DNA damage and repair of human skin keratinocytes concurrently exposed to pyrene derivatives and UVA light

Abstract

Polycyclic aromatic hydrocarbons (PAHs), a class of mutagenic environmental contaminants, insert toxicity through both metabolic activation and light irradiation. Pyrene, one of the most widely studied PAHs, along with its mono-substituted derivatives, 1-amino, 1-bromo, 1-hydroxy, and 1-nitropyrene, were chosen to study the effect of substituents on their phototoxicity, DNA damage and repair. Both alkaline Comet assay, which detects direct DNA damages, and Fpg endonuclease Comet assay, which detects oxidative DNA damages, were conducted at 0, 2, 4, 8, and 24 h of incubation of the cells in minimal growth medium after concomitant exposure to pyrene derivatives and UVA light. All these compounds are photocytotoxic and the phototoxicity is both incubation time and PAH dose dependent; whereas, those without light are not toxic. The LC50 obtained are in the range of 3.5 – 9.3 µM. Cellular DNA damages, both direct and oxidative, are observed immediately after the cells are treated with UVA light and the pyrene derivatives at a concentration of 1.0 µM. The amount of DNA damages (both direct and oxidative) increase from 0 to 4 h of incubation. After 4 hours, subsequent damage induction declines, and this is perceived to be mainly through DNA repair. After longer incubation of 8 h, the damaged cellular DNA start to be repaired, resulting in greatly reduced amount of DNA damages, and the DNA damage reaches the minimum at 24 h of incubation. 1-Amopyrene and 1-hydroxypyrene cause more DNA oxidative damages immediately after the exposure (0 h of incubation), and these damages are repaired within the same timeframe as the other tested compounds. The oxidative DNA damages caused by 1-bromopyrene are repaired starting at 2 h of incubation, earlier than the damages caused by all the other compounds.

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

“…1-NP can also induce oxidative DNA damage as shown, for example, in human umbilical vein endothelial cells at doses <10 μM [ 9 ], or in naked DNA in the presence of NADPH [ 10 ]. In HaCaT keratinocytes, 1-NP induced oxidative DNA damage but only in the presence of UV-light [ 31 ]. In another study, exposure to 1-NP resulted in formation of 8-OH-dG in A549 cells but only after application of very high doses of the compound (above 250 μM).…”

Cytotoxicity of organic surface coating agents used for nanoparticles synthesis and stability

“…Cells were cultured in a humidified incubator with 5% CO 2 at 37 °C. After the cells grew to confluence, they were detached by 25% trypsin/EDTA, and diluted to 3×10 5 cells/mL by their respective complete media as previously reported (Fullove and Yu 2013; McShan and Yu 2014). …”

“…The plate was left at room temperature for 30 min to completely solubilize the formazan salt. The absorbance was read at 540 nm using Multiskan Ascent Plate Reader with Ascent software as it was done before (Wang 2008; Lu 2010; Fullove and Yu 2013). …”

Toxicity of nanoparticle surface coating agents: Structure-cytotoxicity relationship

“…After confluence, the cells were detached by 0.25% trypsin/EDTA, and diluted to 3×10 5 cells/mL with the complete DMEM medium as before 27,28 . A 200 μL cell suspension was added to each well of a 96-well plate and incubated under 5% CO 2 at 37 °C for 24 h for cell adhesion.…”