High-fat or ethinyl-oestradiol intake during pregnancy increases mammary cancer risk in several generations of offspring

Abstract

Maternal exposures to environmental factors during pregnancy influence the risk of many chronic adult-onset diseases in the offspring. Here we investigate whether feeding pregnant rats a high-fat (HF)- or ethinyl-oestradiol (EE2)-supplemented diet affects carcinogen-induced mammary cancer risk in daughters, granddaughters and great-granddaughters. We show that mammary tumourigenesis is higher in daughters and granddaughters of HF rat dams and in daughters and great-granddaughters of EE2 rat dams. Outcross experiments suggest that the increase in mammary cancer risk is transmitted to HF granddaughters equally through the female or male germ lines, but it is only transmitted to EE2 granddaughters through the female germ line. The effects of maternal EE2 exposure on offspring's mammary cancer risk are associated with changes in the DNA methylation machinery and methylation patterns in mammary tissue of all three EE2 generations. We conclude that dietary and oestrogenic exposures in pregnancy increase breast cancer risk in multiple generations of offspring, possibly through epigenetic means.

Figure 1. Multigenerational effect of maternal HF diet.

(a,c,e) Mammary tumour incidence (%) in F1 (control: n=12, HF: n=21), F2 (control: n=22, HF: n=15) and F3 (control: n=18, HF: n=19) generation female offspring of Sprague–Dawley rat mothers (F0) fed a HF diet or a control diet during gestation. (b,d,f) Mammary tumour multiplicity (mean±s.e.m.) in F1 (control: n=7, HF: n=18), F2 (control: n=11, HF: n=12) and F3 (control: n=12, HF: n=10) generation female offspring of Sprague–Dawley rat mothers (F0) fed a HF or a control diet during gestation. (g,h) Mammary tumour incidence (control: n=22, HF×control: n=13, control×HF: n=22) and tumour multiplicity (control: n=11, HF×control: n=9, control×HF: n=15) in F2 generation female (HF×Con) and male (Con×HF) outcrosses. Significant differences versus the control group were determined as follows: log-rank test (tumour incidence), t-test (tumour multiplicty) and one-way ANOVA followed by Dunn's post-hoc test (outcross groups tumour multiplicity). P<0.05 is considered significant; exact P-values are shown in each plot.

Figure 2. Transgenerational effect of maternal EE2-supplemented diet.

(a,c,e) Mammary tumour incidence (%) in F1 (control: n=27, EE2: n=17), F2 (control: n=24, EE2: n=22) and F3 (control: n=23, EE2: n=25) generation female offspring of Sprague–Dawley rat mothers (F0) fed a EE2-supplemented diet or a control diet during gestation. (b,d,f) Mammary tumour multiplicity (mean±s.e.m.) in F1 (control: n=15, EE2: n=14), F2 (control: n=13, EE2: n=11) and F3 (control: n=14, EE2: n=21) generation female offspring of Sprague–Dawley rat mothers (F0) fed EE2 or a control diet during gestation. (g,h) Mammary tumour incidence (control: n=24, EE2×control: n=16, control×EE2: n=12) and tumour multiplicity (control: n=13, HF×control: n=10, control×HF: n=4) in F2 generation female outcross (EE2×Con) and male (Con×EE2) outcrosses. Significant differences versus the control group were determined as follows: log-rank test (tumour incidence), t-test (tumour multiplicty) and one-way ANOVA followed by Dunn's post-hoc test (outcross groups tumour multiplicity). P<0.05 is considered significant; exact P-values are shown in each plot.

Figure 3. Mammary gland morphology in HF or EE2 offspring.

(a) Histological depiction of the fourth abdominal rat mammary gland on PND 21 and (b) terminal end buds (TEBs; indicated by arrows). (c) Number of TEBs on PND21 in F1 (control, HF: n=5), F2 (control: n=6, HF: n=5; outcrosses: n=6) and F3 (control, HF: n=5) generation female offspring of Sprague–Dawley rat mothers (F0) fed HF or a control diet during gestation. (d) Number of TEBs on PND50 in F1 (control, HF: n=5), F2 (control: n=6, HF: n=5, outcrosses: n=5) and F3 (control: n=6, HF: n=5) generation female offspring of Sprague–Dawley rat mothers (F0) fed HF or a control diet during gestation. (e) Number of TEBs on PND21 in F1 (control: n=5, EE2: n=6), F2 (control: n=6, EE2: n=5; outcrosses: n=5) and F3 (control, EE2: n=6) generation female offspring of Sprague–Dawley rat mothers (F0) fed EE2 or a control diet during gestation. (f) Number of TEBs on PND50 in F1 (control, EE2: n=6), F2 (control, EE2: n=5; outcrosses: n=6) and F3 (control: n=5, EE2: n=6) generation female offspring of Sprague–Dawley rat mothers (F0) fed EE2 or a control diet during gestation. All values are expressed as the mean±s.e.m. Significant differences versus the control group were determined as follows: t-test (HF, EE2) and one-way ANOVA (outcross groups) followed by Holm-Sidak post-hoc test. P<0.05 is considered significant; exact P-values are shown in each plot. LN, lymph node; scale bars, 3 mm (a) and 0.5 mm (b).

Figure 4. Effects of maternal HF or EE2 diet on methyltransferases expression.

(a–c) Dnmt1, Dnmt3a and Dnmt3b mRNA expression on PND50 in rat mammary glands of F1 (control: n=6, HF: n=5), F2 (control: n=5, HF: n=4) and F3 (control: n=5, HF: n=3) generation female offspring of Sprague–Dawley rat dams (F0) fed HF or control (Con) diet during gestation. (d–f) Dnmt1, Dnmt3a and Dnmt3b mRNA expression on PND50 in rat mammary glands of F1 (control, EE2: n=6), F2 (control: n=5, EE2: n=6) and F3 (control, EE2: n=5) generation female offspring of Sprague–Dawley rat dams (F0) fed EE2 or control diet during gestation. Quantitative real-time PCR (qPCR) and absolute quantification method was used. Dnmt1, Dnmt3a and Dnmt3b expression of each sample was normalized to rRNA 18S expression. All data are mean±s.e.m. of two independent experiments performed in triplicates. Significant differences versus the control group were determined by two-way ANOVA (after log-transform) followed by Holm-Sidak post-hoc test. P<0.05 is considered significant; exact P-values are shown in each plot.

Figure 5. Differential promoter methylation in mammary glands of EE2 offspring.

(a) Heat maps of differentially methylated gene promoter regions in EE2 offspring (green, n=3) compared with controls (red, n=3). Hypermethylation and hypomethylation are presented separately. MBDCap-seq method was used to access DNA methylation levels. (b) Promoter methylation levels of the polycomb target genes (PcTGs) Foxe3, Gata4, Pax6, Runx3 and Vgf on PND50 rat mammary glands of F1–F3 generation female offspring of Sprague–Dawley rat dams (F0) fed EE2 or control diet during gestation. Data are presented as relative intensity of methylation (mean±s.e.m., n=3 per group), ISIMA statistical approach (see Methods section for details). P<0.05 is considered significant; exact P-values are shown in each plot.