BACKGROUND
butyl benzyl phthalate  is a plasticizer commonly used in pipes, vinyl floor tiles, vinyl foams, and carpet backing, and to a minor extent, in cellulose plastics and polyurethane  <cit> .
this compound has shown to be weakly estrogenic  <cit> , and to induce adverse effects on the development of male reproductive system  <cit> , thus acting as an endocrine disruptor.
adult bbp intake has been estimated at  <dig> μg/kg body weight/day and it has been stated that exposure to infants and children could be up to three-fold higher  <cit> .
environmental endocrine disruptors are of growing concern due to the potential impact on human health.
actually, bbp is one of the phthalates selected by the national toxicology program  center for the evaluation of risks to human reproduction , because of its high production volume, potential for human exposure from their widespread use and occurrence within the environment, concern to the public, and published evidence of reproductive or developmental toxicity  <cit> .
the evaluation concluded that there was negligible concern for adverse reproductive effects in exposed men, but data was insufficient to reach conclusions on possible reproductive effects in exposed women  <cit> .
nevertheless, the european commission has banned bbp from toys and child care items at concentrations of greater than  <dig> % by mass of the plasticized material  <cit>  and new data is providing demonstration of possible developmental effects in humans  <cit> .
urinary concentrations of phthalate metabolites in pregnant mothers, including the major bbp metabolite monobenzyl phthalate, have been inversely correlated to anogenital distance among male infants  <cit> .
moreover an etiological association of serum bbp concentration with endometriosis has been suggested, as severity of endometriosis was strongly correlated with bbp concentration in blood  <cit> .
although more data is needed regarding the possible effects of this compound on human health, experimental studies have given evidence of an antiandrogenic effect of bbp and monobenzyl phthalate.
prenatal and prepubertal exposure of rats to such compounds induced significant alterations on reproductive system of males, like undescended testes, decrease in the anogenital distance and other malformations of the external genitalia, sex accessory glands, epididymis and testes  <cit> .
it has also been reported a decrease in the weight of the ovaries in females exposed during adulthood, as well as changes in the offspring of exposed rats, including a decrease of the body weight of male and female at birth and an increase in the anogenital distance in females  <cit> .
all these changes are compatible with an endocrine disrupting action of bbp.
endocrine disruptors have also been suggested to contribute to the development of hormone-dependent cancers  <cit> .
although the risk for cancer is multifactorial, there is substantial contribution of environmental factors, including diet and environmental chemicals.
breast cancer is an estrogen-dependent malignancy with a significant mortality in women worldwide  <cit> .
changes in the hormonal environment during critical stages of development can modify the architecture and biological characteristics of the mammary gland and thus affect the future susceptibility to develop breast cancer  <cit> .
the effects of environmental endocrine disruptors seem to be dependent on the time when the exposure occurs.
diethylstilbestrol has been associated to increased or decreased susceptibility for chemically-induced mammary cancer in rats depending on the period of exposure  <cit> .
the present study is designed to determine the effects of neonatal and prepubertal exposure to bbp on rat mammary gland at different stages of development using morphological parameters of gland differentiation  <cit> , proliferative index and the genomic expression profile.
here we report that bbp has an effect on the gene expression profile of the mammary gland.
RESULTS
maturation parameters
neonatal/prepubertal exposure to bbp did not affect body weight, uterine weight or day at vaginal opening.
the uterine to body weight ratios were significantly increased in the exposed animals at  <dig> days of age .
bbp exposure did not modify the day at vaginal opening , but decreased significantly  the body weight at that day .
maturation parameters and histoarchitectural study of the mammary gland for controls and rats exposed neonatally/prepubertally to bbp.
results are indicated as mean ± sem.
bw: body weight.
*: significantly different compared to control group .
mammary gland architecture
histoarchitectural analysis of the mammary gland was performed by determining the number of specific epithelial structures at  <dig>   <dig>   <dig> and  <dig> days.
the number of undifferentiated teb decreased from  <dig> to  <dig> days in both control and bbp treated animals, while the tds and lob <dig> increased over time.
neonatal/prepubertal exposure to bbp did not modify significantly the number of these epithelial structures when compared to control groups .
proliferative index of the mammary gland
brdu incorporation into the dna was used as an index of cell proliferation .
teb from bbp exposed rats had significantly  higher proliferation index than controls by  <dig> days, whereas in td and ab the differences were not significant.
the proliferative index of lob <dig> from controls increased from  <dig> to  <dig> days and decreased at  <dig> days, while in bbp exposed rats this structure showed maximum index of proliferation at  <dig> days and decreased thereafter, showing significant differences when compared to controls at all these ages .
gene expression analysis by microarrays
neonatal/prepubertal exposure to bbp induced changes in the gene expression pattern of the mammary gland mainly by  <dig> days, just after the end of the treatment, and the number of modulated genes was low thereafter .
in  <dig> days-old rats, bbp exposure resulted in the up-regulation of  <dig> genes,  <dig> of which were known, and included genes related to morphogenesis and cell differentiation, transcription factors, cell proliferation, response to stress, signal transduction, metabolism, transport and cell organization .
there was only one down-regulated gene .
at  <dig> days of age the number of up-regulated genes decreased significantly to four unknown; two genes were down-regulated .
by  <dig> days of age,  <dig> genes were up-regulated , and  <dig> genes were down-regulated .
in the  <dig> day-old group, two known and one unknown genes were up-modulated .
list of known genes down- and up-regulated in mammary glands of bbp treated rats at  <dig>   <dig>   <dig> and  <dig> days.
average fold change related to control mammary glands is indicated.
up-regulated genes at  <dig> days have been sorted by function according to level  <dig> of gene ontology.
functional analysis of the genes found up-regulated in  <dig> days-old rats showed some categories that were significantly  over-represented: cell proliferation and differentiation, signal transduction  and cell communication, .
we also found over-representation of genes coding for cytoskeletal proteins among the down-modulated by  <dig> days.
validation of microarrays by real time rt-pcr
to validate the results from microarrays we have chosen several genes that were differentially expressed in the mammary glands from bbp treated rats: transcription factors related to proliferation and differentiation , other genes related to differentiation , adhesion  and communication .
we analyzed the gene expression of those genes by real time rt-pcr.
results of the validation are shown in figure  <dig>  in the bbp exposed animals foxg <dig>  hfh <dig>  nfyc and ahr were between  <dig> - and 2-fold up-regulated in comparison to their gene expression in untreated animals.
these differences, although discrete, were statistically significant for hfh <dig> and ahr, and close to significance for the other genes.
we also obtained statistically significant increases in the expression of fabp <dig>  wt <dig> and cadh <dig>  by  <dig> and  <dig> days, bbp exposure induced a significant decrease in the expression of gad <dig>  the decrease of this gene found by microarrays at  <dig> days as well as the increase in tsn observed by  <dig> days of age was not confirmed by real time pcr.
discussion
the objective of the present study was to investigate the effects of neonatal/prepubertal exposure to bbp in the rat mammary gland at different times of development.
we have characterized the genomic signature of the mammary gland, observing modifications in the gene expression profile mainly at the end of the exposure and fewer changes thereafter.
we also studied maturation parameters, finding also alterations compatible with a transitory effect of this environmental compound.
although it is difficult to determine doses for experimental studies, the dose chosen for this study was based on the work of singletary et al.
<cit> , where they reported that treatment of female rats with bbp for  <dig> days prior to dosing with dmba decreased mammary tumor incidence by 37%.
the number of palpable tumors per rat at week  <dig> was significantly inhibited by 58% and 71% for animals administered bbp at  <dig> and  <dig> mg/kg, respectively, compared to controls.
our goal was to determine if neonatal and prepubertal only exposure to bbp would have a direct effect at day  <dig> and if this treatment would leave permanent alterations on mammary gland maturation, cell proliferation and gene expression at later time points.
the offspring were exposed to the bbp via the mother's milk.
considering that the lactating dams were treated with  <dig> mg bbp/kg bw and this is dissipated in the mother before it makes its way via the milk to the  <dig> offspring, hence it is estimated that they receive 1/ <dig> to 1/ <dig> of the mother's dose, i.e.
<dig> – <dig> mg bbp/kg bw/day.
this approximation places the rat bbp exposure near the epa safe dose for humans of  <dig>  mg/kg/day  <cit>  and similar to a  <dig> μg/kg bw/day for adults, and lower than estimated exposure to infants and children   <cit> .
neonatal/prepubertal exposure to bbp increased the relative uterine weight at  <dig> days but not thereafter, and decreased the body weight at day of vaginal opening .
in the literature there are conflicting results regarding the effect of bbp because it showed estrogenic effects in vitro but did not modify the estrogen-modulated gene calbindin-d9k in vivo  <cit> .
however, bbp has also shown to increase the expression of progesterone receptor mrna, a gene regulated by estrogens, in the preoptic area of adult ovariectomized rats  <cit> .
exposure to bbp in other phases of development induced alterations in the onset of puberty.
ashby et al.
reported advanced vaginal opening by  <dig>  days without modifications on uterine weight in rats by effect of prenatal plus prepubertal exposure to bbp, although the authors considered that this result could be related to the variability of the observation or, more interestingly, to an increase in the weight of the bbp treated animals  <cit> .
in two generational studies, aso et al.
reported changes in anogenital distance of female offspring of rats treated with more than  <dig> mg/kg  <cit> .
little is known on the effects of bbp on mammary gland, another target of sexual maturity, and the present work indicates that bbp did not induce significant changes in the morphology of that gland, but changed the proliferative index of teb by  <dig> days and in lob <dig> at  <dig>   <dig> and  <dig> days.
although these modifications are subtle, we cannot rule out that they may have an effect if the mammary gland susceptibility to carcinogenesis, since teb are the most susceptible epithelial structures to malignant transformation  <cit>  and prevention of chemically-induced mammary carcinomas is accompanied by inhibition of cell proliferation  <cit> .
changes in the proliferative index of lob <dig> in bbp exposed animals in comparison to control group can be associated with subtle alterations in mammary development.
the proliferation of lob <dig> is related to the differentiation of these structures to lobules type  <dig>  <cit> .
control group had the highest proliferative index in lob <dig> by  <dig> days, concomitantly with the highest number of lob <dig>  at the age of profound changes that the mammary gland faces as a result of an intense hormonal activity  <cit> .
bbp exposed animals had the maximum mitotic index in lob <dig> by  <dig> days, decreasing by  <dig> days.
in addition, the reduction of proliferation in lob <dig> of bbp group by  <dig> days was not as important as in control group.
the significant changes observed are compatible with slight changes in the timing of the breast tissue development by effect of bbp exposure.
we are the first to report that neonatal/prepubertal exposure to bbp induced modifications in the gene expression of the mammary tissue.
whereas the full relevance of these findings requires tumorigenesis studies, the characterization of the genomic signature of the mammary gland could be used as a predictor of the susceptibility to carcinogenesis.
this statement is based on previous works in which changes in susceptibility to dmba-induced carcinogenesis are accompanied by the induction of a specific genomic profile  <cit> .
neonatal/prepubertal exposure to bbp induced changes in the genomic profile of the mammary gland, mainly at the end of treatment  with a focus on genes involved in cell proliferation/differentiation, communication and signal transduction.
some of those genes related to differentiation were validated by real time rt-pcr, as fabp <dig> and wt <dig>  fabp <dig> belongs to the family of the fatty acid binding proteins, which have a role in fatty acid transport or metabolism.
this member, however, is considered an identical homologue of the mammary-derived growth inhibitor , with a role in the differentiation of the mammary gland  <cit> .
mdgi is a suppressor of tumor growth and an inhibitor of tumor cell proliferation  <cit> .
we have previously observed that this gene is up-regulated in the most differentiated structures of the mammary gland  and silenced in breast cancer progression  <cit> .
another gene found significantly up-regulated by  <dig> days is wt <dig>  which has also been related to development of the mammary gland although its specific function is not well known  <cit> .
this gene is essential for the development of the urogenital system but its role in the breast tissue is controversial.
wt <dig> presents a developmental pattern of expression in normal mammary gland, and changes observed in cell lines and tumors may be indicative of breast-tumor-related perturbations of wt <dig> expression  <cit> .
cadherin  <dig> , also found up-regulated in the exposed animals, is an adhesion molecule with an important role in brain and kidney development and differentiation  <cit> .
we have also observed transitional changes in the expression of these three genes by effect of prepubertal exposure to other endocrine disruptor, bisphenol a  <cit> .
moreover, we also observed changes in the expression of fabp <dig> when this endocrine disruptor was administered in utero.
thus, the exposure during the prenatal period to bisphenol a induced up-regulation or down-regulation of fabp <dig> depending on the age tested, suggesting a "shift" in the normal development of the mammary gland, while prepubertal exposure to this endocrine disruptor induced a transient up-regulation just at the end of the treatment  <cit> .
fabp <dig> mrna has also been reported as sensitive to the action of other hormone interfering agents, since the antiestrogens tamoxifen and ici up-regulate its expression by stabilization of the mrna  <cit> .
these evidences suggest that genes related to differentiation of the mammary gland can be very sensitive to the effects of several compounds that may interfere with hormonal action, suggesting that, even if the effects are not strong enough to modify mammary histoarchitecture, they may change the molecular milieu of the gland.
by  <dig> days of age we also found a moderate increase in the expression of the transcription factors foxg <dig>  hfh <dig> and ahr.
foxg <dig> and hfh <dig> have a role in the development and differentiation of several tissues, but to date their function in the mammary gland is unknown  <cit> .
the aryl hydrocarbon receptor  belongs to the pas  family of transcription factors.
pas proteins control important physiological processes, as toxicant metabolism, circadian rhythms and hormone signaling  <cit> .
we have previously reported a role of ahr in mammary differentiation, as an increased expression in primary cultures of lob <dig> from parous women compared to primary cultures of lob <dig> from nulliparous women breast was observed  <cit> .
ahr is known to interact with signaling pathways that are mediated by estrogen receptor and other hormone receptors.
the mechanisms of ahr-estrogen receptor crosstalk seem to be complex, and recent studies suggest direct activation of estrogen-regulated target genes by ahr  <cit> .
thus, our results suggest that the increase in ahr could be one of the mechanisms by which bbp treatment exerts the observed transitory effects at  <dig> days.
we also observed and validated down-regulation of gad <dig> by  <dig> and  <dig> days of age.
this gene codes for the protein gad <dig>  which catalyzes the production of the neurotransmitter gaba.
gad <dig> can be regulated by estrogens in rat brain  <cit> , and expression changes in discrete regions of the rostral preoptic area during estrous cycle and with age has been described  <cit> .
in addition, gad <dig> has also a role in the control of puberty in nonhuman primates  <cit> .
modifications in the expression of gad <dig> has been investigated in relation to some brain disorders, since gad <dig> mrna decreased in prefrontal cortex and other brain areas of schizophrenia and bipolar disorder patients with psychosis  <cit> .
experiments in rats have found down-modulation of gad <dig> accompanied by hypermethylation of gad <dig> promoter very likely mediated by the overexpression of dna methyltransferase  <dig> in cortical gabaergic interneurons  <cit> .
thus, we can consider the possibility that the observed changes in gad <dig> are due to an effect of bbp on the methylation of gad <dig> promoter.
bbp has also shown an effect on the methylation status of other genes in vitro, as the reported demethylation of erα promoter-associated cpg islands in mcf- <dig> cells  <cit> .
the gaba-ergic system has been involved in hormonal regulation and pathogenesis of breast cancer, since human and mouse tumors have shown higher gaba level and gad activity than normal tissue  <cit> .
hence, although prepubertal exposure to bbp seems to have mainly a transitory effect on the genomic profile of the mammary gland, we cannot discard that subtle changes in the gland may have an effect later in life.
finally, very few genes were regulated by bbp exposure in the oldest age tested .
CONCLUSIONS
in summary, neonatal/prepubertal exposure to bbp had a transitory effect by increasing the relative uterine weight only at the end of exposure.
this compound also changed the gene expression profile of the mammary gland mainly at the same age, although long-term subtle alterations can not be ruled out.

 butyl benzyl phthalate is a plasticizer commonly used in pipes, vinyl floor tiles, vinyl foams, and carpet backing, and to a minor extent, in cellulose plastics and polyurethane. this compound has shown to be weakly estrogenic, and to induce adverse effects on the development of male reproductive system, thus acting as an endocrine disruptor. adult bbp intake has been estimated at μg/kg body weight/day and it has been stated that exposure to infants and children could be up to three-fold higher. environmental endocrine disruptors are of growing concern due to the potential impact on human health. actually, bbp is one of the phthalates selected by the national toxicology program center for the evaluation of risks to human reproduction, because of its high production volume, potential for human exposure from their widespread use and occurrence within the environment, concern to the public, and published evidence of reproductive or developmental toxicity. the evaluation concluded that there was negligible concern for adverse reproductive effects in exposed men, but data was insufficient to reach conclusions on possible reproductive effects in exposed women. nevertheless, the european commission has banned bbp from toys and child care items at concentrations of greater than % by mass of the plasticized material and new data is providing demonstration of possible developmental effects in humans. urinary concentrations of phthalate metabolites in pregnant mothers, including the major bbp metabolite monobenzyl phthalate, have been inversely correlated to anogenital distance among male infants. moreover an etiological association of serum bbp concentration with endometriosis has been suggested, as severity of endometriosis was strongly correlated with bbp concentration in blood. although more data is needed regarding the possible effects of this compound on human health, experimental studies have given evidence of an antiandrogenic effect of bbp and monobenzyl phthalate. prenatal and prepubertal exposure of rats to such compounds induced significant alterations on reproductive system of males, like undescended testes, decrease in the anogenital distance and other malformations of the external genitalia, sex accessory glands, epididymis and testes. it has also been reported a decrease in the weight of the ovaries in females exposed during adulthood, as well as changes in the offspring of exposed rats, including a decrease of the body weight of male and female at birth and an increase in the anogenital distance in females. all these changes are compatible with an endocrine disrupting action of bbp. endocrine disruptors have also been suggested to contribute to the development of hormone-dependent cancers. although the risk for cancer is multifactorial, there is substantial contribution of environmental factors, including diet and environmental chemicals. breast cancer is an estrogen-dependent malignancy with a significant mortality in women worldwide. changes in the hormonal environment during critical stages of development can modify the architecture and biological characteristics of the mammary gland and thus affect the future susceptibility to develop breast cancer. the effects of environmental endocrine disruptors seem to be dependent on the time when the exposure occurs. diethylstilbestrol has been associated to increased or decreased susceptibility for chemically-induced mammary cancer in rats depending on the period of exposure. the present study is designed to determine the effects of neonatal and prepubertal exposure to bbp on rat mammary gland at different stages of development using morphological parameters of gland differentiation, proliferative index and the genomic expression profile. here we report that bbp has an effect on the gene expression profile of the mammary gland.  maturation parameters neonatal/prepubertal exposure to bbp did not affect body weight, uterine weight or day at vaginal opening. the uterine to body weight ratios were significantly increased in the exposed animals at days of age. bbp exposure did not modify the day at vaginal opening, but decreased significantly the body weight at that day. maturation parameters and histoarchitectural study of the mammary gland for controls and rats exposed neonatally/prepubertally to bbp. results are indicated as mean ± sem. bw: body weight. *: significantly different compared to control group. mammary gland architecture histoarchitectural analysis of the mammary gland was performed by determining the number of specific epithelial structures at and days. the number of undifferentiated teb decreased from to days in both control and bbp treated animals, while the tds and lob increased over time. neonatal/prepubertal exposure to bbp did not modify significantly the number of these epithelial structures when compared to control groups. proliferative index of the mammary gland brdu incorporation into the dna was used as an index of cell proliferation. teb from bbp exposed rats had significantly higher proliferation index than controls by days, whereas in td and ab the differences were not significant. the proliferative index of lob from controls increased from to days and decreased at days, while in bbp exposed rats this structure showed maximum index of proliferation at days and decreased thereafter, showing significant differences when compared to controls at all these ages. gene expression analysis by microarrays neonatal/prepubertal exposure to bbp induced changes in the gene expression pattern of the mammary gland mainly by days, just after the end of the treatment, and the number of modulated genes was low thereafter. in days-old rats, bbp exposure resulted in the up-regulation of genes, of which were known, and included genes related to morphogenesis and cell differentiation, transcription factors, cell proliferation, response to stress, signal transduction, metabolism, transport and cell organization. there was only one down-regulated gene. at days of age the number of up-regulated genes decreased significantly to four unknown; two genes were down-regulated. by days of age, genes were up-regulated, and genes were down-regulated. in the day-old group, two known and one unknown genes were up-modulated. list of known genes down- and up-regulated in mammary glands of bbp treated rats at and days. average fold change related to control mammary glands is indicated. up-regulated genes at days have been sorted by function according to level of gene ontology. functional analysis of the genes found up-regulated in days-old rats showed some categories that were significantly over-represented: cell proliferation and differentiation, signal transduction and cell communication,. we also found over-representation of genes coding for cytoskeletal proteins among the down-modulated by days. validation of microarrays by real time rt-pcr to validate the results from microarrays we have chosen several genes that were differentially expressed in the mammary glands from bbp treated rats: transcription factors related to proliferation and differentiation, other genes related to differentiation, adhesion and communication. we analyzed the gene expression of those genes by real time rt-pcr. results of the validation are shown in figure in the bbp exposed animals foxg hfh nfyc and ahr were between - and 2-fold up-regulated in comparison to their gene expression in untreated animals. these differences, although discrete, were statistically significant for hfh and ahr, and close to significance for the other genes. we also obtained statistically significant increases in the expression of fabp wt and cadh by and days, bbp exposure induced a significant decrease in the expression of gad the decrease of this gene found by microarrays at days as well as the increase in tsn observed by days of age was not confirmed by real time pcr. discussion the objective of the present study was to investigate the effects of neonatal/prepubertal exposure to bbp in the rat mammary gland at different times of development. we have characterized the genomic signature of the mammary gland, observing modifications in the gene expression profile mainly at the end of the exposure and fewer changes thereafter. we also studied maturation parameters, finding also alterations compatible with a transitory effect of this environmental compound. although it is difficult to determine doses for experimental studies, the dose chosen for this study was based on the work of singletary et al., where they reported that treatment of female rats with bbp for days prior to dosing with dmba decreased mammary tumor incidence by 37%. the number of palpable tumors per rat at week was significantly inhibited by 58% and 71% for animals administered bbp at and mg/kg, respectively, compared to controls. our goal was to determine if neonatal and prepubertal only exposure to bbp would have a direct effect at day and if this treatment would leave permanent alterations on mammary gland maturation, cell proliferation and gene expression at later time points. the offspring were exposed to the bbp via the mother's milk. considering that the lactating dams were treated with mg bbp/kg bw and this is dissipated in the mother before it makes its way via the milk to the offspring, hence it is estimated that they receive 1/ to 1/ of the mother's dose, i.e. – mg bbp/kg bw/day. this approximation places the rat bbp exposure near the epa safe dose for humans of mg/kg/day and similar to a μg/kg bw/day for adults, and lower than estimated exposure to infants and children. neonatal/prepubertal exposure to bbp increased the relative uterine weight at days but not thereafter, and decreased the body weight at day of vaginal opening. in the literature there are conflicting results regarding the effect of bbp because it showed estrogenic effects in vitro but did not modify the estrogen-modulated gene calbindin-d9k in vivo. however, bbp has also shown to increase the expression of progesterone receptor mrna, a gene regulated by estrogens, in the preoptic area of adult ovariectomized rats. exposure to bbp in other phases of development induced alterations in the onset of puberty. ashby et al. reported advanced vaginal opening by days without modifications on uterine weight in rats by effect of prenatal plus prepubertal exposure to bbp, although the authors considered that this result could be related to the variability of the observation or, more interestingly, to an increase in the weight of the bbp treated animals. in two generational studies, aso et al. reported changes in anogenital distance of female offspring of rats treated with more than mg/kg. little is known on the effects of bbp on mammary gland, another target of sexual maturity, and the present work indicates that bbp did not induce significant changes in the morphology of that gland, but changed the proliferative index of teb by days and in lob at and days. although these modifications are subtle, we cannot rule out that they may have an effect if the mammary gland susceptibility to carcinogenesis, since teb are the most susceptible epithelial structures to malignant transformation and prevention of chemically-induced mammary carcinomas is accompanied by inhibition of cell proliferation. changes in the proliferative index of lob in bbp exposed animals in comparison to control group can be associated with subtle alterations in mammary development. the proliferation of lob is related to the differentiation of these structures to lobules type. control group had the highest proliferative index in lob by days, concomitantly with the highest number of lob at the age of profound changes that the mammary gland faces as a result of an intense hormonal activity. bbp exposed animals had the maximum mitotic index in lob by days, decreasing by days. in addition, the reduction of proliferation in lob of bbp group by days was not as important as in control group. the significant changes observed are compatible with slight changes in the timing of the breast tissue development by effect of bbp exposure. we are the first to report that neonatal/prepubertal exposure to bbp induced modifications in the gene expression of the mammary tissue. whereas the full relevance of these findings requires tumorigenesis studies, the characterization of the genomic signature of the mammary gland could be used as a predictor of the susceptibility to carcinogenesis. this statement is based on previous works in which changes in susceptibility to dmba-induced carcinogenesis are accompanied by the induction of a specific genomic profile. neonatal/prepubertal exposure to bbp induced changes in the genomic profile of the mammary gland, mainly at the end of treatment with a focus on genes involved in cell proliferation/differentiation, communication and signal transduction. some of those genes related to differentiation were validated by real time rt-pcr, as fabp and wt fabp belongs to the family of the fatty acid binding proteins, which have a role in fatty acid transport or metabolism. this member, however, is considered an identical homologue of the mammary-derived growth inhibitor, with a role in the differentiation of the mammary gland. mdgi is a suppressor of tumor growth and an inhibitor of tumor cell proliferation. we have previously observed that this gene is up-regulated in the most differentiated structures of the mammary gland and silenced in breast cancer progression. another gene found significantly up-regulated by days is wt which has also been related to development of the mammary gland although its specific function is not well known. this gene is essential for the development of the urogenital system but its role in the breast tissue is controversial. wt presents a developmental pattern of expression in normal mammary gland, and changes observed in cell lines and tumors may be indicative of breast-tumor-related perturbations of wt expression. cadherin, also found up-regulated in the exposed animals, is an adhesion molecule with an important role in brain and kidney development and differentiation. we have also observed transitional changes in the expression of these three genes by effect of prepubertal exposure to other endocrine disruptor, bisphenol a. moreover, we also observed changes in the expression of fabp when this endocrine disruptor was administered in utero. thus, the exposure during the prenatal period to bisphenol a induced up-regulation or down-regulation of fabp depending on the age tested, suggesting a "shift" in the normal development of the mammary gland, while prepubertal exposure to this endocrine disruptor induced a transient up-regulation just at the end of the treatment. fabp mrna has also been reported as sensitive to the action of other hormone interfering agents, since the antiestrogens tamoxifen and ici up-regulate its expression by stabilization of the mrna. these evidences suggest that genes related to differentiation of the mammary gland can be very sensitive to the effects of several compounds that may interfere with hormonal action, suggesting that, even if the effects are not strong enough to modify mammary histoarchitecture, they may change the molecular milieu of the gland. by days of age we also found a moderate increase in the expression of the transcription factors foxg hfh and ahr. foxg and hfh have a role in the development and differentiation of several tissues, but to date their function in the mammary gland is unknown. the aryl hydrocarbon receptor belongs to the pas family of transcription factors. pas proteins control important physiological processes, as toxicant metabolism, circadian rhythms and hormone signaling. we have previously reported a role of ahr in mammary differentiation, as an increased expression in primary cultures of lob from parous women compared to primary cultures of lob from nulliparous women breast was observed. ahr is known to interact with signaling pathways that are mediated by estrogen receptor and other hormone receptors. the mechanisms of ahr-estrogen receptor crosstalk seem to be complex, and recent studies suggest direct activation of estrogen-regulated target genes by ahr. thus, our results suggest that the increase in ahr could be one of the mechanisms by which bbp treatment exerts the observed transitory effects at days. we also observed and validated down-regulation of gad by and days of age. this gene codes for the protein gad which catalyzes the production of the neurotransmitter gaba. gad can be regulated by estrogens in rat brain, and expression changes in discrete regions of the rostral preoptic area during estrous cycle and with age has been described. in addition, gad has also a role in the control of puberty in nonhuman primates. modifications in the expression of gad has been investigated in relation to some brain disorders, since gad mrna decreased in prefrontal cortex and other brain areas of schizophrenia and bipolar disorder patients with psychosis. experiments in rats have found down-modulation of gad accompanied by hypermethylation of gad promoter very likely mediated by the overexpression of dna methyltransferase in cortical gabaergic interneurons. thus, we can consider the possibility that the observed changes in gad are due to an effect of bbp on the methylation of gad promoter. bbp has also shown an effect on the methylation status of other genes in vitro, as the reported demethylation of erα promoter-associated cpg islands in mcf- cells. the gaba-ergic system has been involved in hormonal regulation and pathogenesis of breast cancer, since human and mouse tumors have shown higher gaba level and gad activity than normal tissue. hence, although prepubertal exposure to bbp seems to have mainly a transitory effect on the genomic profile of the mammary gland, we cannot discard that subtle changes in the gland may have an effect later in life. finally, very few genes were regulated by bbp exposure in the oldest age tested.  in summary, neonatal/prepubertal exposure to bbp had a transitory effect by increasing the relative uterine weight only at the end of exposure. this compound also changed the gene expression profile of the mammary gland mainly at the same age, although long-term subtle alterations can not be ruled out. 
the aim of this study is to determine the effect of neonatal/prepubertal exposure to bbp on maturation parameters and on the morphology, proliferative index and genomic signature of the rat mammary gland at different ages of development. these results suggest that bbp has an effect in the gene expression profile of the mammary gland. moreover, bbp had an effect on the genomic profile of the mammary gland mainly at the end of the exposure, becoming less prominent thereafter. bbp did not induce significant changes on the morphology of the mammary gland, but increased proliferative index in terminal end buds at days and in lobules at several ages. by this age a significant number of genes related to proliferation and differentiation, communication and signal transduction were up-regulated in the glands of the exposed animals. here we show that exposure to bbp increased the uterine weight/body weight ratio at days and decreased the body weight at time of vaginal opening.
 phthalate esters like n-butyl benzyl phthalate are widely used plasticizers. bbp has shown endocrine-disrupting properties, thus having a potential effect on hormone-sensitive tissues. the aim of this study is to determine the effect of neonatal/prepubertal exposure to bbp on maturation parameters and on the morphology, proliferative index and genomic signature of the rat mammary gland at different ages of development.  here we show that exposure to bbp increased the uterine weight/body weight ratio at days and decreased the body weight at time of vaginal opening. bbp did not induce significant changes on the morphology of the mammary gland, but increased proliferative index in terminal end buds at days and in lobules at several ages. moreover, bbp had an effect on the genomic profile of the mammary gland mainly at the end of the exposure, becoming less prominent thereafter. by this age a significant number of genes related to proliferation and differentiation, communication and signal transduction were up-regulated in the glands of the exposed animals.  these results suggest that bbp has an effect in the gene expression profile of the mammary gland. 