Reproductive Toxicology 29 (2010) 99–105

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Reproductive Toxicology

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Reproductive development and function of female rats exposed todi-�-butyl-phthalate (DBP) in utero and during lactation

Marina T. Guerraa, Wellerson R. Scaranoa, Fabíola C. de Toledob,Janete A.A. Franci c, Wilma De G. Kempinasa,∗

a Department of Morphology, Institute of Biosciences, São Paulo State University, UNESP - Botucatu, São Paulo, Brazilb Graduate Program in Cell and Structural Biology, Institute of Biology, State University of Campinas, UNICAMP - Campinas, São Paulo, Brazilc Department of Morphology, Stomatology and Physiology, School of Dentistry, University of São Paulo, USP - Ribeirão Preto, São Paulo, Brazil

a r t i c l e i n f o

Article history:Received 7 May 2009Received in revised form22 September 2009Accepted 10 October 2009Available online 20 October 2009

Keywords:Maternal exposureDi-�-butyl-phthalateFemale offspring

a b s t r a c t

Phthalates are environmental contaminants used in the production of plastics, cosmetics and medicaldevices. Studies on the effects of phthalates on female reproductive health are particularly sparse andmostly restricted to high-dose exposure in rats. In the present study, pregnant rats were treated with100 mg/kg-d of di-�-butyl-phthalate (DBP) or only the vehicle (control group), from GD 12 to GD 20 forevaluation of reproductive outcomes and fetal gonads analysis (F0), and from GD 12 to PND 21 to evaluatereproductive development and function on F1 female offspring. Results showed that all parameters werecomparable between groups, although there was a significant increase in the fetal weight after DBPexposure. However, the body weight at birth was normal. Based on these data we can conclude that, inthese experimental conditions, DBP did not disturb the reproductive development or function of femalerats.

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© 2009 Elsevier Inc. All rights reserved.

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. Introduction

Humans are constantly exposed to a wide range of environ-ental contaminants from industrial processes, through air, food,ater or contact with a variety of consumer products. In recent

ears, one class of chemicals used as plasticizers called phthalatesave attracted special attention from the scientific community andhe general public due to their high production volume, in mil-ions of tons annually, and variety of applications. In addition, theyre suspected of acting as endocrine disruptors which signifieshat they have the potential to modify normal endocrine func-ion [1–4]. Phthalate esters are liquid plasticizers widely used inexible polyvinyl chloride (PVC) products, personal care products,

osmetics (perfume, lotions and nail polish), paints, coatings, someesticide formulations, and pharmaceutical products [5,6] and thusre ubiquitous low-level environmental contaminants [7,8].

∗ Corresponding author at: Department of Morphology, Bioscience Institute,NESP, Distrito de Rubião Jr s/n, Botucatu - São Paulo, CEP 18618-000, Brazil.el.: +55 014 3811 6264; fax: +55 014 3811 6264.

E-mail address: kempinas@ibb.unesp.br (W.D.G. Kempinas).

890-6238/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2009.10.005

Di-�-butyl-phthalate (DBP) is a phthalic acid ester used exten-sively as a plasticizer in many products including medical devices[9], flexible plastics (children’s toys, beverage bottles and feed bot-tles) and some cosmetic formulations [10–12].

Human exposure occurs primarily through contaminated food,especially high-fat foods, which may have been in contact withplastic, adhesives, or other packing materials that contain DBP.Herbal preparations and nutritional supplements may also incor-porate phthalates in their formulations. This is especially truefor di(2-ethylhexyl) phthalate (DEHP) and DBP. These types ofproducts are typically taken by pregnant women. Pharmaceuticalformulations also result in significant human exposure, becausevarious plasticizers are used to coat medicines such as antibiotics,antihistamines and laxatives [13].

Most of the studies related to phthalates are restricted mainlyto high-dose exposure [3,14–17], in which the effects are morepronounced. Human and animal exposures to low concentrationsof phthalates usually occur after the release of phthalate esters

from plastic products and utensils because phthalates are notpermanently bound to the polymer matrix [12,18–21]. Phthalatemetabolites were detected in the urine of children and adults,in serum, seminal fluid, amniotic fluid, breast milk and saliva[22–27]. Estimates for human exposure to DBP range from 0.84

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o 113 �g/kg-d [5,28], but the most critical population comprisesomen of reproductive age in whom exposure may be up to 20

imes greater than in the average person from the general popu-ation. In this case, the highest exposure estimates were above theederal safety standard [5,28,29].

Studies that report effects of phthalates on female reproductiveealth are particularly sparse and mostly restricted to high-dosexposure in rats. The present study aims to evaluate the effectsn female reproductive development and function, in rats whoseothers were exposed to DBP at a dose of 100 mg/kg-d (LOAEL forale rats) during pregnancy and lactation.

. Materials and methods

.1. Animals

Adult female (60 days of age, n = 20) and male (90 days of age, n = 10) Wistarats were supplied by Central Biotherium of State University of São Paulo (Unesp)nd were housed in polypropylene cages (43 cm × 30 cm × 15 cm) with laboratory-rade pine shavings as bedding. Rats were maintained under controlled temperature23 ± 1 ◦C) and lighting conditions (12:12-h photoperiod). Rat chow and filtered tapater were provided at libitum. Two non-gravid female rats were mated with oneale, during the dark portion of the lighting cycle, and the day of sperm detection

n the vaginal smear was considered day 0 of gestation (GD 0). The gravid femalesere randomly assigned between the experimental groups and housed individually

n cages. At birth, the offspring were weighed and reduced to eight pups per litter,n order to maintain the female ones. The experimental protocol followed the Eth-cal Principles in Animal Research of the Brazil College of Animal Experimentationnd was approved by the Bioscience Institute/UNESP Ethics Committee for Animalxperimentation (protocol no. 60/06-CEEA).

.2. Treatment

Gravid rats (n = 5/group) were administered either di-�-butyl-phthalateDBP—Sigma Chemical Co., St. Louis, MO) at 100 mg/kg bw-d, considered the LOAELose for male reproductive effects [10], by gavage (oral route), or corn oil (vehicle)rom GD 12 until GD 20 when the dams were sacrificed and caesarean section waserformed. Reproductive performance data were assessed and the gonads of oneemale offspring (F1) from each litter were also analyzed. Two additional groups ofravid rats (C = 9; T = 10) were exposed to either 100 mg/kg bw-d or vehicle fromD 12 to the end of lactation (post-natal day 21—PND 21) to evaluate the possi-le effects of the treatment on the female offspring. The beginning of the mothers’reatment coincided with the critical period of reproductive system development,hich continues after birth [30,31].

.3. Maternal body weight

Dams were weighed on alternate days from GD 0 until sacrifice on GD20 orhe end of lactation (PND 21) to adjust the volume of DBP to be administered and tovaluate possible maternal toxicity. Pups were weaned on PND 21 and the respectiveothers were sacrificed by decapitation on the following day.

.4. Evaluation of maternal endpoints and analysis of fetal gonads

Control (n = 5) and treated (n = 5) pregnant dams (F0) were killed by decapita-ion on GD 20. After the uterus and ovaries were removed, the number of corporautea were examined by gross morphology, and the number of implantation sites,esorptions and live and dead fetuses were recorded. From female fetuses (oneer litter) the ovaries were collected, fixed in Karnovsky (2.5% glutaraldehyde, 8%araformaldehyde), included in historesin, processed for histological analysis andtained with hematoxylin and eosin. Germ cells were counted, 3 sections per animalith 50 �m of distance among them, from control and treated ovaries and expressed

s number of cells per unit area (no. germ cells/mm2).

.5. Anogenital distance and number of nipples/areola

On PND 4, the anogenital distance (AGD, the distance between the anus and theenital tubercle) was measured in female pups, using a pachymeter. On PND 13, theumber of areolas was recorded. Observations were scored based on the presencer absence of a nipple bud or a discoloration of the skin surrounding the nipple [10].

.6. External signs of puberty onset

Beginning on PND 30, all females (F1) were evaluated daily for vaginal openingVO). The day of complete VO was recorded. From the day of VO, daily vaginal smearsere collected to detect the day of first estrus, characterized by the predominance

f cornified epithelial cells.

xicology 29 (2010) 99–105

2.7. Estrous cycle

On PND 60, in treated (n = 45 females/10 litters) and control (n = 36females/09 litters) groups, the estrous cyclicity of female rats (F1) was assessed oncells from daily vaginal smears, collected over a period of 15 days, as described byMarcondes et al. [32]. Every morning 10 �L of 0.9% saline was instilled into the vaginaand subsequently aspirated. The material was observed under light microscopy andthe estrous cycle phase was determined by cytology: predominance of nucleatedepithelial cells (proestrus); predominance of cornified epithelial cells (estrus); thepresence of cornified and nucleated epithelial cells and leukocytes (metaestrus);predominance of leukocytes (diestrus). The total frequency of each phase for everyrat observed in this period was used to calculate the total length of the proestrus,estrus, metaestrus and diestrus (in days) and the estrous cycle length.

2.8. Analysis of reproductive organs

At PND 60, ovaries and uteri were collected from rats (F1) in estrus phase (n = 5in each group), weighed on precision balance, fixed in Alfac’s solution, dehydrated inethanol and embedded in paraplast. Three sections (5 �m) per animal, with 50 �mof distance among them, were obtained, mounted on glass slides and stained withhematoxylin and eosin.

In each ovary, ovarian follicles and corpora lutea were counted in 3 sectionsper animal and expressed as number per unit area (mm2). Follicles were classifiedaccording to Borgeest et al. [33] and Talsness et al. [34]. Primordial and primaryfollicles were enumerated together; oocytes surrounded by a single layer of eithersquamous or cuboidal epithelial cells were included. Follicles were classified as pre-antral when containing 2–4 layers of granulosa cells with no antral space. Antralfollicles were classified when containing three or more layers of granulosa cells anda clearly defined antral space. Characteristics of atretic follicles included pyknoticgranulosa cells, disorganized granulosa cells, degenerating oocyte and detachmentfrom the basement membrane. In the uterus, the endometrial height was measured,in 3 sections per animal using a light microscope. In each section, five differentregions were analyzed, resulting in a total of 15 measurements per animal.

At PND 75, 8 F1 rats per group in estrus were sacrificed for the determination ofreproductive organ weights (ovaries and uterus) on precision balance and hormonalanalysis.

2.9. Hormonal analysis

Female pups (C = 8; T = 8) were sacrificed on PND 75 (F1), during the estrus phase,between 8:00 and 10:00 a.m. After decapitation, trunk blood was collected andallowed to clot on a refrigerator (4 ◦C) for 30 min. Serum was collected after cen-trifugation and stored at −20 ◦C until analysis. Serum FSH, LH and progesteroneconcentrations were measured using a double-antibody radioimmunoassay (RIA)kit (National Institute of Arthritis, Diabetes and Kidney Diseases–NIADDK, USA). Allthe samples were analyzed at the same assay to avoid inter-assay variability.

2.10. Sexual behavior

On the first estrus after PND 80, control (n = 10) and treated (n = 10) femalerats from the F1 generation were used for the mating test. Rats were maintainedunder controlled temperature conditions on an inverted 12-h light–dark cycle, forat least seven days, with food and water ad libitum. For the evaluation of female sex-ual behavior, sexually experienced males were allowed ten mounts on the femaleand the presence of lordosis was measured. Results were expressed as the lordo-sis quotient (LQ, number of lordosis/10 mounts × 100). All females were used onlyonce.

2.11. Fertility and reproductive performance of F1 female rats

This analysis was performed through natural mating. Rats from control (n = 14)and treated (n = 15) groups were placed with sexually experienced males (1:1), until3 sexual cycles (3 estrus), in the beginning of the morning, during a dark period ofthe cycle. At the end of afternoon, males and females were separated and vagi-nal smears were collected, in which initial sperm detection was determined to beGD 0. On GD 20 females were killed by decapitation. After collection of the uterusand ovaries, the number of corpora lutea (by gross morphology), implantation sites,resorptions, live fetuses and fetal weights were determined. From these results, thefollowing were determined—gestation rate: number of pregnant females/number ofinseminated females × 100; implantation rate (efficiency of implantation): implan-tation sites/corpora lutea × 100; pre-implantation loss rate: number of corporalutea − number of implantations/number of corpora lutea × 100; post-implantationloss rate: number of implantations − number of live fetuses/number of implanta-tions × 100; sex ratio: number of male fetuses/number of female fetuses × 100.

2.12. Statistics

Values are expressed in mean ± SEM and medians (Q1–Q3). For comparison ofresults between the experimental groups, Student’s t-test and Mann–Whitney testwere utilized. Differences were considered significant when p < 0.05.

M.T. Guerra et al. / Reproductive Toxicology 29 (2010) 99–105 101

Table 1Reproductive outcomes of control and DBP-treated groups dams (F0 generation).

Parameters Control (n = 5 litters) Treated (n = 5 litters)

Final body weight (g) 363.78 ± 21.77 371.56 ± 18.50Dam body weight–fetal weight (g) 312.29 ± 21.90 317.72 ± 16.04Uterus weight + fetuses (g) 51.48 ± 1.94 63.23 ± 9.06Body weight of fetuses (g) 3.04 ± 0.16 3.65 ± 0.17*

Number of corpora lutea 12.75 ± 1.03 13.00 ± 0.84Number of implants 12.25 ± 0.75 12.80 ± 0.92Number of resorptions 1.25 ± 0.63 1.20 ± 0.97Number of fetuses 11.00 ± 0.58 11.60 ± 1.70Pre-implantation loss (%)a 0 (0–3.33) 0 (0–0)Post-implantation loss (%)a 7.69 (5.77–11.54) 0 (0–9.09)

Student’s t-test. Values are expressed as mean ± S.E.M. or median (Q1–Q3).* p < 0.05.a Mann–Whitney test.

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ig. 1. Body weight (g) of rats from control (n = 9) and DBP-treated (n = 10) groupshroughout gestation and lactation. Values expressed as mean ± S.E.M.

. Results

.1. Effects on dams (F0)

None of the experimental groups showed visible signs of toxicityuring pregnancy or lactation period. At the dose level tested, DBPreatment had no effects on maternal body weight or body weightain, during pregnancy and lactation (Fig. 1).

DBP exposition, from GD 12 to GD 20, provoked an increasen fetuses’ body weight (F1), when compared to control groupTable 1). All the other reproductive parameters were comparableetween groups.

.2. Effects on the female offspring (F1)

Fig. 2 contains histological sections of fetal ovaries in the con-rol and DBP-treated groups at GD 20, showing normal aspects. Theumber of germ cells per fetal ovarian sections unit area (mm2) wasomparable between groups (n = 5/group) (C = 3.37 ± 0.23 × 103;= 2.83 ± 0.22 × 103).

There was no significant difference between the experimen-

al groups in relation to body weight at birth (g, C = 9; T = 10)C = 6.99 ± 0.14; T = 7.23 ± 0.14).

The absolute and relative anogenital distance, as well as theumber of nipples/areolas did not statistically differ (Table 2).

able 2bsolute and relative anogenital distance (PND 4) and number of nipples/areolas

PND 13) in female offspring (F1) from control and DBP-treated groups.

Parameters Control (n = 9 litters) Treated (n = 10 litters)

Absolute AGD (mm) 1.52 ± 0.03 1.51 ± 0.05Relative AGD (mm/g × 100) 13.69 ± 0.36 13.02 ± 0.58Nipples/areolas 12.08 ± 0.05 12.06 ± 0.03

tudent’s t-test. Values are expressed as mean ± S.E.M.

Fig. 2. Histopathological aspect of longitudinal sections from rat fetal gonads. (A)Control group. (B) DBP-treated group. The primordial follicles are organized in clus-ters (asterisks). 200×, HE.

Administration of DBP at 100 mg/kg-d did not affect the age of

puberty onset on F1 generation, determined by the age (days) atvaginal opening and first estrus (Table 3).

Histopathological analysis of the ovary (Fig. 3) and uterus (Fig. 4)did not reveal significant differences between the groups on F1

Table 3Analysis of puberty onset on F1 generation, determined by the age (days) at vaginalopening (V.O.) and first estrus, from control and DBP-treated groups.

Puberty onset Control (n = 9 litters) Treated (n = 10 litters)

V.O. (days) 35.25 ± 0.28 35.64 ± 0.36First estrus (days) 37.04 ± 0.79 37.46 ± 0.48

Student’s t-test. Values are expressed as mean ± S.E.M.

102 M.T. Guerra et al. / Reproductive Toxicology 29 (2010) 99–105

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Mann–Whitney test]. In the same way, the reproductive perfor-mance was not affected by the treatment with DBP in utero andduring lactation, with exception for the fetal weight, that wasincreased in the treated group, in both sexes (Table 8).

Table 5Body weight and absolute and relative organ weights in the female offspring (F1,

ig. 3. Morphological aspect of longitudinal section from rat ovary. (A) Controlroup. (B) DBP-treated group. CL: corpora lutea; arrow: antral follicle; asterisk:re-antral follicle; v: blood vessels. 100×, HE.

emale rats at PND 60 (n = 5/group). In the same way, there were noignificant differences between groups in relation to the numbersf corpora lutea, primordial/primary, pre-antral, antral and atreticollicles per unit area (mm2). No statistically differences relatedo the thickness (�m) of uterine endometrium, in estrus phase,as observed between the control group and DBP-treated groups

Table 4).Ovary and uterus (with fluid weights) (mg) on PND 60 and PND

5 were unaffected by treatment (Table 5).DBP exposure did not significantly affect serum concentration

f FSH, LH and progesterone (Table 6).Female offspring from control and DBP-treated groups exhibited

normal pattern of estrous cyclicity (Table 7).The lordosis quotient, obtained from sexual behavior test,

as not different between experimental groups (C = 9; T = 10),n PND 80 [median (Q1–Q3): C = 100 (90–100); T = 95 (90–100),

able 4varian follicles, corpora lutea counting per unit area (number/mm2) and thicknessf uterine endometrium (�m) in PND 60 female rats (F1 generation) from controlnd DBP-treated groups.

Structures Control (n = 5 rats) Treated (n = 5 rats)

Primordial and primary follicles 21.42 ± 6.60 18.85 ± 4.79Pre-antral follicles 0.82 ± 0.09 0.91 ± 0.17Antral follicles 1.06 ± 0.19 0.77 ± 0.07Atretic follicles 0.34 ± 0.06 0.32 ± 0.09Corpora lutea 0.63 ± 0.08 0.70 ± 0.09Thickness of uterine endometrium 383.90 ± 10.76 391.26 ± 22.73

tudent’s t-test. Values are expressed as mean ± S.E.M.

Fig. 4. Transverse section of uterus from rats on estrus phase. (A) Control group. (B)DBP-treated group. E: endometrium; M: miometrium; arrows: endometrial glands.100×, HE.

PND 60 and 75) from control and DBP-treated groups.

Parameters Control Treated

PND 60 (n = 5/group)Body weight (g) 202.10 ± 8.23 194.42 ± 8.86Ovaries (mg) 98.0 ± 13.78 93.60 ± 6.59Ovaries/body weight(mg/100 g)

48.00 ± 5.89 47.80 ± 1.71

Uterus + fluid (mg) 345.60 + 26.70 314.20 + 17.04Uterus + fluid/body weight(mg/100 g)

170.60 ± 11.14 162.00 ± 8.66

PND 75 (n = 8/group)Body weight (g) 221.75 ± 6.82 222.65 ± 6.57Ovaries (mg) 72.25 ± 4.12 70.75 ± 3.72Ovaries/body weight(mg/100 g)

32.37 ± 1.58 31.75 ± 1.39

Uterus + fluid (mg) 418.38 ± 30.47 411.88 ± 51.76Uterus + fluid/body weight(mg/100 g)

188.63 ± 12.61 183.88 ± 20.62

Student’s t-test. Values are expressed as mean ± S.E.M.

M.T. Guerra et al. / Reproductive To

Table 6Hormonal analysis (ng/mL) of female offspring (F1) at PND 75, from control andDBP-treated groups.

Serum concentration Control (n = 8 rats) Treated (n = 8 rats)

FSH 5.95 ± 1.08 7.11 ± 1.19LH 3.98 ± 1.03 2.52 ± 0.74Progesterone 4.75 ± 0.72 5.76 ± 1.61

Student’s t-test. Values are expressed as mean ± S.E.M.

Table 7Assessment of estrous cycle length and frequency of each phase over a 15-day periodof evaluation in the female offspring (F1, PND 60) from control and DBP-treatedgroups.

Estrous cycling Control group Treated group

Number of females evaluated/litters 36/9 45/10Estrous cycle length (days) 4.22 ± 0.16 4.35 ± 0.21Frequency of proestrus (days) 1.78 ± 0.26 1.94 ± 0.13Frequency of estrus (days) 4.60 ± 0.19 4.28 ± 0.28Frequency of metaestrus (days) 2.38 ± 0.29 2.31 ± 0.19

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Frequency of diestrus (days) 6.24 ± 0.37 6.46 ± 0.25

tudent’s t-test. Values are expressed as mean ± S.E.M.

. Discussion

Herein we evaluated the reproductive effects of DBP on theemale offspring of rats exposed in utero and during lactation. Dur-ng these critical phases of development, the fetuses and newbornsppear to be more sensitive than adults to endocrine disruption35,36], especially in the male reproductive system [14,37]. Com-ared to males, there is little information on DBP toxicity on theemale reproductive system [6,16,38]. The Centers for Disease Con-rol and Prevention (CDC) found DBP in the bodies of all 289 personsested. The researchers also discovered that the most vulnerable

opulation, women of childbearing age whose fetuses are exposedo DBP in the womb, appears to receive the highest exposures39].

able 8eproductive outcomes of F1 female offspring on PND 80 following in utero and

actation exposure to DBP.

Parameters Control (n = 14 litters) Treated (n = 15 litters)

Gestation rate (%) 93.33 100Final body weight(g)

348.00 ± 9.47 349.76 ± 8.99

Uterus + fetusesweight (g)

52.06 ± 2.59 53.29± 3.49

Ratweight − (uterusweight + fetuses)

295.94 ± 8.14 296.47 ± 6.91

Number of fetuses 11.36 ± 0.59 11.00 ± 0.79Number ofimplantations

12.14 ± 0.84 11.40 ± 0.81

Number of corporalutea

13.71 ± 0.70 12.87 ± 0.43

Fertility potentiala

(%)90.90 (85.71–100) 93.33 (88.34–100)

Pre-implantationallossa (%)

9.09 (0–14.29) 6.67 (0–11.67)

Post-implantationallossa (%)

0 (0–7.92) 0 (0–4.17)

Sex ratio (M:F) 1.30 ± 0.16 1.22 ± 0.17Fetuses weight (g) 2.83 ± 0.08 3.13 ± 0.03**

Males 2.90 ± 0.08 3.21 ± 0.04**Females 2.75 ± 0.08 3.03 ± 0.03**

Placenta weight (g) 0.45 ± 0.01 0.44 ± 0.01

tudent’s t-test (**p < 0.01). Values are expressed as mean ± S.E.M.a Mann–Whitney test, values expressed as median (Q1–Q3).

xicology 29 (2010) 99–105 103

The literature on the effects of DPB administered during ges-tation is controversial. While some authors report that there areno toxic effects to the dams [20,40], others described decreasedweight gain by mothers and significantly lower body weight in thepups [21,38,41]. In the present study DBP had no significant effectson maternal body weight gain during pregnancy and lactation, sug-gesting that the treatment at a dose of 100 mg/kg-d did not provokematernal toxicity.

In the literature there are various reports showing that differ-ent phthalates (DBP, BBP, and DIBP) are fetotoxic, as evidencedby the decrease in fetal body weight after administering the com-pounds to the dams [17,42–44]. On the contrary, the present workfound significant weight gain both in the fetuses obtained fromDBP-treated dams (F0) and, after the fertility test, from the adultoffspring (F1). It is difficult to compare our results with the liter-ature, because different doses and routes of administration wereutilized. Based on preliminary data, in these fetuses, quantita-tive analysis of the ovaries demonstrated no alterations in thenumber of germ cells/unit area. As far as we know, this is thefirst report in which germ cells have been evaluated in femalefetuses exposed to DBP. In this regard, the experiment must bereplicated to obtain more conclusive results. However, the bodyweight of pups at birth was similar between the experimentalgroups.

Several chemicals have been identified as endocrine disruptors,including natural and synthetic hormones, pesticides, plasticizersand industrial by-products [35,36]. Phthalates are considered anti-androgenic agents and also have some estrogenic activity [45–48].AGD and number of nipples are hormonally sensitive developmen-tal measures [21,49]. In the present study, these parameters werenot altered by the treatment. Our results are corroborated by oth-ers, showing that oral exposure to DBP had no significant effects onAGD in female pups [6,21].

Puberty, an event indicated by the age of vaginal opening andfirst estrus [50,51] is characterized by rapid physiological changessuch as growth spurt and maturation of gonads and the brain. Itentails the individual’s transition period from a non-reproductiveto a reproductive state. The main hormone involved in the regula-tion of puberty onset is gonadotropin-releasing hormone (GnRH) inthe hypothalamus that stimulates release of both LH and FSH fromthe pituitary [52], which act on gonads [53], leading to an increase ofthe blood estradiol level. Endocrine disruptor chemicals have beenimplicated in numerous physiological processes affecting normalreproductive health [54].

Salazar et al. [38] verified that females treated with DBP in thediet at doses of 12 and 50 mg/kg for 2.5 months prior to mating,throughout gestation and lactation, and then direct exposure ofthe pups up to 12 weeks of age, presented an evident delay intheir vaginal opening and occurrence of first estrus. Administra-tion of DEHP (di-2-ethylhexyl-phthalate), a phthalate structurallyrelated to DBP, by gavage or inhalation exposure, also delaysthe vaginal opening and first estrus [4,55]. On the other hand,females treated with DBP at doses of 0.5, 5, 50, 100 or 500 mg/kg,from GD 12 to GD 21 [10] and at a dose of 500 mg/kg, by gav-age, beginning on PND 22 [16], did not present alterations inthe age of vaginal opening. In the same manner, Lee et al. [6]demonstrated that DBP at the concentrations of 20, 200, 2000and 10,000 ppm administered through the diet, did not decreasethe vaginal opening age. These results corroborate the presentstudy, since no difference in ages of vaginal opening or first estruswas observed between the DBP-treated group and control ani-

mals, suggesting the absence of treatment effects on pubertyonset.

Phthalates can proliferate peroxisomes by activating the per-oxisome proliferator-activated receptors (PPARs), which regulateapoptosis, differentiation and the cell cycle in the rat ovary, thus

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isrupting the timing of growth and differentiation of ovarian fol-icles [56]. DEHP and DBP are known to cause polycystic ovariesn adult female rats [57], altering estradiol metabolism [58]. Ini-ial studies in vivo demonstrated that the ovary was a target siteor DEHP and that decreased estradiol production was a primaryunctional alteration of DEHP exposure [59]. As a result of the lackf ovulations, there was an absence of corpora lutea, and folli-les became cystic [57]. Nagao et al. [44] treated female rats orallyith BBP and reported that, at high doses, ovaries presented sig-ificantly decreased weight. Grande et al. [60], after administeringEHP to female rats, by daily gavage, from GD 6 to day 21 of lacta-

ion, reported a significant increase in the number of tertiary (withntral formation) atretic follicles at the 405 mg/kg-d dose in the1 generation, but no statistically significant differences related tohe thickness of uterine epithelium. In the present work, no dif-erences were found in weight or histological evaluation of ovariesnd uterus between the DBP-treated group and controls.

Sex steroids influence the growth, function and differentia-ion of female reproduction organs and make them susceptible tondocrine disruption. During the estrous cycle, fluctuating levelsf estrogen and progesterone elicit profound effects on epithelialroliferation and cytodifferentiation [61]. We found no alterations

n uterine endometrial height in the rats exposed to DPB in uterond during lactation, indicating normal hormonal responsivenessf the female reproductive tract, a finding supported by hormonalesults, whereas no difference in LH, FSH and progesterone serumevels between groups was observed. Similarly, Ma et al. [55] andagao et al. [44] also reported normal LH and FSH serum concen-

rations after the administration of two phthalates (DEHP and BBP,espectively) to female rats.

No significant changes were detected in estrous cyclicity oreproductive organ weights due to DBP exposure. These data cor-oborate those of Lee et al. [6], who reported the absence of effectsn the estrous cycle or ovarian and uterine weights in rats thateceived DBP through the diet. Gray et al. [16] also found no alter-tions in the estrous cycle after administering DBP orally to femaleats at 500 mg/kg dose. When DEHP was used at doses increas-ng from 0.015 to 405 mg/kg, no alterations were observed eithern the reproductive organ weights, or in estrous cyclicity, at alloses tested [60]. On the other hand, Nagao et al. [44] demon-trated that females exposed to BBP, by gavage, at doses of 20, 100nd 500 mg/kg, for two weeks, displayed altered estrous cyclicitynd a dose-dependent decrease in ovarian weight. Ma et al. [55]lso reported that inhalational treatment with DBP at 25 mg/m3,ncreased the number of irregular estrous cycles, indicating dis-uptive effects in females. Lee et al. [41] reported that exposure toBP administered in the diet, at different doses (20, 200, 2,000 and0,000 ppm), from GD 15 to PND 21, decreased the lordosis quotient

n rats, the posture adopted by the female in response to a mount-ng male during mating. This fact occurs due to an inappropriatexpression of some specific genes responsible for sexual differenti-tion in the hypothalamus of neonatal rats by exposure to DBP. Leet al. [6] noted disruption of female sexual differentiation as man-fested by hypoplasia of the alveolar buds of the mammary glandst prepubertal stage, decrease in the pituitary weight and changesn cell populations of pituitary hormones in the adult stage in ratsxposed to DBP from late gestation to following lactation. In thisresent study, the normal lordosis quotient, obtained by the sexualehavior test, suggests that DBP did not interfere with the femalerain sexual organization.

Our study is one of the few papers in the literature which report

nvestigation of possible effects on the female reproductive systemollowing exposure to DBP. Although this study must be seen asreliminary, we can conclude that DBP, in these experimental con-itions, did not disturb the reproductive development or functionf female rats.

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xicology 29 (2010) 99–105

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the State of Sao Paulo ResearchFoundation as part of a Masters scholarship (FAPESP, Grant2006/58835-7). The authors are grateful to José Eduardo Bozano,from the Department of Morphology, Bioscience Institute, UNESP,Botucatu/SP–Brazil, for excellent technical assistance.

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