Embryonic toxicokinetic and dynamic differences underlying strain sensitivity to cadmium during neurulation

Abstract

Differences in sensitivity are observed between mouse strains, C57 (sensitive) and SWV (resistant) when exposed to cadmium (Cd) during the neurulation period. In this study, we investigated the toxicokinetics of Cd in relation with toxicodynamic responses to identify factors affecting differential Cd-sensitivity in C57 and SWV. Using a level of exposure which induced developmental toxicity and differential effects between strains, we assessed maternal and embryonic Cd uptake and evaluated biomarkers of response previously linked with Cd exposure, specifically metal ion regulators (Mt1, Mt2, DMT1) and markers of cell cycle arrest/apoptosis induction (p53, Cdkn1a, c-Casp3). Greater Cd uptake was observed in C57 embryos compared to SWV and these observations of differential uptake were associated with increased alterations in expression of biomarkers of metal response (e.g. c-Casp3) and strain sensitivity. Using sensitive and resistant mouse strains, we have identified toxicokinetic and dynamic differences which underlie observed differences in Cd embryonic sensitivity and response.

Figure 1. Cd uptake in maternal liver of C57 and SWV mice

Cd-exposed and control maternal liver of pregnant dams of C57 and SWV were assessed for total Cd uptake via ICPMS 6, 12, 24 h p.i. (GD8.0). Data are shown as mean ± SEM. ANOVA was conducted to determine significant primary and interactive effects across time, exposure, and strain. Asterisks indicates significant Cd uptake in maternal liver (p<0.001 (**)). Livers of Cd-exposed C57 and SWV showed similar Cd concentrations across time (ANOVA, p>0.05)

Figure 2. Differential Cd uptake in embryos of C57 and SWV mice

Cd-exposed and control litters of embryos of C57 and SWV were assessed for total Cd uptake via ICPMS 6, 12, 24h p.i. (GD8.0). Data are shown as mean ± SEM. Asterisks indicate significant (ANOVA) Cd concentrations in Cd exposed embryos compared to controls (p<0.001 (**)). A significant interaction between Cd uptake, time and strain was identified (p<0.05 (#)), indicating differential Cd uptake between strains at specific timepoints. The largest difference between strains was observed at 6h p.i.

Figure 3. RNA expression of biomarkers of Cd response associated with metal ion regulation (Mt1, Mt2, DMT1)in Cd-treated and control C57 and SWV embryos

Homogenized litters of Cd- exposed and control C57 and SWV embryos were assessed for RNA expression at 12 and 24h p.i. via RT-PCR. Raw intensities were adjusted by Bactin and then normalized to the average C57 12h control value to be able to display comparisons between the three probes. Data are shown as mean ± SEM. An asterisk indicates significant differences between Cd exposed versus control at each respective timepoint (Ttest, p<0.05).

Figure 4. Differential RNA expression of Mt1, Mt2 and DMT1 between C57 and SWV embryos during neurulation (GD8.0 – 10)

We conducted RT-PCR for C57 and SWV embryos over the neurulation period (GD8–10) for Mt1 (A), Mt2 (B), and DMT1 (C). Data are shown as mean ± SEM. For Mt1 and Mt2, we observed higher expression in the SWV across time (ANOVA, p<0.05). In contrast, we observed significantly higher DMT1 expression in C57 versus SWV embryos (ANOVA, p<0.001).

Figure 5. Protein expression of biomarkers of Cd response associated with cell cycle arrest and apoptosis in Cd-treated and control C57 and SWV embryos

Homogenized litters (pooled embryos within one litter) of Cd- exposed and control C57 and SWV embryos (n=3–6) were probed for c-Casp3, total p53 and Cdkn1a, 12 and 24h p.i. via Western Blot. Data are shown as geometric mean ± SEM. Asterisks indicate significant differences between Cd exposed versus control at each respective timepoint (Ttest, p<0.05 (*), p<0.005 (**)). Significant differences in c-Casp3 activation were observed between strains (ANOVA, p<0.05).