X-Ray Induced DNA Strand Break Induction and Rejoining in Cultured Bovine Lens Epithelial Cells

Baumstark‐Khan, Christa; Aufderheide, Enno; Rink, H.

doi:10.1159/000267171

Cited by 8 publications

(4 citation statements)

References 13 publications

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“…38 The sensitivity of FADU method is comparable to alkaline elution method of measurement of DNA strand breaks. 52 Fluorometric analysis of DNA unwinding (FADU assay), first reported by Birnboim and Jevcak to detect X-rayinduced DNA damage. 40 It is a fast and reliable technique to detect single-strand DNA breaks as an index of DNA damage induced by genotoxic agents.…”

Section: Discussionmentioning

confidence: 99%

Detection of DNA strand breaks by fluroscence assisted DNA unwinding (FADU) assay in Hela cells treated with berbrine chloride before exposure to various doses of γ – radition

Jagetia¹,

Rao²

2018

IJMBOA

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The DNA damage plays an important role in the cell death and DNA double strand breaks in particular have been implicated in cell mortality. Therefore, the ability of berberine chloride to modulate radiation-induced DNA damage in HeLa cells exposed to different doses of γ-rays was studied by fluorescence assisted DNA unwinding assay. The spontaneous frequency of undamaged DNA remained unaltered in HeLa cells treated with 0, 1, 2, 4, 6 or 8µg/ml of BCL at 0, 0.25, 0.5, 1, 2 or 4h postirradiation. Immediate exposure of HeLa cells to 3 Gy γ-radiation (0h post-irradiation group) caused a drastic rise in the DNA strand breaks as evident by an abrupt reduction in the undamaged double stranded DNA (dsDNA). An elevation in the undamaged ds DNA was observed with time up to 12h post-irradiation in cells exposed to 3Gy irradiation without BCL treatment, indicating repair of radiation-induced DNA damage. However, incubation of HeLa cells with different concentrations of BCL before 3Gy irradiation caused a dose-dependent increase in the DNA strand breaks at all post-irradiation times. The highest DNA strand breaks were observed for 8µg/ml BCL after exposure to 3 Gy. The DNA strand breaks did not show repair up to 12h in cells exposed 3 Gy. The exposure of HeLa cells to 0.25 to 4 Gy γ-radiation resulted in a dose dependent decline in dsDNA at all post-irradiation times and treatment of HeLa cells with 8 µg/ml berberine before irradiation led to a further attrition in the dsDNA. Our study demonstrates that irradiation of HeLa cells resulted in a dose dependent increase in DNA strand breaks and berberine treatment further increased the DNA strand breaks which may be one of the mechanisms of cell death.

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Section: Discussionmentioning

confidence: 99%

Detection of DNA strand breaks by fluroscence assisted DNA unwinding (FADU) assay in Hela cells treated with berbrine chloride before exposure to various doses of γ – radition

Jagetia¹,

Rao²

2018

IJMBOA

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“…The fact that the higher HO fluorescence was observed only in 1% FCS conditions suggests that when HDR cells are incubated immediately after irradiation in a poor medium, they became unable to repair their DNA damage. Indeed, after x‐ray exposure or UV irradiation of bovine lens epithelial cells, DNA damage, repair, and survival are dose‐ and culture type‐dependent [5, 6, 20, 21]. To date, there is no report about the influence of FCS concentration on DNA repair after in vitro lens epithelial cell irradiation.…”

Section: Discussionmentioning

confidence: 99%

“…However, despite the fact that Chalupecky [4] reported a relationship between ionizing radiation and cataract formation in the nineteenth century, cellular mechanisms involved in radiation‐induced cataractogenesis have not been defined. Several authors, using different types of ionizing particle, have focused only on DNA strand break induction in lens epithelial cells [5, 6]. With ultraviolet (UV) B irradiation, Li and Spector [7] reported that lens epithelial cell apoptosis is involved in cataract formation in animal models.…”

Section: Introductionmentioning

confidence: 99%

Ionizing radiation-induced death in bovine lens epithelial cells:Mechanisms and influence of irradiation dose rate

et al. 2000

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We recently reported, in a series of patients receiving total body irradiation before transplant, an influence of dose rate (DR) on cataract formation. The aim of our present in vitro study was to investigate the influence of DR and the mechanisms of lens cell death in a bovine model. After a single fraction of 10 Gy, delivered using low (0.05 Gy/min) or high (2 Gy/min) DR (LDR and HDR, respectively), cells were incubated in media supplemented with two different fetal calf serum (FCS) concentrations (1% and 10%). Cell proliferation was evaluated using Hoechst 33342 (HO) probe and cell viability, with neutral red probe. These fluorimetric assays used a cold light cytofluorimeter. After HO assay, stained cells were examined with fluorescence microscopy to evaluate the nuclear changes related to apoptosis. Global comparison of the mean HO fluorescent values observed with LDR/controls (c) vs. HDR/c revealed a significant difference only after 96 hr (P = 0.036). In 1% FCS conditions, the difference between HDR/c and LDR/c was also statistically significant at 96 hr (P = 0.04). Pairwise multiple comparison using values observed in 1% FCS conditions after 96 hr incubation showed significant difference between HDR vs. c (P = 0.001) and HDR vs. LDR (P = 0.007). This difference, in terms of fluorescence, was correlated to the proportion of cells with nuclear apoptotic morphology. In contrast, cell viability was not influenced by DR whatever the FCS concentration used, from 24 to 96 hr after irradiation. We conclude that our fluorimetric methodology is adapted to evaluate intracellular DNA modifications and cell viability after x‐ray irradiation. We observed that a single fraction of 10 Gy induces in vitro lens epithelial cell apoptosis, which is influenced by DR. In humans, HDR is considered more cataractogenic than LDR. Thus, we speculate that lens cell apoptosis could be one of the major mechanisms of radiation‐induced cataract. Further investigations are necessary to study the other possible mechanisms of cataractogenesis. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 138–144 (2000). © 2000 Wiley‐Liss, Inc.

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“…Since DNA strand breaks may play an important role in mutagenesis and oncogenesis, their dose-effect relationships and their rejoining kinetics are essential indicators reflecting cellular response processes (8)(9)(10)(11). In addition to the primarily induced breaks many other DNA lesions may be transformed to strand breaks by cellular repair processes and can thus easily be measured and quantified.…”

Section: Introductionmentioning

confidence: 99%

Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells¶

Baumstark‐Khan

Hentschel²,

Nikandrova

et al. 2000

Photochem Photobiol

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Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).

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X-Ray Induced DNA Strand Break Induction and Rejoining in Cultured Bovine Lens Epithelial Cells

Cited by 8 publications

References 13 publications

Detection of DNA strand breaks by fluroscence assisted DNA unwinding (FADU) assay in Hela cells treated with berbrine chloride before exposure to various doses of γ – radition

Detection of DNA strand breaks by fluroscence assisted DNA unwinding (FADU) assay in Hela cells treated with berbrine chloride before exposure to various doses of γ – radition

Ionizing radiation-induced death in bovine lens epithelial cells:Mechanisms and influence of irradiation dose rate

Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells¶

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