Cadmium nitrate and DNA methylation in gastropods: comparison between ovotestis and hepatopancreas.

Dietary ingestion is the main route of exposure to hazardous contaminants in land animals. Cadmium, a high-profile toxic metal, affects living systems at different organismal levels, including major storage organs (liver, kidneys), key organs for species survival (gonads), and epigenetic networks regulating gene expression. 5-methylcytosine (5mC) is the most common and best-characterized epigenetic mark among different modified nucleosides in DNA. This important player in methylation-driven gene expression is impacted by cadmium in sentinel terrestrial vertebrates. However, limited information exists regarding its impact on macroinvertebrates, especially land snails commonly used as (eco)toxicological models. We first investigate the methylomic effects of dietary cadmium given as cadmium nitrate on terrestrial mollusks. Mature specimens of the common brown garden snail, Cornu aspersum, were continuously exposed for four weeks to environmentally-relevant cadmium levels. We determined global genomic DNA methylation in hepatopancreas and ovotestis, as well as changes in the methylation status of CG pairs at the 5' region close to the transcription site of gene encoding the Cd-selective metallothionein (Cd-MT). Weight gain/loss, hypometabolism tendency, and survival rates were also assessed. Although this exposure event did not adversely affect survival, gastropods exposed to the highest Cd dose revealed a significant reduction in body weight and a significant increase in hypometabolic behavior. The hepatopancreas, but not the ovotestis, displayed significant hypermethylation, but only for the aforementioned specimens. We also found that the 5' end of the Cd-MT gene was unmethylated in both organs and its methylation status was insensitive to cadmium exposure. Our results are important since they provide scientists, for the first time, with quantitative data on DNA methylation in gastropod ovotestis and refine our understanding of Cd epigenetic effects on terrestrial mollusks.

Short-term effects of very low dose cadmium feeding on copper, manganese and iron homeostasis: A gastropod perspective.

The available information on the interplay between low-dose cadmium intake and copper, manganese, and iron homeostasis in invertebrates is limited. We have currently studied the accumulation of these trace metals in the hepatopancreas of adult snails, Cantareus aspersus, following 14 and 28 days of exposure to low doses of dietary cadmium, up to 1 mg/kg dw (dry weight). The cadmium dose, but not the duration of exposure, had a significant effect on hepatopancreas copper deposition, the values being significantly elevated compared to controls. A significant peak in manganese levels at 14 days was found in snails administered the lowest cadmium dose. These increases occurred even in the absence of cadmium increase in the hepatopancreas. Our data suggest that low dose cadmium feeding can produce a transient disturbance in hepatopancreas copper and manganese homeostasis. Such responses may serve as early biomarkers of physiological changes occurring during the initial stages of cadmium intoxication.

High-level dietary cadmium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas.

5-methylcytosine (5mC) is a key epigenetic mark which influences gene expression and phenotype. In vertebrates, this epigenetic mark is sensitive to Cd exposure, but there is no information linking such an event with changes in global 5mC levels in terrestrial gastropods despite their importance as excellentecotoxicological bioindicators of metal contamination. Therefore, we first evaluated total 5mC content in DNA of the hepatopancreas of adult Cantareus aspersus with the aim to determine whether this epigenetic mark is responsive to Cd exposure. The experiment was conducted under laboratory conditions and involved a continuous exposure, multiple dose- and time-point (14, 28, and 56 days) study design. Hepatopancreas cadmium levels were measured using Flame Atomic Absorption Spectrometry and the percentage of 5-mC in samples using an ELISA-based colorimetric assay. Snail death rates were also assessed. Our results, for the first time, reveal the presence of 5mC in C. aspersus and provide evidence for Cd-induced changes in global 5mC levels in DNA of gastropods and mollusks. Although less sensitive than tissue accumulation, DNA methylation levels responded in a dose- and time-dependent manner to dietary cadmium, with exposure dose having a much stronger effect than exposure duration. An obvious trend of increasing 5mC levels was observed starting at 28 days of exposure to the second highest dose and this trend persisted at the two highest treatments for close to one month, when the experiment was terminated after 56 days. Moreover, a strong association was identified between Cd concentrations in the hepatopancreas and DNA methylation levels in this organ. These data indicate an overall trend towards DNA hypermethylation with elevated Cd exposure. No consistent lethal effect was observed, irrespective of time point and Cd-dosage. Overall, our findings suggest that the total 5mC content in DNA of the hepatopancreas of land snails is responsive to sublethal Cd exposure and give new insights into invertebrate environmental epigenetics.