Abstract
BackgroundDespite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades. Thus, it is pressing to implement tools that allow investigating the capacity of wildlife to respond to excessive UV, particularly species like cetaceans that lack anatomical or physiological protection. One approach is to examine epidermal expression of key genes involved in genotoxic stress response pathways. However, quantitation of mRNA transcripts requires previous standardization, with accurate selection of control and target genes. The latter is particularly important when working with environmental stressors such as UV that can activate numerous genes.ResultsUsing 20 epidermal biopsies from blue, fin and sperm whale, we found that the genes encoding the ribosomal proteins L4 and S18 (RPL4 and RPS18) were the most suitable to use as controls, followed by the genes encoding phosphoglycerate kinase 1 (PGK1) and succinate dehydrogenase complex subunit A (SDHA). A careful analysis of the transcription pathways known to be activated by UV-exposure in humans and mice led us to select as target genes those encoding for i) heat shock protein 70 (HSP70) an indicator of general cell stress, ii) tumour suppressor protein P53 (P53), a transcription factor activated by UV and other cell stressors, and iii) KIN17 (KIN), a cell cycle protein known to be up-regulated following UV exposure. These genes were successfully amplified in the three species and quantitation of their mRNA transcripts was standardised using RPL4 and RPS18. Using a larger sample set of 60 whale skin biopsies, we found that the target gene with highest expression was HSP70 and that its levels of transcription were correlated with those of KIN and P53. Expression of HSP70 and P53 were both related to microscopic sunburn lesions recorded in the whales’ skin.ConclusionThis article presents groundwork data essential for future qPCR-based studies on the capacity of wildlife to resolve or limit UV-induced damage. The proposed target genes are HSP70, P53 and KIN, known to be involved in genotoxic stress pathways, and whose expression patterns can be accurately assessed by using two stable control genes, RPL4 and RPS18.
Highlights
Despite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades
When looking at each species separately, RPS18 had the highest correlation coefficient in all cases, while the second best candidate gene differed amongst species, being ribosomal proteins L4 (RPL4) for fin and sperm whales and phosphoglycerate kinase 1 (PGK1) for blue whales (Table 3)
To study the genotoxic stress pathways used by cetaceans in response to solar UV exposure, we proposed quantifying changes in the expression of key genes, namely those encoding the heat shock protein 70 (HSP70), an indicator of cell stress [22,25], the tumour
Summary
Despite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades. Effectors involved in DNA repair, transcription regulation and cell cycle control, comprise proteins such as the tumour suppressor protein P53 [7,8] This interactive network that involves hundreds of genes is complex [7,9] and to our knowledge has not been studied in wild mammals. Despite the technique’s accuracy, sensitivity and speed [11], variations in the amount and integrity of starting material, transcription- and amplification efficiency rates, as well as the occurrence of inhibitors, can lead to quantitation errors In this context, normalization of data is an essential step that must precede gene expression quantitation [10,11,12]. Accurate selection of reference genes is central to interpreting quantitative PCR results
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