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.]

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

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

First step in the evaluation of the effects of Prestige oil on the shore environment: Availability, bioaccumulation and DNA damage

The Prestige oil tanker shipwrecked off the coast of Galicia (Spain) in November 2002, spilling nearly 63,000 tons of heavy oil, classified by the International Agency for Research on Cancer as possible human carcinogen. In this work, mussels (Mytilus galloprovincialis) were exposed to Prestige oil in the laboratory (1.5 and 3 ppm). Samples of seawater and mussel tissues were taken on days 7 and 14 of the experiment to determine the availability and bioaccumulation of the polycyclic aromatic hydrocarbons (PAH) contained in the oil and the DNA damage induced by the exposure using the comet assay. Seawater was renewed on day 14 and new samples were taken on day 21 to analyze the recovery ability of the mussels. Total PAH (TPAH) contents in seawater from the exposure tanks were higher than in the control tank, and this content was higher in the lower oil-dose tank due to the tendency of PAH to link to particles in water. Exposed mussels had much higher TPAH levels than controls, increasing with time even during the recovery phase. The correlation coefficient obtained demonstrated a clear environmental dose/internal dose relationship. Significant increases in DNA damage were observed in oil-exposed individuals in relation to controls, similar in the two doses tested. The DNA damage was constant during the exposure period but increased during the recovery time, reflecting the strand breakage during the DNA repair processes. The results obtained have shown the importance of determining the presence of pollutants in the environment, their bioaccumulation in organic tissues and their effects on the exposed organisms, in order to perform an integrated approach to evaluate the impact of contamination events in the aquatic environment

Assessment of cellular and molecular toxicity by in vitro tests: the okadaic acid toxin case

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Neuronal cytotoxicity and genotoxicity induced by zinc oxide nanoparticles

Zinc oxide nanoparticles (ZnO NPs) are one of the most abundantly used nanomaterials in consumer products and biomedical applications. As a result, human exposure to these NPs is highly frequent and they have become an issue of concern to public health. Although toxicity of ZnO NPs has been extensively studied and they have been shown to affect many different cell types and animal systems, there is a significant lack of toxicological data for ZnO NPs on the nervous system, especially for human neuronal cells and tissues. In this study, the cytotoxic and genotoxic effects of ZnO NPs on human SHSY5Y neuronal cells were investigated under different exposure conditions. Results obtained by flow cytometry showed that ZnO NPs do not enter the neuronal cells, but their presence in the medium induced cytotoxicity, including viability decrease, apoptosis and cell cycle alterations, and genotoxicity, including micronuclei production, H2AX phosphorylation and DNA damage, both primary and oxidative, on human neuronal cells in a dose- and time-dependent manner. Free Zn(2+) ions released from the ZnO NPs were not responsible for the viability decrease, but their role on other types of cell damage cannot be ruled out. The results obtained in this work contribute to increase the knowledge on the genotoxic and cytotoxic potential of ZnO NPs in general, and specifically on human neuronal cells, but further investigations are required to understand the action mechanism underlying the cytotoxic and genotoxic effects observed

Effects of iron oxide nanoparticles: Cytotoxicity, genotoxicity, developmental toxicity, and neurotoxicity

Iron oxide nanoparticles (ION) with superparamagnetic properties hold great promise for use in various biomedical applications; specific examples include use as contrast agents for magnetic resonance imaging, in targeted drug delivery, and for induced hyperthermia cancer treatments. Increasing potential applications raise concerns over their potential effects on human health. Nevertheless, very little is currently known about the toxicity associated with exposure to these nanoparticles at different levels of biological organization. This article provides an overview of recent studies evaluating ION cytotoxicity, genotoxicity, developmental toxicity and neurotoxicity. Although the results of these studies are sometimes controversial, they generally indicate that surface coatings and particle size seem to be crucial for the observed ION-induced effects, as they are critical determinants of cellular responses and intensity of effects, and influence potential mechanisms of toxicity. The studies also suggest that some ION are safe for certain biomedical applications, while other uses need to be considered more carefully. Overall, the available studies provide insufficient evidence to fully assess the potential risks for human health related to ION exposure. Additional research in this area is required including studies on potential long-term effects

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.Research funded by Xunta de Galicia (ED431B 2016/013) and by EU COST Action IS1402. V. Valdiglesias was supported by a Xunta de Galicia postdoctoral fellowship (reference ED481B 2016/190-0). D. Marcos-Pérez and M. Sánchez-Flores were supported by INDITEX-UDC fellowships. The work of S. Bonassi was supported by grants funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC)

Genotyping an ALAD polymorphism with real-time PCR in two populations from the Iberian Peninsula

Lead-induced toxicity varies among individuals partly because of genetic differences in their susceptibility to the metal's effects. One genetic polymorphism associated with lead toxicity is a G-to-C transversion at position 177 in the coding region of delta-aminolevulinic acid dehydratase (ALAD), originating from two codominant alleles (ALAD1 and ALAD2). We examined the distribution of this single nucleotide polymorphism in two populations from the Iberian Peninsula. Genomic DNA was extracted from whole blood, and a real-time PCR assay was designed to determine ALAD polymorphic distribution. The distribution of MspI polymorphism was similar in the two populations studied, and allelic frequencies were comparable to those obtained in other studies of Caucasians. Further studies are needed to assess fully the functional significance of this polymorphism and its influence on the toxicokinetics of lead