Modified comet assays for the detection of cyclobutane pyrimidine dimers and oxidative base damages

The comet assay (also known as single-cell gel electrophoresis) is a technique for the detection of DNA damage at the level of the individual cell. It is a versatile, relatively simple to perform and sensitive method. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of cyclobutane pyrimidine dimers (CPDs), crosslinks, base damage, and apoptotic nuclei. Many investigators also interested in examining the DNA damage as a function of time after exposure to a known genotoxic agent. Here, we present a procedure of comet assay for the detection of DNA strand breaks, base damages, and CPDs that can be used to measure DNA damage during toxicity, oxidative stress, and ultraviolet radiation exposure and it can be applied in human toxicological biomonitoring scenarios

Effect of linalool on UVB-induced inflammatory markers expression in HDFa.

<p>(A) Western blot analysis of TNF-α, IL-6, IL-10 and COX-2 expression in HDFa. (B) The quantification of protein was performed by densitometric analysis using Image-studio software (LI COR, USA). The densitometry data represent means ± SD from three immunoblots and are shown as relative density of protein bands normalized to ß-actin level. Values not sharing a common marking (# and *) differ significantly at P < 0.05 (DMRT).</p

Linalool prevents oxidative stress activated protein kinases in single UVB-exposed human skin cells

<div><p>Ultraviolet-B radiation (285–320 nm) elicits a number of cellular signaling elements. We investigated the preventive effect of linalool, a natural monoterpene, against UVB-induced oxidative imbalance, activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling in HDFa cells. We observed that linalool treatment (30 μM) prevented acute UVB-irradiation (20 mJ/cm²) mediated loss of activities of antioxidant enzymes in HDFa cells. The comet assay results illustrate that linalool significantly prevents UVB-mediated 8-deoxy guanosine formation (oxidative DNA damage) rather than UVB-induced cyclobutane pyrimidine (CPD) formation. This might be due to its ability to prevent UVB-induced ROS formation and to restore the oxidative imbalance of cells. This has been reflected in UVB-induced overexpression of MAPK and NF-κB signaling. We observed that linalool inhibited UVB-induced phosphorylation of ERK1, JNK and p38 proteins of MAPK family. Linalool inhibited UVB-induced activation of NF-κB/p65 by activating IκBa. We further observed that UVB-induced expression of TNF-α, IL6, IL-10, MMP-2 and MMP-9 was modulated by linalool treatment in HDFa cells. Thus, linalool protects the human skin cells from the oxidative damages of UVB radiation and modulates MAPK and NF-κB signaling in HDFa cells. The present findings substantiate that linalool may act as a photoprotective agent against UVB-induced skin damages.</p></div

Effect of linalool on UVB induced DNA damage in HDFa cells.

<p>(A) Linalool on UVB induced CPDs formation was analyzed by T4 modified comet assay. Fluorescence microscopic images (20X) were recorded using fluorescence microscope (Cell imaging station, life technologies) under red fluorescence lamb. (B) Linalool on UVB induced 8-OxoG was analyzed by 8-OxoG modified comet assay. Fluorescence microscopic images (20X) were recorded using fluoresece microscope (Cell imaging station, life technologies) under red fluorescence lamb. The percentage tail DNA was calculated by CASP software for both CPD and 8-OxoG modified assay. Values are given as means ± SD of six experiments in each group. Values not sharing a common marking (# and *) differ significantly at P = 0.05 (DMRT).</p

Effect of linalool on UVB-induced lipid peroxidation and antioxidant status in skin cells.

<p>(A) Lipid peroxidation status of linalool and/or UVB irradiated HDFa cells. TBARS expressed as nmol/mg protein (B) GSH level of linalool and/or UVB irradiated HDFa cells. GSH level was expressed as mg/g protein (C) Enzymatic antioxidants status of linalool and/or UVB irradiated HDFa cells. *Enzyme concentration required for 50% inhibition of nitroblue tetrazolium reduction in one minute. **μmol of hydrogen peroxide consumed per minute. ***μg of glutathione consumed per minute. Values are given as means ± SD of six experiments in each group. Values not sharing a common marking (# and *) differ significantly at P = 0.05 (DMRT).</p

Effect of linalool on UVB-induced MAPKs and NF-κB signaling in HDFa.

<p><b>(A)</b> Western blot analysis of p-ERK1, p-JNK and p-p38 expression in HDFa. <b>(B)</b> The quantification of protein was performed by densitometric analysis using Image-studio software (LI COR, USA). The densitometry data represent means ± SD from three immunoblots and are shown as relative density of protein bands normalized to ß-actin level. (<b>C)</b> Western blot analysis of cytosolic NF-κB and IκBa expression in HDFa. <b>(D)</b> Band intensities were analyzed by Image studio software and normalized to ß-actin level. <b>(E)</b> Western blot analysis of NF-κB nuclear expression in HDFa. <b>(F)</b> Band intensities were analyzed by Image studio software and normalized to Lamin A level. The densitometry data represent means ± SD from three immunoblots and are shown as relative density of protein bands normalized to Lamin A level. Values not sharing a common marking (# and *) differ significantly at P < 0.05 (DMRT).</p