Sexually immature male ERE-Luc reporter mice to assess low dose estrogen-like effects of CdCl2 versus dietary Cd

Abstract

CdCl2 salt is widely used in exposure oriented studies, while the biological exposure of Cadmium (Cd) occurs mostly through diet. Hence, we designed a in vivo imaging methodology with sexually immature male ERE-Luc reporter mice to test the estrogen-like (EL) effects of Cd as a natural component in wheat and flax bread based diets (containing 17.57 and 49.22 ug/kg Cd concentrations respectively) and CdCl2 per-oral dose of 1 ug/kg/bw/day. Total exposure of ingested and % bioaccumulation of Cd in selected organs were estimated as 547 ng (4.4%), 776 ng (0.3%) and 2131.8 ng (0.1%) corresponding to CdCl2, wheat and flax bread based diet treatments respectively. Cd from CdCl2 bioaccumulated more readily, despite the exposure of Cd is higher with bread based diets. Longitudinal in vivo imaging did not reveal significant changes in luciferase activity. White adipose tissue (WAT) and prostate were identified as novel target organs of Cd. Indeed, the rest of the observed EL effects, endogenous target gene expression and necropsy findings are not consistent to any particular organ or treatment. This implies that, the observed EL effects due to low doses of Cd (either from CdCl2 or dietary form) occur only as subtle changes at the molecular level, but inadequate to cause significant changes at the anatomo-pathological level during the 21 day exposure period. The study demonstrates the sensitivity of the methodology to assess EL effects of food embedded Cd and underlines the limitations of directly extrapolating the results of suspected chemicals in their pure form to dietary exposure scenarios.

Keywords: CdCl2; Reporter mice; bioavailability; dietary Cd; endocrine disruptors; estrogenicity; in vivo imaging.

Figure 1

Cd accumulation in liver, small intestine and kidney of male treated for 21 days with CdCl₂ or two bread based diets. A: Fresh liver and kidney weight were measured after dissection. B: Tissue cadmium content was measured in liver, small intestine and kidney and data was expressed as ng/g of tissue. Data represents mean ± SEM (n=5) and asterisk indicates a significant difference from control, as assessed by one-way ANOVA plus Bonferroni post hoc test (*p<0.05), (***p<0.001).

Figure 2

Bioluminescence in vivo imaging of male ERE-Luc mice treated with CdCl₂ and two bread based diets for a period of 21 days. A: Representative images of ERE-Luc mice at 0, 1, 7, 14 and 21 days of treatment. B: Quantitative analysis of photon emission from chest, thymus and abdomen of control and treated mice. Data represent the average of determinations made in groups of 5 animals each.

Figure 3

CdCl₂ or two bread based diets showing tissue specific pattern of ER activation in 21 day old male ERE-Luc mice. (A) Representative images of photon emission exposure in freshly excised liver, small intestine, kidney and brain as measured by CCD camera at the end of the 21 day long treatment. (B) Tissues were dissected from euthanized mice at the end of the treatment and ex vivo imaging analysis were measured in isolated tissues to identify the tissue specific localization of the luciferase signal. The emitted photons are counted and expressed as Cts/Cm²/s. (C) Luciferase content of the dissected tissues were measured by enzymatic assay in tissue homogenates. Data in (A) and (B) were obtained from the same experiment; data represents mean ± SEM (n=5) and asterisk indicates a significant difference from control, as assessed by one-way ANOVA plus Bonferroni post hoc test (*p<0.05), (**p<0.01).

Figure 4

Effect of CdCl₂ or two bread based diets on expression pattern of ER-α and target genes in 21 day old male ERE-Luc mice. Data show relative levels of ER-α, PR and PTMA mRNAs in brain, kidney, thymus, WAT and prostate. The expression is shown relative to the ribosomal 18S RNA. data represents mean ± SEM (n=5) and asterisk indicates a significant difference from control, as assessed by one-way ANOVA plus Bonferroni post hoc test (*p<0.05).