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

Abstract

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°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).

Phototoxicity of Ultraviolet (UV) Radiation: Evaluation of UV-Blocking Efficiency of Intraocular Lens (IOL) Materials Using Retinal Cell Culture and in vitro Bioassays~!2009-06-08~!2010-02-10~!2010-03-26~!

“…Thus, more detailed studies are necessary to establish whether cell damage induced by UV radiation is apoptotic or non-apoptotic. The measurement of condensed pycnotic nuclei from Hoechst 33342 fluorescent micrographs has been used in in vitro photocytotoxicity studies of rat hepatocytes, Chinese hamster ovary cells, Xenopus XTC-2 cells, and B-cell hybridoma [50][51][52][53]. Findings from these studies support the use of Hoechst 33342 as a sensitive nuclui morphological marker for monitoring UV-induced cell damage.…”

Effect of Amalaki rasayana on DNA damage and repair in randomized aged human individuals

“…Results of mass spectrometry analysis are provided in Table 1 and Total ion chromatogram of rasayana injection is provided as supplementary figure 1S. proportional to DNA strand breaks and inversely proportional to the strand breaks repair (Baumstark- Khan et al, 2000). The initial kinetic studies have shown that the exposure of PBMCs of aged (55 -80.years; N= 16) and young (22 -25 years; N=16) subjects with 1 J/cm 2 of UVC resulted in an increase in the strand breaks up to 3 h repair time.…”

NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR

“…Exposure to UVB (28-315 nm) induces CPD and 6-4PP photolesions (36,37). These premutagenic DNA lesions are shown to be detrimental to replication, capable of causing replication fork collapse (38)(39)(40). In this study, we investigated the DSB repair kinetics by visualizing 53BP1 foci in melanoma Figure 6.…”