Control and target gene selection for studies on UV-induced genotoxicity in whales

BACKGROUND: Despite 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. RESULTS: Using 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. CONCLUSION: This 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.