Findings obtained in this study indicate that maternal exposure to HF or EE2 during pregnancy increases mammary cancer risk in multiple generations of offspring. We observed that maternal HF diet increases mammary cancer risk in the daughters and granddaughters. Maternal EE2 exposure increased the risk in three consecutive generations (daughters, granddaughters and great-granddaughters), indicating that the effects are transgenerational for the maternal EE2 exposure, but multigenerational for the maternal HF exposure.
Our outcross experiments suggested that increased breast cancer risk in granddaughters can be equally transmitted through in utero HF-exposed fathers or mothers, but this effect was not transgenerational as the F3 generation (great-granddaughters) did not exhibit an increase in mammary cancer risk. A different pattern of inheritance in offspring of dams exposed to EE2 during pregnancy was revealed by outcross experiments: breast cancer risk in granddaughters was determined by whether their mother (increased risk) or father (reduced risk) had been exposed in utero to EE2. These opposing changes may explain why no difference in mammary cancer risk was seen in the F2 generation offspring when both parents had been exposed to EE2 in utero.
The breast cancer risk-lowering effect on F1 EE2 male germ line, however, was transient and not transgenerational, because F3 generation females, produced by mating F2 EE2 females with F2 EE2 males, exhibited increased mammary tumour susceptibility. Our findings indicating a difference in mediation of effects of EE2 exposure through female and male germ lines are in agreement with other reports showing that certain in utero
exposures have opposite outcomes if transmitted through the male or female germ line. For instance, prenatal under-nutrition leads to reduced birth weights in the second generation offspring of exposed males, but to overweight in the offspring of exposed females35
Differences in inheritance through the male and female germ lines in the F2 EE2 generation likely reflect the fact that, in females, germ cells mature before birth, whereas in males these cells are only produced after puberty onset36
. In both genders, primordial germ cells enter genital ridges on gestation day 10.5. By gestation day 12.5, the cells enter a premeiotic stage in mice37
and undergo rapid DNA demethylation and sex-specific de novo
. In male mice, the primordial cells then undergo mitotic arrest until they start proliferating again at puberty. Premeiotic cells in the female genital ridge, in contrast, enter meiotic prophase as oocytes and reach full maturity as germ cells at the time of birth39
. Two recent studies show that transmission of DNA methylation occurs mainly through maternal gametes40
Global DNA demethylation, followed by cell- and gender-specific remethylation, occurs twice during fetal development: first in the fertilized egg and then in primordial germ cells when they reach genital ridge. Thus, the multigenerational and transgenerational effects on mammary tumourigenesis of the maternal HF and EE2-supplemented diets, respectively, may be caused by the different durations of these in utero
exposures. The HF diet was fed to pregnant dams before conception and throughout pregnancy, whereas the EE2-supplemented diet was fed from day 14 to 20 of pregnancy, as an earlier EE2 exposure would disrupt pregnancy. It is possible that in order for breast cancer risk to be transmitted in a transgenerational manner, the exposures need to occur within a certain window of development, as shown for other diseases22
Mammary gland levels of Dnmt1
mRNA were increased in all three EE2 generations, but only in F1 HF generation. Our results are in agreement with other studies showing that in utero
exposures to endocrine disruptors alter the expression of DNA methyltransferases in adult target tissues19
. In addition, our results suggest that an increase in mammary cancer risk following maternal exposure to EE2 during pregnancy (F0 dams) may involve an increase in Dnmt1
in each generation of offspring. Further studies will be needed to elucidate how the expression of Dnmt
genes in mammary tissue is regulated in the context of in utero
oestrogenic exposure, and how these changes are transmitted from one generation to another.
The global methylation analysis identified 375 differentially hyper- or hypomethylated gene promoter regions in mammary glands of EE2 daughters, granddaughters and great-granddaughters, hinting at an epigenetic transgenerational effect of EE2. Some of the hypermethylated promoter regions involve the PcTGs Pax6, Runx3, Foxe3, Gata4 and Vgf. These genes regulate stem cell differentiation and are often methylated in these34
and in cancer cells31
. The epigenetic trait observed in the EE2 offspring might have been induced and maintained by high levels of Dnmt1, Dnmt3a
. However, whether the changes in DNA methylation are inherited through gametes or re-established in each generation needs further verification. In addition, it remains to be determined whether the relationship between increased breast cancer risk and differential mammary gland DNA methylation in the EE2 offspring is a causal one or simply an association. It is also possible that other epigenetic mechanisms, such as aberrant histone modification and changes in microRNA expression are involved in mediating the transgenerational effects of EE2 and multigenerational effects of HF on breast cancer risk, but this remains to be investigated.
Our results show that an increase in TEB number was closely linked to increased risk of mammary tumourigenesis. These undifferentiated mammary structures are the sites of malignant transformation in the rat mammary gland26
; the corresponding structures in the human breast are the terminal ductal lobular units44
. Studies have shown that higher number of TEBs correlates with higher risk of mammary cancer26
. Alteration in TEB numbers may also reflect epigenetic modifications in the mammary tissue45
. In cultures of human mammary cells, for instance, epithelial differentiation is regulated by the expression of genes involved in chromatin remodelling and DNA methylation46
This study demonstrates, for the first time, that maternal dietary exposure to HF directly affects two consecutive generations of offspring and causes a multigenerational increase in breast cancer risk, whereas maternal dietary exposure to EE2 during pregnancy initiates a transgenerational increase in the offspring's breast cancer risk that persists up to three consecutive generations and is associated with changes in the DNA methylation machinery and DNA methylation patterns. We did not investigate whether the maternal exposures caused mutations or other genetic alterations in the offspring. However, this is unlikely because no genetic abnormalities have been found in DES daughters47
and most familial breast cancer cases exhibit no known mutations1
. If confirmed in humans, our findings represent a novel perspective on how breast cancer risk can be transmitted across generations and could have marked repercussions for breast cancer prevention and treatment. This study also has important public health implications, as intake of HF diets and chronic exposure to low levels of EDCs found in foods and drinking water can lead to adverse effects on human health48
beyond a single generation.