Maternal nutrient supplementation counteracts bisphenol A-induced DNA hypomethylation in early development

Abstract

The hypothesis of fetal origins of adult disease posits that early developmental exposures involve epigenetic modifications, such as DNA methylation, that influence adult disease susceptibility. In utero or neonatal exposure to bisphenol A (BPA), a high-production-volume chemical used in the manufacture of polycarbonate plastic, is associated with higher body weight, increased breast and prostate cancer, and altered reproductive function. This study shows that maternal exposure to this endocrine-active compound shifted the coat color distribution of viable yellow agouti (Avy) mouse offspring toward yellow by decreasing CpG (cytosine-guanine dinucleotide) methylation in an intracisternal A particle retrotransposon upstream of the Agouti gene. CpG methylation also was decreased at another metastable locus, the CDK5 activator-binding protein (CabpIAP). DNA methylation at the Avy locus was similar in tissues from the three germ layers, providing evidence that epigenetic patterning during early stem cell development is sensitive to BPA exposure. Moreover, maternal dietary supplementation, with either methyl donors like folic acid or the phytoestrogen genistein, negated the DNA hypomethylating effect of BPA. Thus, we present compelling evidence that early developmental exposure to BPA can change offspring phenotype by stably altering the epigenome, an effect that can be counteracted by maternal dietary supplements.

Fig. 1.

Maternal BPA exposure shifts offspring coat color distribution toward yellow. (A) Genetically identical A^vy/a offspring representing the five coat color phenotypes. (B) Coat color distribution of A^vy/a offspring born to 16 control (n = 60) and 17 BPA-exposed (n = 73) litters (50-mg BPA/kg diet).

Fig. 2.

Maternal BPA exposure reduces DNA methylation at nine CpG sites within the A^vy IAP. (A) The A^vy allele contains a contra-oriented IAP insertion within pseudoexon 1A (PS1A) of the Agouti gene. A cryptic promoter (short arrowhead labeled “A^vy ectopic”) drives constitutive ectopic Agouti expression. Transcription of the Agouti gene normally initiates from a developmentally regulated hair cycle-specific promoter in exon 2 (short arrowhead labeled “A, a wild type”). (B) The IAP sequence containing nine CpG sites (gray boxes) is located between the cryptic Agouti promoter and the IAP promoter and the downstream 3′ genomic sequence. Bold text represents IAP sequence, and nonbold text represents genomic sequence. The location of the bisulfite-converted genomic reverse primer for amplifying the A^vy IAP is underlined. (C) Box plots representing the percentage of cells methylated at CpG sites 1–9 in control (n = 60) and BPA-exposed (n = 73) A^vy/a offspring (diet group t test; ##, P < 0.01; *, P < 0.05). (D) Average methylation across CpG sites 1–9 in d22 tissues derived from ectodermal (brain and tail), mesodermal (kidney), and endodermal (liver) tissues from BPA-exposed A^vy/a offspring (n = 10) representing the five coat color phenotypes is highly correlated (Pearson's r > 0.9 and P < 0.05 for each correlation). T, tail; B, brain; K, kidney; L, liver.

Fig. 3.

Maternal BPA exposure decreases offspring methylation at the Cabp^IAP metastable epiallele. (A) The Cabp^IAP metastable epiallele (17) contains a contra-oriented IAP insertion within intron 6 of the murine CDK5 activator-binding protein (Cabp) gene, resulting in short aberrant transcripts originating from the 5′ LTR of the IAP (short arrowhead labeled “Cabp AT1a,b”). Short aberrant transcripts also originate at the normal transcription start site (short arrowhead labeled “Cabp wild type”) because of premature truncation upstream of the IAP insert (Cabp AT2 and AT3). Normal Cabp transcription covers 14 exons, resulting in a 2-kb transcript. (B) The IAP sequence containing nine CpG sites located between the cryptic Cabp promoter and the IAP promoter (bold text) and the downstream 3′ genomic sequence (nonbold text). The location of the bisulfite-converted genomic reverse primer for amplifying the 5′ Cabp^IAP locus is underlined. (C) Box plots representing the percentage of cells methylated at CpG sites 6–9 in control (n = 39) and BPA-exposed (n = 39) A^vy/a offspring (diet group t test; ##, P < 0.01).

Fig. 4.

Maternal nutritional supplementation counteracts BPA-induced DNA hypomethylation and the shift in coat color distribution to yellow. (A) Coat color distribution of A^vy/a offspring born to 14 BPA-exposed/methyl donor-supplemented mothers (n = 54), 16 control mothers (n = 60), and 17 BPA-exposed mothers (n = 73). (B) Coat color distribution of A^vy/a offspring born to 13 BPA-exposed/genistein-supplemented mothers (n = 39), 16 control mothers (n = 60), and 17 BPA-exposed mothers (n = 73). (C) Box plots representing the percentage of cells methylated at CpG sites 1–9 in BPA-exposed/methyl donor-supplemented (n = 54) and control (n = 60) offspring (P = 0.25). (D) Box plots representing the percentage of cells methylated at CpG sites 1–9 in BPA-exposed/genistein-supplemented (n = 39) and control (n = 60) offspring (P = 0.46).