Genome-Wide Analysis of Cadmium-Induced, Germline Mutations in a Long-Term Daphnia pulex Mutation-Accumulation Experiment

Abstract

Background: Germline mutations provide the raw material for all evolutionary processes and contribute to the occurrence of spontaneous human diseases and disorders. Yet despite the daily interaction of humans and other organisms with an increasing number of chemicals that are potentially mutagenic, precise measurements of chemically induced changes to the genome-wide rate and spectrum of germline mutation are lacking.

Objectives: A large-scale Daphnia pulex mutation-accumulation experiment was propagated in the presence and absence of an environmentally relevant cadmium concentration to quantify the influence of cadmium on germline mutation rates and spectra.

Results: Cadmium exposure dramatically changed the genome-wide rates and regional spectra of germline mutations. In comparison with those in control conditions, Daphnia exposed to cadmium had a higher overall $A:T\to G:C$ mutation rates and a lower overall $C:G\to G:C$ mutation rate. Daphnia exposed to cadmium had a higher intergenic mutation rate and a lower exonic mutation rate. The higher intergenic mutation rate under cadmium exposure was the result of an elevated intergenic $A:T\to G:C$ rate, whereas the lower exon mutation rate in cadmium was the result of a complete loss of exonic $C:G\to G:C$ mutations-mutations that are known to be enriched at 5-hydroxymethylcytosine. We experimentally show that cadmium exposure significantly reduced 5-hydroxymethylcytosine levels.

Discussion: These results provide evidence that cadmium changes regional mutation rates and can influence regional rates by interfering with an epigenetic process in the Daphnia pulex germline. We further suggest these observed cadmium-induced changes to the Daphnia germline mutation rate may be explained by cadmium's inhibition of zinc-containing domains. The cadmium-induced changes to germline mutation rates and spectra we report provide a comprehensive view of the mutagenic perils of cadmium and give insight into its potential impact on human population health. https://doi.org/10.1289/EHP8932.

Figure 1.

Overview of mutation-accumulation (MA) experiment. A single Daphnia pulex genotype from Buck Lake, Dorset, Ontario, Canada, was the progenitor of the sublines for both the control conditions and Cd exposure. “S” refers to individual sublines. Circles containing a vertical line represent the germline genome. Horizontal red dashes in the “germline genome” represent mutations. Whole-genome sequencing with Illumina was undertaken at “ ${\text{Generation}}_{\mathrm{x}}$” for all sublines (Table 1).

Figure 2.

(A, B) Comparison of the proportion of total mutations in each genome region to the random expectation (i.e., if mutations were randomly distributed throughout the genome). Gray dashed lines represent the proportion of total mutations in each region that are expected if mutations are randomly distributed across the genome. Solid lines represent observed mutation proportions in each genome region for control conditions (A) and Cd exposure (B). *** corresponds to $p<5.0\times {10}^{-4}$, Exact Binomial Test. (C) Comparison of the proportion of total mutations in each genome region between control conditions (solid lines) and Cd exposure (dashed lines). Arrows (2C) indicate the direction regional mutation rate were significantly different between Cd exposure and control conditions. * Corresponds to $p<5.0\times {10}^{-2}$; ** corresponds to $p$ < $5.0\times {10}^{-3}$; Fisher’s Exact Test. Summary data, including the expected and observed proportions, for the mutation rates can be found in Table S2 (control and Cd). Actual $p$-values for control vs. Cd can be found in Table S4.

Figure 3.

(A) Genome-wide conditional mutation rates of control conditions and Cd exposure. For control (solid black boxes) and Cd exposure (white boxes), the rates of each of the six base-substitution classes are plotted. Error bars are included (SE; gray). Significant differences between control and Cd are denoted by asterisks. The data for 3A are located in Tables S5–S6. Values represent 12 (control) and 9 (Cd) sublines (B) conditional base-substitution rates of intergenic and genic regions. The conditional mutation rates are plotted independently for intergenic and genic regions for the six mutation classes. Solid boxes represent control condition results, and white boxes represent Cd exposure results. The data for 3B are in Table S7 (C) Mutation cluster analysis. The proportion of total mutations for each mutational class are plotted for control conditions and Cd exposure. The data for 3C are in Table S10. (D) Heat map of $p$-values from Fisher’s exact test for all possible mutation contexts. The Fisher’s exact test for Cd exposure is in Table S8, and the results for control conditions are in Table S11. $p$-Values are plotted for control conditions and Cd exposure, independently. The z-axis is the premutation nucleotide. The y-axis is the nucleotide that is 5′ adjacent to the mutation site. The x-axis is the nucleotide that is 3′ adjacent to the mutation site. Note: Cd, cadmium.

Figure 4.

(A) Measurement of 5-hydroxymethylation levels in control conditions and Cd exposure. The mean and corresponding SEM are plotted. The data plotted correspond to three to four replicates for each experimental condition. The measurements used to generate Figure 4A are provided in Table S9. (B) Proposed mechanism for lower $\mathrm{C}:\mathrm{G}\to \mathrm{G}:\mathrm{C}$ mutation rate in Cd exposure. $5-\text{mC}=5-\text{methylcytosine}$. $5-\text{hmC}=5-\text{hydroxymethylcytosine}$. “TET” is TET protein, which converts 5–mC to5–hmC. Note: Cd, cadmium; SEM, standard error of the mean.