γH2AX prefers late replicating metaphase chromosome regions

Reyes-Ábalos, Ana Laura; Liddle, Pablo; Folle, Gustavo A.; Tomaso, María Vittoria Di

doi:10.1016/j.mrgentox.2018.06.001

Cited by 4 publications

(4 citation statements)

References 89 publications

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“…However, Khoury et al reported that aneugens caused significant phosphorylation of H2AX in HepG2 cells by incell Western technique, 37 which was different from our results. Because γ-H2AX is known to mainly form during DNA replication, that is, interphase of the cell cycle, 43 while aneugens act on the mitosis phase, but not interphase, and thus prefer not to cause phosphorylation of H2AX, the technique here, that is, LC-MS/MS, is supported as more specific and unlikely to yield false positive results than Western technique, though the latter is commonly used in in vitro genotoxicity assays.…”

Section: ■ Discussionmentioning

confidence: 99%

Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of Genotoxic Chemicals Revealed by Mass Spectrometry Analysis

Chen

et al. 2020

Chem. Res. Toxicol.

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Genotoxic chemicals act by causing DNA damage, which, if left unrepaired, can have deleterious consequences for cell survival. DNA damage response (DDR) gets activated to repair or mitigate the effects of DNA damage. Histone H2AX and H3 phosphorylation biomarkers (γ-H2AX and p-H3) have attracted great attention as they play pivotal roles in the DDR. Simultaneous quantitation of γ-H2AX and p-H3 in exposed cells may monitor the toxicity of genotoxic chemicals and to some extent reflect the subsequent DDR process. Reported here is the first comprehensive characterization of distinct orchestration and dynamic processes on cellular γ-H2AX and p-H3 for two major types of genotoxic chemicals, clastogens and aneugens, by stable isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). We find that clastogens significantly induce an increase in γ-H2AX and a decrease in p-H3; aneugens have no obvious effect on γ-H2AX, whereas induce either an increase or a decrease in p-H3. In addition, the specific profiles of clastogens and aneugens affecting DNA damage may be dynamically observed, which in turn provides insights into the processes involving DNA damage repair as well as transcription. Taken together, these results suggest that robust LC-MS/MS analysis of γ-H2AX and p-H3 can not only quantitatively differentiate mechanistic information on clastogens and aneugens but also dynamically present the detail profiles of DNA damage and repair processes.

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Section: ■ Discussionmentioning

confidence: 99%

Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of Genotoxic Chemicals Revealed by Mass Spectrometry Analysis

Chen

et al. 2020

Chem. Res. Toxicol.

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“…Our present data provided evidence of the sensitivity of the cell-signaling repertoire of murine hippocampal astrocytes to the genetic damage induced by acute exposure to the drug of human-wide consumption EtOH and/or the stress response hormone CTS (Figure 1). Genomic DNA damage was assessed by γH2AX foci, which are produced a few minutes after its induction by the early phosphorylation of the H2A histone variant, H2AX [48][49][50][51][52][53]. γH2AX immunoreactivity detected in astrocyte nuclei also indicated the early activation of the DDR cell pathways [41,[44][45][46][47].…”

Section: Discussionmentioning

confidence: 99%

“…Genetic damage and DDR were assessed by analyzing the rapid phosphorylation of the histone variant H2AX (termed γH2AX foci) around sites of DNA damage. γ-H2AX recruits a series of proteins involved in the downstream DDR pathway [48][49][50][51][52][53], including connections with the DNA repair [44,54,55]. To detect evolution of DDR and early signs of apoptosis, the DDR-related apoptosis, which operates via the regulation of the proapoptotic bax gene [56][57][58], was assessed by recognizing BAX (proapoptotic effectors BCL-2-associated X protein or BCL-2-like protein 4) immunoreactivity.…”

Section: Introductionmentioning

confidence: 99%

Acute Genetic Damage Induced by Ethanol and Corticosterone Seems to Modulate Hippocampal Astrocyte Signaling

Reyes-Ábalos,

Álvarez-Zabaleta,

Olivera-Bravo

et al. 2024

International Journal of Cell Biology

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Astrocytes maintain CNS homeostasis but also critically contribute to neurological and psychiatric disorders. Such functional diversity implies an extensive signaling repertoire including extracellular vesicles (EVs) and nanotubes (NTs) that could be involved in protection or damage, as widely shown in various experimental paradigms. However, there is no information associating primary damage to the astrocyte genome, the DNA damage response (DDR), and the EV and NT repertoire. Furthermore, similar studies were not performed on hippocampal astrocytes despite their involvement in memory and learning processes, as well as in the development and maintenance of alcohol addiction. By exposing murine hippocampal astrocytes to 400 mM ethanol (EtOH) and/or 1 μM corticosterone (CTS) for 1 h, we tested whether the induced DNA damage and DDR could elicit significant changes in NTs and surface-attached EVs. Genetic damage and initial DDR were assessed by immunolabeling against the phosphorylated histone variant H2AX (γH2AX), DDR-dependent apoptosis by BAX immunoreactivity, and astrocyte activation by the glial acidic fibrillary protein (GFAP) and phalloidin staining. Surface-attached EVs and NTs were examined via scanning electron microscopy, and labeled proteins were analyzed via confocal microscopy. Relative to controls, astrocytes exposed to EtOH, CTS, or EtOH+CTS showed significant increases in nuclear γlH2AX foci, nuclear and cytoplasmic BAX signals, and EV frequency at the expense of the NT amount, mainly upon EtOH, without detectable signs of morphological reactivity. Furthermore, the largest and most complex EVs originated only in DNA-damaged astrocytes. Obtained results revealed that astrocytes exposed to acute EtOH and/or CTS preserved their typical morphology but presented severe DNA damage, triggered canonical DDR pathways, and early changes in the cell signaling mediated by EVs and NTs. Further deepening of this initial morphological and quantitative analysis is necessary to identify the mechanistic links between genetic damage, DDR, cell-cell communication, and their possible impact on hippocampal neural cells.

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“…After three washes (5 min each), cells were incubated with 1.5 mg/L 4.6-diamidino-2-phenylindole (DAPI, ab2629482, Abcam) that was used as the nuclear label. Then, the cells were mounted in ProLong Gold antifade (P36930, Invitrogen) and sealed with colorless nail enamel ( Liddle et al, 2014 ; Reyes-Ábalos et al, 2018 ). The preparations were protected from light and preserved at 4°C until confocal microscopy imaging.…”

Section: Methodsmentioning

confidence: 99%

Astrocyte DNA damage and response upon acute exposure to ethanol and corticosterone

Reyes-Ábalos,

Álvarez-Zabaleta,

Olivera-Bravo

et al. 2024

Front. Toxicol.

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Introduction: Astrocytes are the glial cells responsible for brain homeostasis, but if injured, they could damage neural cells even deadly. Genetic damage, DNA damage response (DDR), and its downstream cascades are dramatic events poorly studied in astrocytes.Hypothesis and methods: We propose that 1 h of 400 mmol/L ethanol and/or 1 μmol/L corticosterone exposure of cultured hippocampal astrocytes damages DNA, activating the DDR and eliciting functional changes. Immunolabeling against γH2AX (chromatin DNA damage sites), cyclin D1 (cell cycle control), nuclear (base excision repair, BER), and cytoplasmic (anti-inflammatory functions) APE1, ribosomal nucleolus proteins together with GFAP and S100β plus scanning electron microscopy studies of the astrocyte surface were carried out.Results: Data obtained indicate significant DNA damage, immediate cell cycle arrest, and BER activation. Changes in the cytoplasmic signals of cyclin D1 and APE1, nucleolus number, and membrane-attached vesicles strongly suggest a reactivity like astrocyte response without significant morphological changes.Discussion: Obtained results uncover astrocyte genome immediate vulnerability and DDR activation, plus a functional response that might in part, be signaled through extracellular vesicles, evidencing the complex influence that astrocytes may have on the CNS even upon short-term aggressions.

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γH2AX prefers late replicating metaphase chromosome regions

Cited by 4 publications

References 89 publications

Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of Genotoxic Chemicals Revealed by Mass Spectrometry Analysis

Distinct Orchestration and Dynamic Processes on γ-H2AX and p-H3 for Two Major Types of Genotoxic Chemicals Revealed by Mass Spectrometry Analysis

Acute Genetic Damage Induced by Ethanol and Corticosterone Seems to Modulate Hippocampal Astrocyte Signaling

Astrocyte DNA damage and response upon acute exposure to ethanol and corticosterone

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