Ageing-associated effects of a long-term dietary modulation of four trace elements in mice

Abstract

Trace elements (TEs) are essential for diverse processes maintaining body function and health status. The complex regulation of the TE homeostasis depends among others on age, sex, and nutritional status. If the TE homeostasis is disturbed, negative health consequences can result, e.g., caused by impaired redox homeostasis and genome stability maintenance. Based on age-related shifts in TEs which have been described in mice well-supplied with TEs, we aimed to understand effects of a long-term feeding with adequate or suboptimal amounts of four TEs in parallel. As an additional intervention, we studied mice which received an age-adapted diet with higher concentrations of selenium and zinc to counteract the age-related decline of both TEs. We conducted comprehensive analysis of diverse endpoints indicative for the TE and redox status, complemented by analysis of DNA (hydroxy)methylation and markers denoting genomic stability maintenance. TE concentrations showed age-specific alterations which were relatively stable and independent of their nutritional supply. In addition, hepatic DNA hydroxymethylation was significantly increased in the elderly mice and markers indicative for the redox status were modulated. The reduced nutritional supply with TEs inconsistently affected their status, with most severe effects regarding Fe deficiency. This may have contributed to the sex-specific differences observed in the alterations related to the redox status and DNA repair activity. Overall, our results highlight the complexity of factors impacting on the TE status and its physiological consequences. Alterations in TE supply, age, and sex proved to be important determinants that need to be taken into account when considering TE interventions for improving general health and supporting convalescence in the clinics.

Keywords: Ageing; Genome stability (maintenance); Redox status; Sex; Trace elements.

Graphical abstract

Fig. 1

Changes in Zn status as well as related biomarkers and genes. Markers for the evaluation of the Zn status were determined in serum and liver of adult (30 weeks) and old (66 weeks) male and female C57BL/6Jrj mice (n = 8–10) receiving a -TE, +TE, or +TE_aa diet for 26 weeks. Total Zn and free Zn concentrations were analysed in serum (A, B) and liver (C, D) by ICP-MS/MS and via fluorescent probes, respectively. To further evaluate the Zn status, relative expression levels of the Zn regulators Mt1 (E) and Mt2 (F) as well as the Zn transporters Zip3 (G), Zip5 (H), and ZnT10 (I) were examined via qRT-PCR analysis. 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). 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.

Fig. 2

Changes in Se status as well as related biomarkers and genes. Markers for the evaluation of the Se status were determined in serum and liver of adult (30 weeks) and old (66 weeks) male and female C57BL/6Jrj mice (n = 8–10) receiving a -TE, +TE, or +TE_aa diet for 26 weeks. Se concentrations were analysed in serum (A) and liver (D) by ICP-MS/MS. To further evaluate the Se status, GPX enzyme activity was analysed by a NADPH-consuming assay in serum (B) and liver (E). Additionally, serum Selenop concentrations (C) were analysed by affinity-HPLC-ICP-MS/MS and hepatic Selenop transcription level was examined by qRT-PCR analysis, whereby 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). 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.

Fig. 3

Changes in Cu status as well as related biomarkers and genes. Markers for the evaluation of the Cu status were determined in serum and liver of adult (30 weeks) and old (66 weeks) male and female C57BL/6Jrj mice (n = 8–10) receiving a -TE, +TE, or +TE_aa diet for 26 weeks. Cu concentrations were analysed in serum (A) and liver (B) by ICP-MS/MS. On the basis of the analysed TE concentrations, serum Cu/Se (E) and serum Cu/Zn ratio (F) were calculated. To further evaluate the Cu status, relative hepatic expression levels of Cu transporters Ctr1 (C) and Atp7b (D) were examined by qRT-PCR analysis. 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). 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.

Fig. 4

Changes in Fe and Mn status, as well as related biomarkers and genes. Markers for the evaluation of the Fe and Mn status were determined in serum and liver of adult (30 weeks) and old (66 weeks) male and female C57BL/6Jrj mice (n = 8–10) receiving a -TE, +TE, or +TE_aa diet for 26 weeks. Hepatic Fe (B) as well as serum (H) and hepatic (I) Mn concentrations were determined via ICP-MS/MS. To further evaluate the Fe status, serum transferrin (A) concentrations were analysed via ELISA and relative hepatic expression levels of genes involved in Fe metabolism including Dmt1 (C), Fech (D), Fpn (E), Hamp (F), and Hmox1 (G) were examined by qRT-PCR analysis. 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). 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.

Fig. 5

Changes in markers indicative for the redox status in murine liver. Various markers indicative for the redox status 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 + TE_aa diet for 26 weeks. Enzyme activities of the Nrf2 targets NQO1 (A), TXNRD (B), and GST (C) were determined by activity assays. Relative expression levels of Nqo1 (D) and Txnrd1 (E) mRNA were examined by qRT-PCR analysis. 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). 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.

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 + TE_aa 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.