Aging affects sex- and organ-specific trace element profiles in mice

Abstract

A decline of immune responses and dynamic modulation of the redox status are observed during aging and are influenced by trace elements such as copper, iodine, iron, manganese, selenium, and zinc. So far, analytical studies have focused mainly on single trace elements. Therefore, we aimed to characterize age-specific profiles of several trace elements simultaneously in serum and organs of adult and old mice. This allows for correlating multiple trace element levels and to identify potential patterns of age-dependent alterations. In serum, copper and iodine concentrations were increased and zinc concentration was decreased in old as compared to adult mice. In parallel, decreased copper and elevated iron concentrations were observed in liver. The age-related reduction of hepatic copper levels was associated with reduced expression of copper transporters, whereas the increased hepatic iron concentrations correlated positively with proinflammatory mediators and Nrf2-induced ferritin H levels. Interestingly, the age-dependent inverse regulation of copper and iron was unique for the liver and not observed in any other organ. The physiological importance of alterations in the iron/copper ratio for liver function and the aging process needs to be addressed in further studies.

Keywords: aging; biomarkers; epigenetic markers; homeostasis; trace elements.

Figure 1

Age- and sex-related changes of serum TE profiles and biomarkers. Concentrations of Mn (A), I (B), Cu (C), Fe (D), Se (G), and Zn (K) were analyzed in the serum of adult (24 weeks) and old (109-114 weeks) male and female C57BL/6Jrj mice (n = 4-5) receiving chow diet. Serum concentrations were determined using ICP-MS/MS (A-D, G, K). Further biomarkers were detected by ELISA (E, F) and fluorescent probes (L) to assess the Fe marker ferritin (E) and transferrin (F) as well as free Zn (L), respectively. The Se status was further validated by GPX activity (H) and relative Selenop levels (I), based on NADPH-consuming glutathione reductase coupled assay and Dot blot analysis, respectively. Statistical testing based on Two-Way ANOVA and post hoc analysis using Bonferroni’s test with * p < 0.05, *** p < 0.001 vs. adult and ^# p < 0.05, ^## p < 0.01 vs. male.

Figure 2

TE profile analysis in the liver of mice. Liver tissue of adult (24 weeks) and old (109-114 weeks) male and female C57BL/6Jrj mice (n = 4-5) receiving chow diet were analyzed for their concentrations of Mn (A), Zn (B), Se (C), Fe (E), and Cu (F) using ICP-MS/MS. Furthermore, Se-sensitive GPX activity was assessed by NADPH-consuming assay (D). Statistical testing based on Two-Way ANOVA and post hoc analysis using Bonferroni’s test revealed no significant differences for age and sex.

Figure 3

TE changes in various organs in relation to age and sex. TEs in various organs of adult (24 weeks) and old (109-114 weeks) male and female C57BL/6Jrj mice receiving a chow diet ad libitum were analyzed by ICP-MS/MS. The heat map indicates changes of TE content in murine organs of old mice compared to adult animals (A; n=9-10) or of female mice in comparison to male animals (B; n=9-10) given in % (100 % represents no change). Each row represents one organ, whereas each column represents one element. Statistical testing based on Two-Way ANOVA and post hoc analysis using Bonferroni’s test with * p < 0.05, whereas grey * indicates p < 0.1.

Figure 4

Expression analysis of various TE-related genes in liver. Relative expression levels of TE-related genes in the liver of adult (24 weeks) and old (109-114 weeks) male and female mice (n = 4-5) fed with a chow diet ad libitum. Expression levels were normalized by a composite factor based on the house-keeping genes Hprt and Rpl13a. Finally, variances are expressed as fold change compared to adult males (mean adult males = 1). Statistical testing based on Two-Way ANOVA and post hoc analysis using Bonferroni’s test with * p < 0.05.

Figure 5

Proinflammatory cytokines and DNA and protein modifications in relation to age and sex. Serum (A) and liver extracts (B–G) of adult (24 weeks) and old (109-114 weeks) male and female mice (n = 4-5) fed with a chow diet ad libitum were subjected to enzyme-linked immunosorbent assay (A), qRT-PCR analysis (B–D), tandem mass spectrometry (E, F), and immunoblotting (G). This way, proinflammatory cytokines (A–D), global DNA methylation (mdC/dC; E), and hydroxymethylation (hmdC/dC; F), as well as 3-nitrotyrosine (3-NT, G) protein modifications were determined. Hepatic transcription levels (B–D) were normalized by a composite factor based on the house-keeping genes Hprt and Rpl13a, whereas 3-NT-modified proteins were normalized to GAPDH (G). Except for (E) and (F), where data is given in %, data is presented as fold change compared to male adults (A–D, G). Statistical testing based on Two-Way ANOVA and post hoc analysis using Bonferroni’s test with * p < 0.05, ** p < 0.01 vs. adult and ^# p < 0.05, ^### p < 0.001 vs. male.

Figure 6

Activity and expression levels of Nrf2 target genes. The enzyme activities of the Nrf2 targets NQO1 (A) and total GST (C) were determined by activity assays, whereas the relative protein levels of FTH (B) normalized to the house-keeping gene GAPDH were analyzed by Western blot in liver tissue of adult (24 weeks) and old (109-114 weeks) male and female mice (n = 4-5). Statistical testing based on Two-Way ANOVA and post hoc analysis using Bonferroni’s test with ** p < 0.01 vs. adult and ^# p < 0.05, ^### p < 0.001 vs. male.