Epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice

Abstract

Background: Developmental effects of exposure to endocrine disruptors can influence adult characters in mammals, but could also have evolutionary consequences. The aim of this study was to simulate an environmental exposure of an experimental population of mice to high amounts of nutritional phytoestrogens and to evaluate parameters of relevance for evolutionary change in the offspring. The effect of a continuous pre- and post-natal exposure to high levels of dietary isoflavones was evaluated on sexual maturity, morphometric parameters and DNA methylation status in mice. Adult mice male/female couples were fed ad libitum either with control diet (standard laboratory chow) or ISF diet (control diet plus a soy isoflavone extract at 2% (w/w) that contained the phytoestrogens genistein and daidzein). In the offspring we measured: i) the onset of vaginal opening (sexual maturation) in females, ii) weight and size in all pups at 7, 14, 21 and 42 days post-natal (dpn) and iii) DNA methylation patterns in skeletal alpha-actin (Acta1), estrogen receptor-alpha and c-fos in adults (42 dpn).

Results: Vaginal opening was advanced in female pups in the ISF group, from 31.6 +/- 0.75 dpn to 25.7 +/- 0.48. No differences in size or weight at ages 7, 14 or 21 dpn were detected between experimental groups. Nevertheless, at age 42 dpn reduced size and weight were observed in ISF pups, in addition to suppression of normal gender differences in weight seen in the control group (males heavier that females). Also, natural differences seen in DNA methylation at Acta1 promoter in the offspring originated in the control group were suppressed in the ISF group. Acta1 is known to be developmentally regulated and related to morphomotric features.

Conclusion: This study demonstrates in mammals that individuals from a population subjected to a high consumption of isoflavones can show alterations in characters that may be of importance from an evolutionary perspective, such as epigenetic and morphometric characters or sexual maturation, a life history character.

Figure 1

(a) scheme shows the distribution of CpG sites in the region analyzed for methylation changes in the promoter of Acta1 and their distance to the starting of transcription, describing also the sequence analyzed. (b) and (c) correspond to sequencing of PCR products amplified from bisulphite treated DNA that includes the 8 CpG sites analyzed, which are indicated with arrows. (b) shows electrophenograms with direct sequencing data and (c) shows raw data electrophenograms from which CpG DNA methylation was quantified; shown sequences were obtained with the reverse primer; CpG sites in the forward strand appear in the reverse strand as CG when methylated and as CA when unmethylated.

Figure 2

Scheme shows the distribution of CpG sites in the region analyzed for methylation changes in the promoter of ERα, the distance to the starting of transcription of the first translated exon and the sequence analyzed; the region includes 6 CpG sites.

Figure 3

Timing of female mice sexual maturation observed in the offspring of a population of mice subjected to ISF or control diet, expressed as (a) distribution of occurrence of vaginal opening along days after birth and as (b) mean day of occurrence of vaginal opening after birth ± SE ± 1,96*SE (P < 0.001).

Figure 4

Comparison of morphometric parameters between pups 42 days old. Variation in adult weight and size observed in the offspring of a population of mice subjected to ISF or control diet is shown. (a) indicates the general trend of decreased size (P = 0.03, *) and weight (P = 0.06, #) in the ISF group with regard to controls, when pooling males and females. Size differences in (a) are explained by variations in both sexes, as shown in (b). Weight differences in (a) are accounted for by variations only in males, which are heavier in the control group than in the ISF group (P = 0.04, ‡), as can be seen in (c). Also, in the control group males are heavier that females (P < 0.001; c, right, ±), difference that is suppressed in the ISF group (P = 0.3; c, left).

Figure 5

Methylation profiles of the analyzed region of the Acta1 promoter in liver from offspring generated in the control or ISF group. No differences in DNA methylation in liver were detected among treatments when comparisons were performed pooling male and female data (P = 0.3), as shown in (a). Nevertheless, gender methylation differences seen in (b), the controls (P = 0.0006), are suppressed in (c), the ISF group (P = 0.4). Significant changes in individual CpG sites are shown with *, P values are indicated in the text.

Figure 6

Methylation profiles of the analyzed region of the Acta1 promoter in pancreas from offspring generated in the control or ISF group. DNA methylation gender differences are not observed in the population maintained on (a), control diet (P = 0.93), or on (b) ISF diet (P = 0.837). Nevertheless, pooling male and female data together shows a nearly significant difference due to the ISF treatment (P = 0.08), in which CpG site specific changes were detected in sites 5 (P = 0.004) and 8 (P = 0.045), as seen in (c), indicated with *. Using only control animals (both males and females) we found that methylation is increased in liver regarding to pancreas (P = 0.007), which shows the occurrence of tissue specific differences in DNA methylation for Acta1, as seen in (d).

Figure 7

Methylation profiles of the analyzed region of the ERα promoter in liver from offspring generated in the control or ISF group. No treatment effects were detected within males, within females or pooling male and female data, as seen in (a), (b) and (c).

Figure 8

Scheme showing two hypothetical population structures subjected to different consumption of dietary isoflavones.

Figure 9

Scheme of a proposed mechanism of action through which endocrine disruptors could alter methylation patterns acting from the mother to the embryo.