PMC

DNA damages visualized on stretched chromatin fibres. Quantification of single and clustered signals of 8-oxo-dG (A) and ARP (B), respectively. Grey bars represent the number of single signals (left y-axis,) whereas black bars represent clusters (right axis). At least 10 fibres were analysed per data point and SDs are calculated. A detail of representative fibres is shown below the corresponding columns, open arrowheads highlight singles, filled arrowheads clusters. In green the total DNA is stained with YOYO-I. DNA lesions are shown in red. The reduction in both single damage sites as well as clusters after Naringin treatment is significant on the P < 0.05 level as calculated by unpaired t-test with Welch’s correction.

DNA fragmentation quantification by Comet-assay. (A and B) Total DNA damage (ssbs + dsbs) detected by the alkaline Comet-assay (•). Naringin-treated cells show lower levels of DNA fragmentation (open square). Split-dose irradiation (filled inverted triangle) leads to a DNA damage comparable to a single exposure of 200 kJ/m², for details see text. (C and D) Neutral Comet-assay shows a dose-dependent induction of DNA breaks (•). The number of breaks can again be reduced by the antioxidant (open square). In contrast to the alkaline Comet-assay, the neutral version reveals an increase of the number of dsbs if the irradiation is performed in split dose (filled inverted triangle). All measurements are means of medians together with SD. Representative micrographs for comet specimens are shown for alkaline Comet-assay (top) and neutral Comet-assay (bottom) for the indicated treatments.

Oxidatively induced clustered DNA lesions (OCDLs) were measured with the neutral Comet-assay and FPG as an enzymatic probe. (A) Sample comets with (+FPG) and without (−FPG) enzymatic processing. An increase in DNA fragmentation can be seen for all FPG-treated comets as partly separated DNA species in the direction of electrophoresis. (B) Quantification of OCDL. Grey bars represent the FPG-treated cells, and the black bars represent the mock treated ones. Shown are the means of medians; error bars represent the SD. The black line above the bars shows the difference between FPG- and mock-treated comets for each time point. (C) Repair kinetics for cells exposed to UVA irradiation and repair incubated at 4°C (filled inverted triangle) and 37°C (filled square). The overall level of DNA fragmentation is higher in cells kept at 37°C.

Replication-independent formation of γH2AX foci in UVA-exposed HaCaT cells and primary human fibroblasts. (A and B) Dose–response curves for the formation of γH2AX foci in G₁ arrested cells after continuous UVA exposure (filled circle). Split-dose exposure leads to an increased number of foci in both cell types (filled inverted triangle). Cells pre-treated with the antioxidant Naringin show a reduced number of foci (open square). Means and SD are given. (C) Typical sample micrographs of G₁ arrested HaCaT cells (controls: top), acute irradiation (middle: 1 × 200 and 1 × 400 kJ/m²) as well as after split-dose irradiation (bottom: 2 × 400 kJ/m²). (D–F) H2AX phosphorylation was quantified in the nuclear extracts following acute irradiation. A dose-dependent increase in the amount of γH2AX can be found up to 600 kJ/m². With higher doses, saturation in the amount of phosphorylated H2AX was found. In contrast, pre-incubation with the antioxidant Naringin reduces the amount of phosphorylated H2AX. Data represent the mean of two experiments and the SD. (G) Nuclear ROS levels at two different dose rates as measured by Flow cytometry in HaCaT cells using chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester. A dose-dependent, but dose-rate independent increase of the ROS levels was detected with UVA doses used in this study and environmental relevant doses and dose rates.