There is increasing evidence that UVA radiation, which makes up approximately 95% of the solar UV light reaching the Earth's surface and is also commonly used for cosmetic purposes, is genotoxic. However, in contrast to UVC and UVB, the mechanisms by which UVA produces various DNA lesions are still unclear. In addition, the relative amounts of various types of UVA lesions and their mutagenic significance are also a subject of debate. Here, we exploit atomic force microscopy (AFM) imaging of individual DNA molecules, alone and in complexes with a suite of DNA repair enzymes and antibodies, to directly quantify UVA damage and reexamine its basic mechanisms at a single-molecule level. By combining the activity of endonuclease IV and T4 endonuclease V on highly purified and UVA-irradiated pUC18 plasmids, we show by direct AFM imaging that UVA produces a significant amount of abasic sites and cyclobutane pyrimidine dimers (CPDs). However, we find that only approximately 60% of the T4 endonuclease V-sensitive sites, which are commonly counted as CPDs, are true CPDs; the other 40% are abasic sites. Most importantly, our results obtained by AFM imaging of highly purified native and synthetic DNA using T4 endonuclease V, photolyase, and anti-CPD antibodies strongly suggest that CPDs are produced by UVA directly. Thus, our observations contradict the predominant view that as-yet-unidentified photosensitizers are required to transfer the energy of UVA to DNA to produce CPDs. Our results may help to resolve the long-standing controversy about the origin of UVA-produced CPDs in DNA.
This in vitro study characterized the temporal cytokine expression profile from human monocytes exposed to phagocytosable Ti particles (0.78+/-0.12 microm) and to Ti discs of comparable surface roughness. Human THP-1 monocytes were cultured in six well tissue culture polystyrene (TCPS) plates. Each well was either bare, contained Ti particles (the particles were clearly engulfed by the monocytes), or contained a Ti disc. Half of the wells were treated with 1 microg/mL lipopolysaccharide (LPS), while the other half were left unstimulated. Unstimulated and LPS-stimulated cells in bare wells were the negative and positive controls, respectively. Supernatant was sampled from each well at 1, 6, 24, 48, and 72 h and assayed for the expression of nine different cytokines using a Luminex system. Three cytokines (IL-1beta, GM-CSF and IL-13) gave little to no response under all conditions, while six cytokines (TNF-alpha, IL-6, MIP-1alpha, MCP-1, VEGF, and IL-1ra) were clearly detectable. Expression levels generally increased with culture time, particle concentration, and LPS stimulation. Most significantly, it was found that cells treated by Ti discs produced in many instances a higher cytokine expression than did particles.