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.This work was supported by Xunta de Galicia [ED431B 2019/02], Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142 to V.V], and Deputación Provincial de A Coruña [to M.S.-F. and N.F.-B.]

Multiparametric in vitro genotoxicity assessment of different variants of amorphous silica nanomaterials in rat alveolar epithelial cells

The hazard posed to human health by inhaled amorphous silica nanomaterials (aSiO2 NM) remains uncertain. Herein, we assessed the cyto- and genotoxicity of aSiO2 NM variants covering different sizes (7, 15, and 40 nm) and surface modifications (unmodified, phosphonate-, amino- and trimethylsilyl-modified) on rat alveolar epithelial (RLE-6TN) cells. Cytotoxicity was evaluated at 24 h after exposure to the aSiO2 NM variants by the lactate dehydrogenase (LDH) release and WST-1 reduction assays, while genotoxicity was assessed using different endpoints: DNA damage (single- and double-strand breaks [SSB and DSB]) by the comet assay for all aSiO2 NM variants; cell cycle progression and γ-H2AX levels (DSB) by flow cytometry for those variants that presented higher cytotoxic and DNA damaging potential. The variants with higher surface area demonstrated a higher cytotoxic potential (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_15_Phospho). SiO2_40 was the only variant that induced significant DNA damage on RLE-6TN cells. On the other hand, all tested variants (SiO2_7, SiO2_15_Unmod, SiO2_15_Amino, and SiO2_40) significantly increased total γ-H2AX levels. At high concentrations (28 µg/cm2), a decrease in G0/G1 subpopulation was accompanied by a significant increase in S and G2/M sub-populations after exposure to all tested materials except for SiO2_40 which did not affect cell cycle progression. Based on the obtained data, the tested variants can be ranked for its genotoxic DNA damage potential as follows: SiO2_7 = SiO2_40 = SiO2_15_Unmod > SiO2_15_Amino. Our study supports the usefulness of multiparametric approaches to improve the understanding on NM mechanisms of action and hazard prediction

Suitability of salivary leucocytes to assess DNA repair ability in human biomonitoring studies by the challenge-comet assay

The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the kinetics of repair process. Peripheral blood mononuclear cells (PBMC) are the cells most frequently employed in human biomonitoring studies using the challenge-comet assay, but having a validated alternative of non-invasive biomatrix would be highly convenient for certain population groups and circumstances. The objective of this study was to validate the use of salivary leucocytes in the challenge-comet assay. Leucocytes were isolated from saliva samples and challenged (either in fresh or after cryopreservation) with three genotoxic agents acting by different action mechanisms: bleomycin, methyl methanesulfonate, and ultraviolet radiation. Comet assay was performed just after treatment and at other three additional time points, in order to study repair kinetics. The results obtained demonstrated that saliva leucocytes were as suitable as PBMC for assessing DNA damage of different nature that was efficiently repaired over the evaluated time points, even after 5 months of cryopreservation (after a 24 h stimulation with PHA). Furthermore, a new parameter to determine the efficacy of the repair process, independent of the initial amount of damage induced, is proposed, and recommendations to perform the challenge-comet assay with salivary leucocytes depending on the type of DNA repair to be assessed are suggested. Validation studies are needed to verify whether the method is reproducible and results reliable and comparable among laboratories and studies. © 2022 The AuthorsFunding text 1: This work was funded by the Spanish Ministry of Science and Innovation : MCIN/AEI/10.13039/501100011033 (Grants PID2020-113788RB-I00 and PID2020-114908 GA-I00 ), NanoBioBarriers project (PTDC/MED-TOX/31162/2017), Xunta de Galicia (ED431B 2022/16), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds ( FEDER / FNR ), Spanish Ministry of Education, Culture and Sport [ BEAGAL18/00142 to V.V.], and Spanish Ministry of Economy and Competitiveness , co-financed by the European Social Fund [ RYC-2015-18394 to L.L,-L,]. Funding for open access charge: Universidade da Coruña/CISUG. ; Funding text 2: This work was funded by the Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 (Grants PID2020-113788RB-I00 and PID2020-114908 GA-I00), NanoBioBarriers project (PTDC/MED-TOX/31162/2017), Xunta de Galicia (ED431B 2022/16), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR), Spanish Ministry of Education, Culture and Sport [BEAGAL18/00142 to V.V.], and Spanish Ministry of Economy and Competitiveness, co-financed by the European Social Fund [RYC-2015-18394 to L.L,-L,]. Funding for open access charge: Universidade da Coruña/CISUG

Exploring frailty-related biomarkers and potential influence of environmental factors

Recent evidence advocates that healthy ageing may be possible, with morbidity compressed to later years. One area of concern is the burden of environmentally induced disease in susceptible populations. Older adults are a well-recognized susceptible population due to the decline of immune defences and the burden of multiple chronic diseases. As a susceptible population, the burden of environmentally induced disease and lifestyle factors is an increasing concern. Frailty is an age-related syndrome expected to increase over the next decades given the observed demographic shift. This syndrome has been identified to be the most common condition leading to disability, institutionalization and death in the older adults and the risk factors associated with its development are yet to be clarified. The main aim of the present study is to investigate a relation between frailty status, biomarkers and environmental exposures

Effects of Zinc Oxide Nanoparticle Exposure on Human Glial Cells and Zebrafish Embryos

Zinc oxide nanoparticles (ZnO NPs) are among the most widely used nanomaterials. They have multiple applications in cosmetics, textiles, paints, electronics and, recently, also in biomedicine. This extensive use of ZnO NPs notably increases the probability that both humans and wildlife are subjected to undesirable effects. Despite being among the most studied NPs from a toxicological point of view, much remains unknown about their ecotoxicological effects or how they may affect specific cell types, such as cells of the central nervous system. The main objective of this work was to investigate the effects of ZnO NPs on human glial cells and zebrafish embryo development and to explore the role of the released Zn2+ ions in these effects. The effects on cell viability on human A172 glial cells were assessed with an MTT assay and morphological analysis. The potential acute and developmental toxicity was assessed employing zebrafish (Danio rerio) embryos. To determine the role of Zn2+ ions in the in vitro and in vivo observed effects, we measured their release from ZnO NPs with flame atomic absorption spectrometry. Then, cells and zebrafish embryos were treated with a water-soluble salt (zinc sulfate) at concentrations that equal the number of Zn2+ ions released by the tested concentrations of ZnO NPs. Exposure to ZnO NPs induced morphological alterations and a significant decrease in cell viability depending on the concentration and duration of treatment, even after removing the overestimation due to NP interference. Although there were no signs of acute toxicity in zebrafish embryos, a decrease in hatching was detected after exposure to the highest ZnO NP concentrations tested. The ability of ZnO NPs to release Zn2+ ions into the medium in a concentration-dependent manner was confirmed. Zn2+ ions did not seem entirely responsible for the effects observed in the glial cells, but they were likely responsible for the decrease in zebrafish hatching rate. The results obtained in this work contribute to the knowledge of the toxicological potential of ZnO NPs.This research was funded by the Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 (grant PID2020-114908GA-I00), Xunta de Galicia (ED431B 2022/16 and ED481A 2019/003 to A.A-G.), CICA-Disrupting Project 2021SEM-B2, and Ministry of Education, Culture and Sport (BEAGAL18/00142 to V.V.)

Toxoplasma gondii IgG Serointensity Is Positively Associated With Frailty

Background: Persistent inflammation related to aging (inflammaging) is exacerbated by chronic infections and contributes to frailty in older adults. We hypothesized associations between Toxoplasma gondii (T. gondii), a common parasite causing an oligosymptomatic unremitting infection, and frailty, and secondarily between T. gondii and previously reported markers of immune activation in frailty.Methods: We analyzed available demographic, social, and clinical data in Spanish and Portuguese older adults [N = 601; age: mean (SD) 77.3 (8.0); 61% women]. Plasma T. gondii immunoglobulin G (IgG) serointensity was measured with an enzyme-linked immunosorbent assay. The Fried criteria were used to define frailty status. Validated translations of Mini-Mental State Examination, Geriatric Depression Scale, and the Charlson Comorbidity Index were used to evaluate confounders. Previously analyzed biomarkers that were significantly associated with frailty in both prior reports and the current study, and also related to T. gondii serointensity, were further accounted for in multivariable logistic models with frailty as outcome.Results: In T. gondii-seropositives, there was a significant positive association between T. gondii IgG serointensity and frailty, accounting for age (p = .0002), and resisting adjustment for multiple successive confounders. Among biomarkers linked with frailty, kynurenine/tryptophan and soluble tumor necrosis factor receptor II were positively associated with T. gondii serointensity in seropositives (p < .05). Associations with other biomarkers were not significant.Conclusions: This first reported association between T. gondii and frailty is limited by a cross-sectional design and warrants replication. While certain biomarkers of inflammaging were associated with both T. gondii IgG serointensity and frailty, they did not fully mediate the T. gondii-frailty association.This work was supported in part by the Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033(grant PID2020-113788RB-I00); Xunta de Galicia (grant ED431B 2022/16); Ministry of Education, Culture and Sport (grant BEAGAL18/00142 to V.V.); and Ministry of Economy and Competitiveness, cofinanced by the European Social Fund (grant RYC-2015-18394 to L.L.-L.). Additionally supported, in part, by the University of Maryland School of Medicine Center for Research on Aging in Baltimore, Maryland; a Clinical Science Research & Development Service Merit Award, Office of Research and Development, U.S. Department of Veterans Affairs, Washington, District of Columbia (grant 1 I01 CX001310-01 to T.T.P.); a R01 grant from the National Institute on Aging, National Institutes of Health, Bethesda, Maryland (grant NIA R01 AG018859 to E.J.K.); and by the Military and Veteran Microbiome: Consortium for Research and Education in Aurora, Colorado (L.A.B., A.J.H., C.A.L., T.T.P.). The opinions expressed in the article belong to the authors and cannot be construed as official positions or opinions of the funders, including the U.S. Veterans Affairs Administration and the National Institutes of Health. Data collected and used for the analyses reported in this article are not available because the initial consent did not include this sharing and because other primary analyses have not been completed. Funding for open access charge: Universidade da Coruna/CISUG

Association of Torquetenovirus Viremia with Physical Frailty and Cognitive Impairment in Three Independent European Cohorts

Introduction: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. Methods: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). Results: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p &lt; 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p &lt; 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. Conclusions: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals

Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron–sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.Ataxia UK, FARA Australasia and FARA US

DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death

The comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan-Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06-1.90) for overall mortality, and 1.94 (1.04-3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases