In this study we found that in utero exposure to BBP significantly affects post-natal maturation of female rats, including delayed vaginal opening, changes in morphology of the mammary gland, and changes in gene expression of the mammary gland at puberty. These effects were dose-dependent, and the alterations of the mammary gland were age-specific.
Our findings demonstrate that prenatal exposure of BBP causes a significant delay in female sexual maturity as determined by the day of vaginal opening associated with a delayed pubertal onset. Other authors have reported changes in the time of vaginal opening by prenatal plus prepubertal exposure to BBP by administration to the mothers of 1g/L in drinking water , as well as modifications in the anogenital distance of rats exposed prenatally by oral gavage to the pregnant dams with 100 mg BBP/kg body weight . Another indicator of sexual maturity is the morphological and cellular changes of mammary gland. We observed that rats exposed prenatally to the higher dose of BBP had a higher number of undifferentiated epithelial structures in the mammary gland mainly at the age of pubertal onset (35 days). This is in accordance with the delay in the time of vaginal opening and may suggest changes in the normal development of the mammary gland. Moreover, we found that in all epithelial structures, except in TD, the proliferative index in BBP-exposed animals was maximal by 35 days, in contrast to control animals, which had the highest proliferation indexes by 50 days. More importantly, exposure to the high dose of BBP induced more proliferating TEB by 35 days when compared to unexposed animals. The TEB is the least differentiated epithelial structure in the mammary gland, and the most susceptible to malignant transformation . Thus, alterations in number and/or proliferation of TEB may cause the changes in susceptibility of this gland to malignant transformation. For instance, we have previously demonstrated that both pregnancy and treatment of virgin rats with the placental hormone human chorionic gonadotropin (hCG) protected rat mammary gland from chemically-induced malignant transformation and that this effect was resulted from elimination of undifferentiated TEB and inhibition of cell proliferation [16, 17]. It has been described that the age of highest susceptibility of this gland to chemically-induced carcinogenesis is around 50 days, when the TEB are more proliferative . The fact that the BBP-exposed animals showed more TEB and AB and more proliferating cells by 35 days suggests that BBP can induce alterations in the development of the mammary tissues, expanding or shifting the window of susceptibility of the gland to transformation.
The changes in the susceptibility of the mammary gland to chemically-induced carcinogenesis are likely resulted from the alterations in specific genomic signature. Thus, we have studied the gene expression profiles of the mammary glands from the rats exposed prenatally to the low and the high doses of BBP. In accordance to the results of puberty onset, morphology and proliferative index of the mammary gland, we found changes in the gene expression profile of the glands by 35 days of age. The effect of xenobiotics with a putative endocrine-disrupting influence seems to depend on time of exposure. Thus, we previously observed that treatment with the same high dose of BBP during the neonatal and lactational periods caused transitory changes in the gene expression profile of the gland, inducing a high number of up-modulated genes by 21 days of age (just after the end of the exposure) but not at other ages, and no changes in the day of vaginal opening . In utero exposure to 250 μg of bisphenol A (BPA), another endocrine disruptor widely used, also induced time-specific changes in the gene expression profile of the mammary gland , protein expression , and shifted the windows of susceptibility for mammary carcinogenesis . Different effects were observed when the same dose was administrated during the lactational period (neonatal + prepubertal), finding increased proliferation and decreased apoptosis in the mammary glands at 50 days, but not at 21 days .
The function of genes dysregulated at puberty by effect of in utero exposure to BBP included developmental and immune system processes, metabolism, signaling or apoptosis. For example, we observed a down-modulation of the immune-related genes Cd5, Cd3d, Ptprc, especially by effect of the higher dose. Other authors have also suggested an influence of phthalates on immunity. BBP inhibited lipopolysaccharide-induced TNF-alpha in a mouse macrophage cell line , and human case-control data have associated the concentration of BBP in house dust with allergic symptoms . We also observed at 35 days a down-modulation of genes related to developmental processes in the BBP-exposed rats, including mammary gland differentiation markers (casein kappa -Csn3-, lactalbumin alpha -Lalba-, whey acidic protein -Wap- and milk fat globule-EGF factor 8 protein -Mfge8 or lactadherin-). Other genes annotated to "cell proliferation and differentiation" category have been found down-regulated at that age. That is the case of Bhlhb3, a transcription factor that is involved in circadian rhythm, differentiation, and that has been reported to repress the transcription of cyclin D1 and thus inhibit proliferation . These results are in agreement with the ones obtained in the proliferation analysis (more proliferative structures by 35 days in the BBP-exposed groups). In relation to this, we have also found a down-modulation of genes related to apoptosis (such as lactalbumin ) and up-modulation of Cryab, which has been described to have anti-apoptotic effects . Hence, our results are compatible with a decreased apoptosis, as it has been described for other endocrine disruptors such as BPA . All these data suggest a proliferation/apoptosis balance tilted to proliferation in the mammary glands of the exposed rats, and that TEB may be more susceptible to malignant transformation at 35 days of age.
Prenatal BBP treatment also induced modifications in the expression of genes related to response to stress (Ddit4, Cryab) and metabolism. The down-modulation of Thedc1, with a role in lipid metabolism, was not confirmed by real time PCR at 35 days although we found a significant increase in its expression at 50 days (data not shown). Moreover, some of the genes studied, such as Cryab, had also a role in metabolism . Modifications in the expression of similar genes (related to metabolism or oxidative stress) have been also described in testis of rats exposed prenatally with BBP. Such modifications are suggested to be related with changes in the hormonal balance of developing fetal testis . Considering such evidence, our results could also be a reflection, at least in part, of changes in the hormonal balance in the BBP treated rats.
Therefore, our data suggest that prenatal exposure to BBP can have an effect on normal post-natal development of the rat. Delayed vaginal opening could be the result of an altered maturation of the hypothalamic-gonadal axis for the timing of the onset of puberty. While the development of the mammary gland undergoes a critical phase with the initiation of puberty, modifications in the morphology of the gland and in the quantity of each specific structure begins before puberty and continues for a long period of time. The changes in the morphology and the proliferative index of the mammary gland, more than advanced or retarded growth, suggest a different pattern in the normal development of the gland that would affect the windows of susceptibility of this gland. This would increase or decrease the susceptibility to malignant transformation depending of the moment when a carcinogenic insult occurs. Such changes can be supported, at least in part, by modifications in the gene expression profile of the gland. BBP exposure down-modulated the expression of genes related to functions like immunity, apoptosis and also differentiation markers, which would be in accordance with a less differentiated gland. Our results also points out the possibility of a metabolic disruption of this compound. In fact, there is growing evidence that several pollutants, some of them considered endocrine disruptors such as phthalates, may interact with members of the PPAR superfamily and interfere with lipid and carbohydrate metabolism . Such pollutants are tentatively named "metabolic disrupters", and are currently of interest since several human diseases, such as atherosclerosis, hyperlipidemia and obesity, often reflect subtle deregulations of complex metabolic pathways and are manifested after many years .
On the other hand, we need to consider the different methodological approaches in the interpretation of the data obtained. We have visualized the number of the epithelial structures and the proliferation index of such structures. However, because stromal-epithelial interactions are essential for the development of the mammary gland, in addition to methodological reasons, gene expression profile was determined in the whole mammary tissue of the forth gland (with the exception of the lymph node). Thus, the changes in gene expression observed can be related to modifications in the biology of the epithelial (as suggested by the mammary differentiation markers), and stromal cells, such as the fibroblasts or adipocytes. Actually, changes in lipid metabolism may be occurring in the adipose tissue, and changes in the immune related genes may be a reflection of modifications in the number, type or function of infiltrated immune cells. In any case, all these cell types have an essential role in the development of the mammary gland, and thus changes in their expression profile may be of importance in the biology of such tissue [42, 43].
As mentioned above, we have previously reported that the prenatal exposure to another endocrine disruptor, bisphenol A (BPA), also induced changes in the mammary gland that were time- and dose-specific . Both doses tested (25 and 250 μg/kg) caused modification of the expression of similar clusters of genes (e.g. several differentiation markers, immune-related genes) that have also been reported as differentially modulated by BBP exposure in this work. These evidences suggest that some genes involved in general and essential processes may be especially sensitive to the effect of endocrine disruptors, and taken together, data regarding mammary gland morphology, proliferative index and gene expression profile analyses suggest changes in the biology of the gland that may change its susceptibility to breast cancer. Even when caution must be applied when comparing animal studies with human data, these studies give further evidence of the potential effect of BBP on human health. Although the action of this and other xenobiotics may not be as strong as other steroidal endocrine disruptors, they are produced in much larger quantities and population is continuously exposed to them. Actually BBP exposure in adults has been estimated at 2 μg/kg body weight/day, and three-old higher in infants and children . Moreover, urinary concentrations of BBP have been detected in > 75% of reference population, with significantly higher levels in children and adolescents . The mechanisms by which this xenobiotic alters mammary gland biology and gene expression are not still well determined. New studies are currently underway to elucidate if there are modifications in the hormonal balance causing such changes and those are related to the risk of mammary gland malignant transformation.