Fig. 6.
Changes in genomic stability markers and global DNA (hydroxy)methylation in murine liver. Genomic stability markers were analysed in liver tissue of adult (30 weeks) and old (66 weeks) male and female C57BL/6Jrj mice (n = 8–10) receiving a -TE, +TE, or + TEaa diet for 26 weeks. Regarding DNA damage, the total amount of DNA strand breaks as well as alkali-labile sites (A), and relative 8-oxodG levels (B) were analysed by alkaline comet assay and applying an ELISA kit, respectively. PARylation levels were determined via HPLC-MS/MS (C), and BER incision activity was determined towards an AP site analogue (D), an 8-oxodG (E), and a 5-OHdU (F) containing oligonucleotide by a non-radioactive incision activity assay. Relative expression levels of Polβ (G), Aptx (I), and Lig1 (J) mRNA were examined by qRT-PCR analysis and hepatic transcription levels were normalised to a composite factor based on the house keeper genes Hprt and Rpl13a. Variances are expressed as fold changes compared to + TE male adults (mean + TE male adult = 1). Polβ protein quantification was performed via western blot analysis using β-actin for normalisation of determined protein levels. DNA (hydroxy)methylation was quantified via HPLC-MS/MS (K, L). Statistical testing based on Three-Way ANOVA and Bonferroni's post-test with * p < 0.05, **p < 0.01, ***p < 0.001. Detailed results of statistical testing are summarised in Table 2.