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

Martinez-Levasseur LM, Gendron D, Knell RJ, Acevedo-Whitehouse K - BMC Res Notes (2013)

Bottom Line: Despite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Zoology, Regent's Park, London NW1 4RY, UK.

ABSTRACT

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.

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HSP70, P53 and KIN expression levels and correlations. A) Mean levels of expression (in ΔCt, y axis inverted) of HSP70 (n=60), P53 (n=59) and KIN (n=60). Bars = ± 95% CI. B) Correlation between KIN and HSP70 expression levels (in ΔCt). C) Correlation between P53 and HSP70 expression levels (in ΔCt). The lines show regression lines. Lower ΔCt values represent higher levels of expression.
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Figure 2: HSP70, P53 and KIN expression levels and correlations. A) Mean levels of expression (in ΔCt, y axis inverted) of HSP70 (n=60), P53 (n=59) and KIN (n=60). Bars = ± 95% CI. B) Correlation between KIN and HSP70 expression levels (in ΔCt). C) Correlation between P53 and HSP70 expression levels (in ΔCt). The lines show regression lines. Lower ΔCt values represent higher levels of expression.

Mentions: The gene with the highest expression level was the gene coding for the heat shock protein 70 (HSP70; ΔCt mean = 5.22 ± 0.21 SE, n = 60). Expression levels for HSP70 were 1.31 times the levels observed for the tumour protein P53 gene (ΔCt mean = 6.72 ± 0.12 SE, n = 59) and 1.69 times that of the gene coding for the KIN protein (ΔCt mean = 8.85 ± 0.11 SE, n = 60) (Figure 2A). To investigate whether gene expression levels were correlated, we fitted three mixed effects models, one for each of the target genes (Table 4). Direct relationships were observed between the expressions of KIN and HSP70 and the expression of P53 and HSP70 (Table 4; Figure 2B and C, respectively). To investigate whether gene expression levels were related to the presence of UV-induced microscopic damage such as intracellular oedema and cytoplasmic vacuolation previously recorded (see [4] for details), we constructed three mixed effect models, one for each of the target genes (Table 5). Interestingly, the models showed that expression of P53 and HSP70 was lower when oedema was present (Table 5; Figure 3A). Vacuolation did not significantly predict gene expression and thus was not retained in the final models (Table 5). However, when observed graphically, there appears to be a slight trend where higher levels of gene expression tend to be observed when vacuoles are present (Figure 3B). KIN expression was not significantly correlated with any of the epidermal lesions included in the full model (Table 5).


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

Martinez-Levasseur LM, Gendron D, Knell RJ, Acevedo-Whitehouse K - BMC Res Notes (2013)

HSP70, P53 and KIN expression levels and correlations. A) Mean levels of expression (in ΔCt, y axis inverted) of HSP70 (n=60), P53 (n=59) and KIN (n=60). Bars = ± 95% CI. B) Correlation between KIN and HSP70 expression levels (in ΔCt). C) Correlation between P53 and HSP70 expression levels (in ΔCt). The lines show regression lines. Lower ΔCt values represent higher levels of expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716943&req=5

Figure 2: HSP70, P53 and KIN expression levels and correlations. A) Mean levels of expression (in ΔCt, y axis inverted) of HSP70 (n=60), P53 (n=59) and KIN (n=60). Bars = ± 95% CI. B) Correlation between KIN and HSP70 expression levels (in ΔCt). C) Correlation between P53 and HSP70 expression levels (in ΔCt). The lines show regression lines. Lower ΔCt values represent higher levels of expression.
Mentions: The gene with the highest expression level was the gene coding for the heat shock protein 70 (HSP70; ΔCt mean = 5.22 ± 0.21 SE, n = 60). Expression levels for HSP70 were 1.31 times the levels observed for the tumour protein P53 gene (ΔCt mean = 6.72 ± 0.12 SE, n = 59) and 1.69 times that of the gene coding for the KIN protein (ΔCt mean = 8.85 ± 0.11 SE, n = 60) (Figure 2A). To investigate whether gene expression levels were correlated, we fitted three mixed effects models, one for each of the target genes (Table 4). Direct relationships were observed between the expressions of KIN and HSP70 and the expression of P53 and HSP70 (Table 4; Figure 2B and C, respectively). To investigate whether gene expression levels were related to the presence of UV-induced microscopic damage such as intracellular oedema and cytoplasmic vacuolation previously recorded (see [4] for details), we constructed three mixed effect models, one for each of the target genes (Table 5). Interestingly, the models showed that expression of P53 and HSP70 was lower when oedema was present (Table 5; Figure 3A). Vacuolation did not significantly predict gene expression and thus was not retained in the final models (Table 5). However, when observed graphically, there appears to be a slight trend where higher levels of gene expression tend to be observed when vacuoles are present (Figure 3B). KIN expression was not significantly correlated with any of the epidermal lesions included in the full model (Table 5).

Bottom Line: Despite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Zoology, Regent's Park, London NW1 4RY, UK.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus