Sexually immature male ERE-Luc reporter mice to assess low dose estrogen-like effects of CdCI 2 versus dietary CD

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 per kg Cd concentrations respectively) and CdCl2 per-oral dose of 1 ug per kg per bw per day. Total exposure of ingested and per cent bioaccumulation of Cd in selected organs were estimated as 547 ng (4.4 per cent), 776 ng (0.3 per cent) and 2131.8 ng (0.1 per cent) 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 anatomopathological 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. (Authors abstract)

Emerging evidences show that, Cd is clearly a potent non-steroidal estrogen in vivo and in vitro due to its ability to bind with high affinity to estrogen receptor alpha (ER alpha), independently of estrogen binding and to activate this receptor. Cd is present in virtually all foods at variable concentrations, depending on the type of commodity and the extent of environmental contamination. Bread, accounting for 36 per cent of the weekly intake, has been identified as the biggest single source of Cd in diet in Sweden. Cd absorption after dietary exposure in adults is relatively low (5 to 10 per cent), but may be higher in infants. Females are reported to retain higher liver and kidney concentrations than males, males are less vulnerable to Cd related bone effects compared to females, but are much prone to Cd related reproductive damage affecting semen quality, testis and epididymis.

Oral and chronic Cd intake was shown to induce neoplastic and proliferative lesions in adrenal, kidney, prostate, pituitary and testis of experimental animals. Cd was reported to produce neoplasm in the prostate of rats or changes in sperm counts in a dose dependent fashion at a low dose exposure that is far below the threshold for significant testicular toxicity. Low dose effects occur at a range of human exposure and at a range below the no adverse effect level (NOAEL) where, various irreversible effects are reported.

A methodology to screen even subtle effects during long term exposure of low doses of contaminants in its inherent form, at physiological level is lacking. Luc reporter mouse was generated for the rapid, quantitative and systemic analysis of the ER activity in living mice in the presence or absence of exogenous estrogenic stimuli. Using sexually immature male ERE Luc reporter mice this study generated a non invasive bioluminescent in vivo imaging methodology to measure the changes in estrogenic activity in response to treatment for a period of 21 days. It also investigated the EL effects of two cereal based diets which are known to concentrate environmental Cd and compared its effects with CdCl2. The present methodology was generated to investigate the effect of 21 day administration of Cd, as CdCl2 or as an inherent bread contaminant, on ER signalling via ERE containing promoters. The results demonstrate that a cereal based diet or CdCl2 may affect ER signalling in brain, prostate and WAT, in sexually developing immature male mice. We also show that the observed Cd related effects occur only at the molecular level and not at the anatomo-pathological level, due to various treatments.

The study of luciferase accumulation in prostate indicates that only flax bread diet induced significant changes, however, the quantitative measurement of ER alpha mRNA showed that CdCl2 and dietary Cd have a strong influence on the accumulation of this receptor in this organ. PR mRNA was significantly modulated by all the treatments. Previous reports have indicated an involvement of this heavy metal in the development of prostate cancer. Altogether, WAT and prostate were identified as targets of Cd in sexually immature male mice and the rest of the observed EL effects are not consistent to a particular organ or treatment. In conclusion, this study undertakes to analyse the Cd related EL effects and shows a imaging-based methodology to investigate the effect of extremely low concentrations of Cd embedded in food matrix and emphasizes the novel possibility to carry out long term exposure oriented studies of suspected chemicals in their inherent presence in the food chain or environment. The methodology further exemplifies the power of reporter systems-imaging technology offering a surrogate end point in a physiological set up and pro-sensitivity to acquire a global view of potential organs affected by the compound of interest.(Editors comments)