Coupling of Random Amplified Polymorphic DNA Profiles Analysis and High Resolution Capillary Electrophoresis System for the Assessment of Chemical Genotoxicity

Cadmium (Cd) can be toxic to terrestrial snails, but few data are available about its genotoxic effects on early life stages (ELS). The aim of this study was to investigate the genotoxic potential of Cd in embryos of Helix aspersa using a new approach that couples Random Amplified Polymorphic DNA (RAPD) and a high-resolution capillary electrophoresis system (HRS). Clutches of H. aspersa were exposed to Cd solutions (2, 4, and 6 mg/L) from the beginning of their embryonic development. In addition to a dose-dependent effect of Cd on hatching rate, DNA fragmentation was observed in embryos that were exposed to 6 mg Cd/L. The analysis of RAPD products with HRS showed differences between the profiles of exposed and nonexposed embryos, starting at 2 mg Cd/L. In comparison to the profiles of the control samples, all profiles from the exposed snails exhibited an additional 270 bp DNA fragment and lacked a 450 bp DNA fragment. These profile modifications are related to the genotoxic effect of Cd on the ELS of H. aspersa. Our study demonstrates the efficacy of coupling RAPD and HRS for a rapid and efficient screening of the effects of chemicals on DNA

Geographical localization of the 16 house sparrow populations sampled in this study.

<p>I - Paris (Jardin des Plantes), II - Cachan, III - Wissous, IV - Cosne-Cours sur Loire, V - Rully, 6 - Paris (Cité internationale universitaire), 7 - Gennevilliers, 8 - Le Mans, 9 - Crégy les Meaux, 10 - Berck, 11 - Thieux, 12 - Seninghem, 13 - Ceffonds, 14 - Crennes, 15 - Réaup-Lisse and 16 - Arles. The five populations in roman numbers were sampled for parasite prevalence and intensity. The zoomed region, in the upper right corner, corresponds to the region Ile de France. Scale bar, 100 km for France and 30 km for the region Ile de France.</p

(a) Negative correlation between Pb concentration in feathers and the axis 1 of the principal component analysis on environmental variables (see materials and methods for more details).

<p>(b) Positive correlation between Cd concentration in feathers and the axis 3 of the principal component analysis. Each point represents a house sparrow population.</p

Pearson’s correlation coefficient matrix among the environmental variables used to characterize 16 house sparrow populations in France. * and ** indicates P values ≤0.05 and ≤0.01, respectively.

<p>Pearson’s correlation coefficient matrix among the environmental variables used to characterize 16 house sparrow populations in France. * and ** indicates P values ≤0.05 and ≤0.01, respectively.</p

Study site localisation, Cd contamination (µg/g DM) in soils and landscape types.

<p>Study site located around the former Metaleurop Nord smelter (Nord-Pas de Calais, France); maps present the landscape types in each square and the Cd concentrations in the study area soils. Selected squares for wood mice sampling are bolded in both maps.</p

Pollution level, median Cd and Pb soil concentrations (µg/g DM), and the number of sampling squares for each landscape type studied.

<p>Pollution level, median Cd and Pb soil concentrations (µg/g DM), and the number of sampling squares for each landscape type studied.</p

Model outputs presenting partial R_LR² and <i>p</i>-values in brackets (F-statistics) for each variable and for the entire model.

<p>Models were constructed as follows: Index ∼ age+gender+[TM]soil+landscape+[TM]organ+age:gender+age:[TM]organ+gender:[TM]organ+[TM]soil:[TM]organ+landscape:[TM]organ. Because landscape and two-way interactions between [TMs] in organs and age or gender were not significantly related to the studied indices in all tested models (LRT, p>0.050), these variables were not included in the table. When a two-way interaction was significant, all terms (even not significant ones) included in this interaction are presented in the table.</p

Predicted relationships between indices and metal concentrations in organs (µg/g DM).

<p>Predictions between indices and TM concentrations are performed on wood mice from the former Metaleurop Nord smelter; (a) between SMI and [Cd]_liver; (b) between SMI and [Cd]_kidneys; (c) between SLI and [Cd]_liver; (d) between SLI and [Cd]_kidneys; (e) between SLI and [Pb]_liver; and (f) between SKI and [Cd]_kidneys. For all figures, the age of individuals was fixed (7 mg of crystalline lens mass, which corresponds to adulthood). For graphical representation, the “highly polluted” class was subdivided into two classes of contamination, “highly polluted” (10≤ [Cd]_soil ≥20 µg/g DM) and “extremely polluted” (20≤ [Cd]_soil ≥70 µg/g DM) in figures (a) to (d). For figures (a) and (b), predicted relationships are shown only for males. For figures (a) to (d), relationships between indices and TM concentrations are represented at different levels of soil contamination (lightly, moderately, highly and extremely polluted). Because interactions between concentrations in organs and soil were not significant in figures (e) and (f), predicted relationships are drawn for all contaminations levels.</p

For each soil contamination level, concentrations of Cd and Pb (minimum, mean, and maximum values, µg/g DM) in the liver and kidneys and number and percentage (in brackets) of wood mice at risk for metal-induced stress from the surroundings of the former Metaleurop Nord smelter.

<p>Individuals are considered at risk for metal-induced stress when TM concentrations were above the thresholds defined by Shore and Douben <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066399#pone.0066399-Shore1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066399#pone.0066399-Shore2" target="_blank">[23]</a>.</p

Scheme of exposure and sampling conditions during embryonic development of control and Cd-exposed eggs of <i>Cantareus aspersus</i> applied to the present study.

<p>(A) Illustration of developmental stages of snail eggs with specification of the albumen (A) and the embryo (E) (above), followed by details of exposure regimes and sampling dates applied for (I) the assessment of Cd concentrations in snail embryos, (II) RT and qRT-PCR detection of embryonic <i>MT</i> isoform genes, and (III) evaluation of successful hatching rates during development along the time axis (below), starting immediately after egg laying. n is the number of clutches used for each exposure regime. (B) Exposure scheme of eggs from each clutch (left-hand) treated with control solution (C) or with increasing nominal concentrations (5, 10 and 15 mg/L) of Cd; also shown are the numbers of eggs pooled (right-hand) to one sample at each of the days selected during the development.</p