Crosstalk of Nrf2 with the Trace Elements Selenium, Iron, Zinc, and Copper

Abstract

Trace elements, like Cu, Zn, Fe, or Se, are important for the proper functioning of antioxidant enzymes. However, in excessive amounts, they can also act as pro-oxidants. Accordingly, trace elements influence redox-modulated signaling pathways, such as the Nrf2 pathway. Vice versa, Nrf2 target genes belong to the group of transport and metal binding proteins. In order to investigate whether Nrf2 directly regulates the systemic trace element status, we used mice to study the effect of a constitutive, whole-body Nrf2 knockout on the systemic status of Cu, Zn, Fe, and Se. As the loss of selenoproteins under Se-deprived conditions has been described to further enhance Nrf2 activity, we additionally analyzed the combination of Nrf2 knockout with feeding diets that provide either suboptimal, adequate, or supplemented amounts of Se. Experiments revealed that the Nrf2 knockout partially affected the trace element concentrations of Cu, Zn, Fe, or Se in the intestine, liver, and/or plasma. However, aside from Fe, the other three trace elements were only marginally modulated in an Nrf2-dependent manner. Selenium deficiency mainly resulted in increased plasma Zn levels. One putative mediator could be the metal regulatory transcription factor 1, which was up-regulated with an increasing Se supply and downregulated in Se-supplemented Nrf2 knockout mice.

Keywords: Nrf2; copper; homeostasis; iron; selenium; zinc.

Figure 1

Fe (A–C), Se (D–F), Zn (G–I), and Cu (J–L) concentrations in the jejunum (A,D,G,J), liver (B,E,H,K), and plasma (C,F,I,L) of six-month-old male and female Nrf2 KO and WT mice fed a standard chow diet with 0.3 ppm Se. The TE profile was analyzed using ICP-MS/MS. Scatter dot plots with mean (n = 6–7). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. WT and ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 vs. male (two-way ANOVA with Bonferroni’s post-test).

Figure 2

Biomarkers of the Se status. Se concentrations in the plasma (A) and liver (B) of Nrf2 KO and WT male mice fed diets with defined Se contents (–Se: 0.03 ppm; +Se: 0.15 ppm; ++Se: 0.6 ppm). The TE profile was analyzed using ICP-MS/MS. Enzyme activity of TXNRD (C) and GPX (D) was analyzed together with mRNA expression of Selenow (E) from liver samples of male Nrf2 KO and WT mice. Bars represent means + SD (n = 4-5). * p < 0.05; *** p < 0.001 vs. WT and ^## p < 0.01; ^### p < 0.001 vs. –Se (two-way ANOVA with Bonferroni’s post-test).

Figure 3

Hepatic enzyme activities as markers for Nrf2 activity. The Nrf2 target gene NQO1 was measured by an activity assay (A). Total enzyme activity of all GST isoforms was measured by an activity assay (B). Samples were the liver of Nrf2 KO and WT male mice fed diets with defined Se contents (–Se: 0.03 ppm; +Se: 0.15 ppm; ++Se: 0.6 ppm). Bars represent means + SD (n = 4–5). *** p < 0.001 vs. WT and ^# p < 0.05; ^### p < 0.001 vs. –Se (two-way ANOVA with Bonferroni’s post-test).

Figure 4

Biomarkers of the Fe status. Fe (A), ferritin (B), and transferrin (C) concentrations in the plasma of male Nrf2 KO and WT mice fed diets with defined Se contents (–Se: 0.03 ppm; +Se: 0.15 ppm; ++Se: 0.6 ppm). Additionally, Fe concentration in the liver (D) as well as mRNA and protein expression of Fe-related genes/proteins in the liver were determined by qPCR or western blot, respectively (E,G–L). Ferroportin mRNA was measured in the duodenum (F). The TE profile was analyzed using ICP-MS/MS (A,D). Further Fe plasma parameters were detected by ELISA (B,C). Bars represent means + SD (n = 4–5). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. WT and ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 vs. –Se (two-way ANOVA with Bonferroni’s post-test).

Figure 5

Biomarkers of the Cu and Zn status. Cu and Zn concentrations in the plasma (A,C) and liver (B,D) of male Nrf2 KO and WT mice fed diets with defined Se contents (–Se: 0.03 ppm; +Se: 0.15 ppm; ++Se: 0.6 ppm), analyzed by ICP-MS/MS. Additionally, mRNA (F–H) and protein expression (C, I–J) of Cu- and Zn-related genes/proteins in the liver of these animals were determined. Western blots were normalized to the Ponceau staining (P). Bars represent means + SD (n = 4–5). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. WT and ^# p < 0.05; ^## p < 0.01; ^### p < 0.001 vs. –Se (two-way ANOVA with Bonferroni’s post-test). C = Ctr1.