Effects on specific promoter DNA methylation in zebrafish embryos and larvae following benzo[a]pyrene exposure

Abstract

Benzo[a]pyrene (BaP) is an established carcinogen and reproductive and developmental toxicant. BaP exposure in humans and animals has been linked to infertility and multigenerational health consequences. DNA methylation is the most studied epigenetic mechanism that regulates gene expression, and mapping of methylation patterns has become an important tool for understanding pathologic gene expression events. The goal of this study was to investigate aberrant changes in promoter DNA methylation in zebrafish embryos and larvae following a parental and continued embryonic waterborne BaP exposure. A total of 21 genes known for their role in human diseases were selected to measure percent methylation by multiplex deep sequencing. At 96hpf (hours post fertilization) compared to 3.3hpf, dazl, nqo1, sox3, cyp1b1, and gstp1 had higher methylation percentages while c-fos and cdkn1a had decreased CG methylation. BaP exposure significantly reduced egg production and offspring survival. Moreover, BaP decreased global methylation and altered CG, CHH, and CHG methylation both at 3.3 and 96hpf. CG methylation changed by 10% or more due to BaP in six genes (c-fos, cdkn1a, dazl, nqo1, nrf2, and sox3) at 3.3hpf and in ten genes (c-fos, cyp1b1, dazl, gstp1, mlh1, nqo1, pten, p53, sox2, and sox3) at 96hpf. BaP also induced gene expression of cyp1b1 and gstp1 at 96hpf which were found to be hypermethylated. Further studies are needed to link aberrant CG, CHH, and CHG methylation to heritable epigenetic consequences associated with disease in later life.

Keywords: Benzo[a]pyrene; DNA methylation; Embryo; Larvae; Zebrafish.

Figure 1

BaP effects on parental zebrafish egg production and cumulative survival in offspring. (A) Eggs were collected before and after 42.0 ± 1.9 μg/L (ppb) BaP waterborne exposure to adult zebrafish. The average number of eggs collected per day per tank was calculated after 7 to 11 days of exposure (n = 12 tanks pre-exposure, n = 6 tanks/treatment group during exposure, p<0.05 one-way ANOVA and Neumann Keuls multiple comparison tests). (B) Percent offspring survival was determined at 24, 48, 72, 96 hpf. Sample sizes (n=3 or 4) were 30 embryos/pool with a total of 90–120 fertilized eggs per treatment group; p<0.05, one-way ANOVA followed by Tukey’s post hoc test.

Figure 2

Global DNA methylation effects in the offspring after parental and continued embryonic BaP (42.0 ± 1.9 μg/L) exposure. Three pools of embryos (each pool 200 embryos) or larvae (each pool 30 larvae) were used to extract DNA and determine 5-methylcytosine percentage (n=3, * p<0.05, Student t-test).

Figure 3

Constitutive percent promoter methylation in CG, CHG, and CHH sites of 21 genes at 3.3 and 96 hpf in zebrafish. Data is presented from highest to lowest % CG DNA methylation. Percent CHG and CHH methylation at 3.3 hpf (not shown) was similar to that at 96 hpf.

Figure 4

Site-specific % CG methylation profile at 3.3 hpf in zebrafish embryos. Blue = methylated cytosine, yellow = unmethylated cytosine, and in parenthesis is the mean % CG DNA methylation change for each gene due to BaP exposure.

Figure 5

Site-specific % CG methylation profile at 96 hpf in zebrafish larvae. Blue = methylated cytosine, yellow = unmethylated cytosine, and in parenthesis is the mean % CG DNA methylation change for each gene due to BaP exposure.

Figure 6

Constitutive mRNA expression in 3.3 and 96 hpf zebrafish as measured by qRT/RT-PCR. Fold change of expression was normalized to 18S rRNA expression and relative to cyp1b1 expression at 3.3 hpf (n = 3 pools, 200 or 30 embryos or larvae, respectively/pool; *p<0.05; Student T-test between 3.3 and 96 hpf within each gene).

Figure 7

BaP induced effects on mRNA expression in embryos at 3.3 hpf (A) and in larvae at 96 hpf (B) as measured by qRT/RT-PCR. Fold change was normalized to 18S rRNA expression and relative to controls (n = 2–3 pools, 200 or 30 embryos or larvae, respectively/pool; *p < 0.05).