS: None of the markers investigated revealed a higher risk in the treatment group, supporting the safety of IDN 5933 at doses prescribed and for duration of six months. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03004508 , December 20, 2016. Trial retrospectively registered.

Frailty Status in Older Adults Is Related to Alterations in Indoleamine 2,3-Dioxygenase 1 and Guanosine Triphosphate Cyclohydrolase I Enzymatic Pathways.

BACKGROUND: Frailty is a multidimensional syndrome correlated to the loss of homeostasis and increased vulnerability to stressors, which is associated with increase in the risk of disability, comorbidity, hospitalization, and death in the elderly. It is based on the interplay of physiological, psychological, social, and environmental factors. OBJECTIVES: Because aging involves a detrimental immune response, this work aimed to assess the possible role of chronic low-grade immune stimulation on frailty status in the elderly. METHODS: Biomarkers involved in indoleamine 2,3-dioxygenase 1 and guanosine triphosphate cyclohydrolase I enzymatic pathways (namely neopterin, tryptophan, kynurenine, phenylalanine, tyrosine, and nitrite) were analyzed in a population of Spanish older adults aged 65 years and above, and their relationships with frailty status were evaluated. RESULTS: Significant increases in neopterin levels, kynurenine/tryptophan ratio, and phenylalanine/tyrosine ratio, and significant decreases in tryptophan, nitrite and tyrosine concentrations in frail individuals compared with nonfrail persons were obtained. Significant correlations were also observed between immune biomarkers, indicating they change in parallel, thus, pointing to interrelated causes. Besides, reference ranges for a number of immune biomarkers in the population of robust older adults were established for the first time. CONCLUSIONS: Results obtained in the present study are consistent with the idea that frailty status in the elderly is associated with an additional degree of immune stimulation, manifested in a more intense disturbance of indoleamine 2,3-dioxygenase 1 and guanosine triphosphate cyclohydrolase I pathways than in nonfrail or prefrail older adults.

Oxidative stress, genomic features and DNA repair in frail elderly: A systematic review.

Frailty is an emerging geriatric syndrome characterized by higher vulnerability to stressors, with an increased risk of adverse health outcomes such as mortality, morbidity, disability, hospitalization, and institutionalization. Although it is generally recognized to have a biological basis, no particular biological trait has been consistently associated to frailty status so far. In this work, epidemiological studies evaluating association of frailty status with alterations at cellular level - namely oxidative stress, genomic instability and DNA damage and repair biomarkers -were revised and compared. A total of 25 studies fulfilled inclusion/exclusion criteria and, consequently, were included in the review. Variations of oxidative stress biomarkers were often associated to frailty status in older people. On the contrary, genomic instability seems not to be linked to frailty. The only study which addressed the possible relationship between DNA repair modulations and frailty status also failed in finding association. Despite the large number of cellular alterations known to be associated with frailty, studies on this issue are still very scarce and limited to some of the possible cellular targets. The established link between DNA repair, genomic instability, and age and age-related disorders, encourage deeper investigations on this line.

Immune biomarkers in older adults: Role of physical activity.

Aging is associated with a decline in the normal functioning of the immune system. Several studies described the relationship between immunological alterations, including immunosenescence and inflammation, and aging or age-related outcomes, such as sarcopenia, depression, and neurodegenerative disorders. Physical activity is known to improve muscle function and to exert a number of benefits on older adult health, including reduced risk for heart and metabolic system chronic diseases. However, the positive influence of physical activity on the immune system has not been elucidated. In order to shed light on the role of physical activity in immune responses of older individuals, a number of immunological parameters comprising % lymphocyte subsets (CD3+, CD4+, CD8+, CD19+, and CD16+56+) and serum levels of neopterin and tryptophan metabolism products were evaluated in peripheral blood samples of older adults performing normal (N = 170) or reduced (N = 89) physical activity. In addition, the potential influence of other clinical and epidemiological factors was also considered. Results showed that subjects with reduced physical activity displayed significantly higher levels of CD4+/CD8+ ratio, kynurenine/tryptophan ratio, and serum neopterin, along with lower %CD19+ cells and tryptophan concentrations. Further, some immunological biomarkers were associated with cognitive impairment and functional status. These data contribute to reinforce the postulation that physical activity supports healthy aging, particularly by helping to protect the immunological system from aging-related changes.

Lymphocyte Subsets in a Population of Nonfrail Elderly Individuals.

Age-related frailty is characterized by increased vulnerability to stress due to decline in homeostatic reserve, which results in increased risk of adverse health outcomes including disability, hospitalization, and death. The relationship between frailty and immunological system alterations is well established. Thus, analysis of immunological changes, such as alterations in lymphocyte subsets, during senescence may provide useful markers for frailty and associated pathologies. Since reference ranges currently used for lymphocyte subsets do not specifically differentiate the elderly group, the aim of this study was to (1) establish reference ranges in nonfrail elderly individuals and (2) assess the evolution of these parameters with age. Further, the influence of other physiological and lifestyle factors was also evaluated. The study was performed on 144 elderly individuals (aged 65-95) from Galicia (in northwestern Spain). Percentages of lymphocyte subpopulations (CD3(+) T lymphocytes, CD4(+) T-helper lymphocytes, CD8(+) T-cytotoxic lymphocytes, CD19(+) B lymphocytes, and CD56(+)16(+) natural killer cells) were analyzed in peripheral blood by flow cytometry, and reference ranges were calculated. The individual status as nonfrail or prefrail did not markedly affect the immunological parameters, but an apparent influence of age was obtained for %CD3(+), %CD4(+), and %CD19(+) cells, all of which fell with increasing age. Women showed higher levels of %CD19(+) lymphocytes. No significant influence of smoking habits, physical activity, or drinking alcohol or caffeine beverages was observed. The results obtained may serve as a basis to establish comparisons between frail and nonfrail elderly individuals, in order to determine the usefulness of lymphocyte subsets as immunological biomarkers of frailty.

γH2AX assay as DNA damage biomarker for human population studies: defining experimental conditions.

H2AX histone phosphorylation represents an early event in the cellular response against DNA double-strand breaks (DSBs), and plays a central role in sensing and repairing DNA damage. Therefore, the analysis of H2AX phosphorylated (γH2AX) may be possibly used as biomarker of genotoxicity and genomic instability with a number of applications in human epidemiology. However, the lack of an experimental standard leads to a wide heterogeneity in the results obtained and their interpretation, affecting the reliability of the assay. To address the most critical issues limiting the use of the γH2AX assay in human population studies, a flow cytometry analysis was performed to establish differences in γH2AX levels between fresh or cryopreserved peripheral blood lymphocytes, and to assess the influence of phytohemagglutinin (PHA) stimulation. To this purpose, cells were treated with 4 known genotoxic chemicals with different mechanisms of DSB induction, ie, bleomycin, methyl methanesulfonate, camptothecin, and actinomycin. According to our results, both unstimulated and stimulated fresh lymphocytes can be efficiently employed to evaluate γH2AX levels, but the sensitivity of the assay is depending upon the kind of damage observed. On the other hand, cryopreserved lymphocytes require PHA stimulation since unstimulated cells showed too high basal damage. Consequently, the protocol conditions will depend on the expected mechanism of production of DSB and the characteristics of the study design (sample collection and storage conditions, type of epidemiological study). Further studies are required to standardize the protocol of γH2AX assay to be employed as biomarker of genotoxicity or genomic instability in human population studies.