Monitoring the biochemical changes occurring to human keratinocytes exposed to solar radiation by Raman spectroscopy

Abstract

Solar radiation exposure is recognised to be a significant contributor to the development of skin cancer. Monitoring the simultaneous and consecutive mechanisms of interaction could provide a greater understanding of the process of photocarcinogenesis. This work presents an analysis of the biochemical and morphological changes occurring to immortalised human epithelial keratinocyte (HaCaT) cell cultures exposed to simulated solar radiation (SSR). Cell viability was monitored with the aid of the Alamar Blue assay, morphological examination was done with haematoxylin and eosin staining (H&E) and changes to the biochemical constituents (nucleic acids and proteins) as a result of the radiation insult were demonstrated through a combination of Raman microspectroscopy and multivariate analysis of spectral patterns. The spectral results suggest that SSR induces changes to the conformational structure of DNA as an immediate result of the radiation, whereas alteration in the protein signature is mostly seen as a later response.

Biochemical impact of solar radiation exposure on human keratinocytes monitored by Raman spectroscopy; effects of cell culture environment

“…The instrument is routinely calibrated everỹ 1000 hours. The integrated spectral distribution over the range 280 to 400 nm constitutes a total UV intensity of 63.63 W m À2 , proportioned as 62.30 W m À2 within 315 to 400 nm (UVA) and 1.33 W m À2 in the range 280 to 315 nm (UVB) [12]. The Q-sun simultaneously delivers 400 W m À2 over the range 400 to 700 nm [23].…”

“…In the presentation of the results, the exposures are given in terms of exposure time. These values can be easily converted to UV, or full spectral dose, noting that 1 W m À2 equals 1 J m À2 s À1 [12].…”

“…In previous studies, it was shown that simulated solar radiation (SSR) exposure can produce short and longterm detrimental effects on keratinocytes (HaCaT) cultured in 2D models [12,13]. The radiation and cell interaction induces a series of immediate and later biochemical responses through the interaction with endogenous photosensitizers, which can be translated in the formation of reactive oxygen and nitrogen species (ROS and RNS), single strand break, DNA-protein cross links and the formation of cyclobutane pyrimidine dimers [12,14,15]. Such reactive species can be generated by radiation across the solar spectrum, highlighting the importance of not only the UV wavelengths in the study of the effects of solar radiation [16,17].…”

“…In a previous study of SSR of HaCaT, in addition to conventional cytotoxicity assay screening of cellular responses, Raman microspectroscopy was demonstrated to be an ideal technique to identify variations in cellular metabolism as a result of the external insult [12,18,19]. This technique allows rapid, non-destructive and high spatial resolution measurements (~0.5-1.5 μm) in tissues or single cells.…”

“…proteins, lipids, nucleic acids) or specific molecules in these groups (e.g. phenylalanine, amide I, adenine, cytosine, tyrosine) which can be altered upon exposure to external stimuli such as solar radiation [12,[19][20][21]. Raman spectroscopy is relatively insensitive to water, compared to, for example, the complementary technique of infrared absorption spectroscopy, and little or no sample preparation is required [22].…”