Toxicology 205 (2004) 131–137

Effect of oral intake of dibutyl phthalate on reproductiveparameters of Long Evans rats and pre-pubertal

development of their offspring

Veronica Salazar, Carmen Castillo, Carmen Ariznavarreta,Rocıo Campon, Jesus A.F. Tresguerres∗

Laboratory of Experimental Endocrinology, Department of Physiology, School of Medicine,Complutense University of Madrid, Avda. Complutense s/n, 28040 Madrid, Spain

Available online 3 August 2004

Abstract

To investigate the influence of dibutyl phtalate (DBP) given in a soy-free rat chow on pre-pubertal development, 46 LongEvans female rats 2-month-old were divided into three experimental groups and fed three different chows: (1) control; (2) DP0.61 g/kg chow (12 mg/kg rat/day); (3) DP 2.5 g/kg chow (50 mg/kg rat/day) for 2 months. While under this treatment, they weremated and their offspring studied. Litter size and female:male ratio were recorded. At 14 days of age 6, male pups of eachgroup were sacrificed and testis and thymus were excised and weighed. Pups were weaned at 22 days of age and continued intothree experimental groups according to diet. From day 22 onwards, vaginal opening, occurrence of first estrous, and pre-putials nce waso l showed ad (r werw openingw ecrease ina t delay (0 eent BP byt©

K ct; Rat

f teructs

0

eparation were recorded.Results:The percent of pregnancies showed a marked decrease in group 3, while no differebserved between groups 1 and 2. Sex prevalence and litter size were not affected by the different diets. Pup survivaecrease when mothers were fed diet 2, but it was similar in diets 1 and 3. Pup weights on day 2 showed an evidentP < 0.05)eduction in groups 2 and 3, the decrease being more marked (P < 0.001) in group 3. On day 6, pups of group 2 showed loeights (P < 0.01) as compared with the other groups. Weight gain was significantly higher in pups of group 3. Eyeas not affected by the different diets. Fourteen-day-old male pups’ relative weight of thymus and testis showed a dnimals whose mothers had been fed diets 2 and 3. Vaginal opening and occurrence of first estrous showed an evidenP <.05;P < 0.01) in females fed diets 2 and 3. Significant differences (P < 0.001) in pre-putial separation were observed betw

reated and untreated groups.Conclusion:Offspring pre-pubertal development seems to be affected by oral intake of Dheir mothers during pregnancy, the effects being more evident in the reproductive development of male pups.

2004 Elsevier Ireland Ltd. All rights reserved.

eywords: di-n-butyl phthlate (DBP); Maternal exposure; Embryonic loss; Male reproductive tract malformation; Antiandrogenic effe

∗ Corresponding author. Tel.: +349 13941484;ax: +349 13941628.

E-mail address:guerres@med.ucm.es (J.A.F. Tresguerres).

1. Introduction

Dibutyl phthalate (DBP) is a phthalic acid es(PAEs) with extensive use in industry in such prod

300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2004.06.045

132 V. Salazar et al. / Toxicology 205 (2004) 131–137

as plastic (PVC) piping, various varnishes and lacquers,safety glass, nail polishes, paper coatings, dentalmaterials, pharmaceuticals, and plastic food wrap.Nevertheless, the largest market for these esters isin plasticizing agents for polyvinyl chloride products(Autian, 1973). The plasticizers are not irreversiblybound to the polymer matrix and, under certain useor disposal conditions, may migrate from the plas-tic to the external environment (Marx, 1972). PAEsare ubiquitous environmental pollutants because oftheir widespread manufacture, use, and disposal (Marx,1972; Mayer et al., 1972; Marsman, 1995).

Recently, PAEs such as DBP were reported to beestrogenic in a recombinant yeast screening and inestrogen-responsive human breast cancer cells (Joblinget al., 1995; Sonnenschein et al., 1995; Soto et al.,1995; Harris et al., 1997). Additionally, differentauthors have reported developmental toxicity in bothrats and mice (Ema et al., 1993, 1994, 1997, 1998;Wine et al., 1997; Saillenfait et al., 1998; Shiota etal., 1980; Lamb et al., 1987). It has been describedthat DBP exerts adverse effects on the developmentof the reproductive system of male rat offspringfollowing maternal exposure throughout pregnancyand lactation (Ema et al., 1998; Mylchreest et al.,1998). DBP administration showed antiandrogeniceffects in male offspring, such as decreased anogenitaldistance (AGD), hypospadias, non-scrotal testes,decreased testicular size, underdeveloped or absentepididymis, and decreased weight of the seminalvF ngesw hadb hichm ales P-i ,2

antfi temi P inf medc

om-p ofe dro-g del,t pro-

ductive parameters of pregnant mothers were observed.On a second stage, long-term effects of DBP admin-istered during pre- and postnatal ontogeny have beenstudied in rat reproductive organs as well as in othersystems at different stages of the postnatal develop-ment of their offspring.

2. Materials and methods

2.1. Animals

Forty-five female and ten male rats 2-month-old ofthe Long Evans strain were used in this experiment.They were maintained under controlled light on a 12-hlight–dark cycle at approximately 21–24◦C tempera-ture conditions. Animals had ad libitum access to tapwater. Female rats were divided into three differentexperimental groups (n = 15) depending on the typeof chow they were fed. Male rats were fed the samecontrol chow received by the female control group.After being fed for 2.5 months, animals of the dif-ferent groups were mated and during pregnancy, fe-males kept receiving their corresponding chows andwere housed individually. Both male and female pupswere weaned on postnatal day (PND) 22 and were di-vided again according to the chow their mothers hadbeen fed with, into three experimental groups withfree access to chow and water. Sacrifice of animalson PND14 and week 12 was performed by decapita-t thiss rac-t RD2

2

en-t dif-f d byN ya-f (2)w ntra-t e)o ratc /kgc daya

esicles and prostate gland (Mylchreest et al., 1998).urthermore, it has been observed that same chaere reported in male offspring whose motherseen given DBP on days 15–17 of pregnancy, weans that this 3 day–day period of pre-natal m

exual differentiation is a sensitive window for DBnduced reproductive tract malformations (Ema et al.000a,b).

Existing literature does not report any significndings in the development of the reproductive sysn females after chronic exposure of dams to DBood. However, a deeper insight of this aspect seeonvenient.

The present study was conducted to carry out a carative analysis of the developmental toxicologynvironmental chemicals, such as DBP, with antianenic and/or estrogenic action in a mammalian mo

he Long Evans rat. On a first stage of the study, re

ion. The experimental procedures employed intudy are in accordance with the principles and pices of the 1986 Animals Act, published in Spain (23/1988).

.2. Treatments

Female rats were given DBP at different concrations mixed with their chows. Therefore, threeerent kinds of soya-free rat chows were prepareutreco (Toledo, Spain): type (1) was a control so

ree rat chow without any kind of additives; typeas a soya-free rat chow that contained a conce

ion of DBP (D2270; Sigma-Aldrich Chimie, Francf 0.61 g/kg chow; and type (3) was a soya-freehow that contained a concentration of DBP of 2.5 ghow, corresponding to (2) 12 mg/kg body weight/nd (3) 50 mg/kg body weight/day.

V. Salazar et al. / Toxicology 205 (2004) 131–137 133

2.3. Observations

Percentage of pregnant rats was studied once mat-ing males were separated from dams. Animals’ weightswere recorded weekly. Different readings on the off-spring started on the day of delivery, that from nowonwards, will be considered postnatal day 0. OnPND0, the number of pups in each litter was recorded;on PND2, the number of pups, their sex, and theirweight were observed; on PND6, same parameterswere considered. Litter size and female:male ratio wererecorded. From PND6 onwards, eyes’ opening wasstudied. On PND14, six males from each group wereweighted and then sacrificed by decapitation; testis,thymus, and plasma were collected and processed aslater described. On PND22, pups were weaned ontodiets containing DBP at the same concentrations fedto dams. From PND22 onwards, vaginal opening andoccurrence of first estrous were studied in females andmeasurement of pre-putial separation was recorded inmales.

2.4. Tissue collection and processing

Testis and thymus were collected on PND14 of 18male pups (six for each diet). Both organs were weighedand immediately frozen in liquid nitrogen and stored at−80◦C. Plasma was also stored at−80◦C for futurehormonal determinations.

2

e wasm wasp nfi-d fi-c

3

ntalc an-i 3).T reasei an-i aveb

Table 1Maternal and reproductive findings in dams and neonatal litter

Control DBP 0.61 DBP 2.5

Weight gain (g/3 months) 69.0± 7.1 51.3± 4.8 51.2± 4.3Pregnancy (%) 81.8 81.8 58.3Litter size (number of 8.6± 0.8 8.9± 1.1 8± 1.4

animals)Pups survival (%) 72.3 60 71.4Sex prevalence 1.1 0.9 1.1

(female:male)

No statistically significant difference in the off-spring as far as litter size, percentage of pups survival,and sex prevalence in litters (female:male ratio) was de-tected. However, pups belonging to group 2 presenteda lower survival percentage (Table 1).

Pups weight was recorded on PND2 and PND6, andweight gain between PND6 and PND2 was later ob-tained. On PND2, groups 2 and 3 showed significantlylower weights (P < 0.05 andP < 0.001) as comparedwith pups born from control dams, the difference beingmore evident with the highest dose of DBP (P < 0.001versus control group).On PND6, a significant decreasewas observed only in pups from group 2 (P < 0.01versus control group). Pups from group 3 presented aweight similar to controls. Weight gain (from PND2 toPND6) was significantly higher in pups from dams fedthe high DBP concentration (Table 2).

Time for eyes’ opening did not seem to be affectedby DBP diets, and all pups opened their eyes aroundPND12. Data obtained on males sacrificed on PND14showed that body weight and thymus relative weightwere not affected by the DBP diets (Table 2), whereas

F e onP

.5. Data statistical analysis

The results are presented as the means± standardrror of the mean (S.E.M.). Comparison of groupsade using a one-way ANOVA and Duncan testerformed postoc if differences were noted. A coence level≥ 95% (P < 0.05) was considered signiant.

. Results

Weight gain of dams that received the experimehows during 2 months was lower in the group ofmals fed the highest concentration of DBP (grouphe pregnancy rate showed also a significant dec

n this group. No significant differences betweenmals that were fed either control diet or diet 2 heen found (Table 1).

ig. 1. Testis relative weight (mg/g) of male pups after sacrificND14 (∗∗∗P < 0.001 vs. control).

134 V. Salazar et al. / Toxicology 205 (2004) 131–137

Table 2Offspring pre-pubertal findings

Control DBP 0.61 DBP 2.5

Pups weight PND2 (g) 7± 0.1 6.3± 0.3∗ 5.4± 0.2∗∗Pups weight PND6 (g) 11.3± 0.2 10± 0.3∗∗∗ 11.2± 0.4Pups weight gain PND2–PND6 (g/4 days) 4.4 3.7 5.7Eyes’ opening (days) 12± 0.1 12.3± 0.1 12.1± 0.3Male pups weight PND14 (g) 24.3± 0.5 23.2± 0.7 24.8± 0.5Male pups thymus relative weight PND14 (mg/g) 4.09± 0.39 4.13± 0.32 3.69± 0.06

∗ P < 0.05 vs. control.∗∗ P < 0.001 vs. control.

∗∗∗ P < 0.01 vs. control.

testis relative weight was dramatically decreased (P< 0.001 versus control group) in pups coming fromgroups 2 and 3 (Fig. 1). No differences were observedamong the two different concentrations of DBP on thechows.

After weaning, pubertal parameters were studied inboth male and female pups. Pre-putial separation inmales was severely affected by DBP and animals thatwere born from dams fed the highest dose experienceda dramatic (P< 0.001) delay, on PND41 approximatelyversus PND38 and PND36 in lower concentrations ofDBP and control chow, respectively (Fig. 2). Femalepups presented an evident delay in their vaginal open-ing and occurrence of first estrous when they came frommothers fed DBP diets. Vaginal opening was delayedfrom approximately PND35 in control group to ap-proximately PND37 in DBP diets groups (P < 0.001versus control group) (Fig. 3). The occurrence of firstestrous was significantly delayed (P< 0.05) on femalesof the group with the highest dose of DBP, presenting a

Fv

value of approximately PND40 versus approximatelyPND38 in control animals (Fig. 4).

4. Discussion

Adverse effect of DBP on pregnant rats has been evi-denced by two parameters studied in dams: weight gainand pregnancy rate. As for the weight gain, it showed apattern of decrease in the group of dams that receivedthe highest dose of DBP. Previous studies (Ema andMiyawaki, 2001a,b) with monobutyl phthalate (MBP),a common metabolite of DBP, showed that significantdecrease in weight gain was observed in those animalsthat were given a toxic dose of MBP of 500 mg/kg.The dose administered to the animals in our studiedwas 50 mg/kg, and therefore, that’s probably the causefor just observing a slight pattern of decrease of weightgain that does not prove to be statistically significant.

F ex-p la

ig. 2. Pre-putial separation (days) in male offspring (∗∗∗P < 0.001s. control).

ig. 3. Vaginal opening (days) in female offspring from damsosed to DBP before and during pregnancy (∗P < 0.05 vs. contrond∗∗P < 0.001 vs. control).

V. Salazar et al. / Toxicology 205 (2004) 131–137 135

Fig. 4. First estrous (days) of female offspring (∗P< 0.05 vs. control).

Pregnancy rate was lower in animals that receiveda dose of 50 mg/kg of DBP. The effects of DBP onreproductive function of pregnant animals may be ex-plained by the studies ofEma et al. (2000a,b), in whichrats given DBP by gastric intubation at 0, 250, 500,750, 1000, 1250, or 1500 mg/kg showed a significantincrease in both pre- and postimplantation incidences.It was also observed by the group of Ema that uter-ine decidualization was significantly decreased. There-fore, all these findings may suggest that there is indeedearly embryonic loss due to DBP, and that it may bemediated, at least in part, via the suppression of uter-ine decidualization, an impairment of uterine function.Similar findings were reported byEma and Miyawaki,2001a,b, after administration of MBP under identicalconditions. These data suggest that MBP is also in-volved in the suppression of uterine decidualization,and that, therefore, DBP may exert its effects on em-bryonic loss via its metabolite MBP. It is also inter-esting to notice that the administration of DBP in ourstudy covered the susceptible window for resorption offetuses in females, which is days 12–17 of pregnancyas shown byEma et al. (2000a,b). However, it has beendemonstrated (Ema, 2002) that the doses of DBP thatproduce both maternal toxicity and embryonic loss aremuch higher than those producing malformation in thereproductive system of male offspring.

Apparently, the male reproductive system may bemore susceptible than other organ systems to DBPand MBP toxicity after maternal exposure. In fact, ourr ups’r de-v ed a

dramatically decreased testis relative weight whengiven any of the two doses of DBP. This decrease intesticular size has been produced by the oral intake ofDBP at two relatively low doses. These findings arein accordance with previous results (Ema et al., 1998;Mylchreest et al., 1998) that described not only adecrease in testicular weight, but also other sex-ual malformations in males such as a decrease inanogenital distance, reduced testosterone levels, in-crease in the incidence of undescended testes, hy-pospadias, non-scrotal testes, underdeveloped or ab-sent epididymis, and decreased weight of the sem-inal vesicles and prostatic gland in postnatal maleoffspring. Androgens, testosterone, and dihydrotestos-terone are critical determinants of the male phe-notype (Jost et al., 1973). Pre-natal testicular de-scent is controlled by testosterone (Spencer et al.,1991), and masculinization of external genitalia, AGD,in males is controlled by dihydrotestosterone.

Our study presented also a significant delay of ap-proximately 5 days in pre-putial separation in malepups that had been given the highest dose of DBP ascompared with control animals. It is well known thatpre-putial separation is an androgen-dependent event,and this data together with the previous findings suggestthat testosterone- and dihydrostestosterone-dependentevents are the most affected by daily in utero exposureto DBP.

The underlying mechanism of DBP-induced disrup-tion of male reproductive development is still unknown.I xe cedb ) an-t er-t ever,G ef-f me-d inceD inga . Inc c ef-f pro-d ctlyw -g ivityb th-w ingd the

esults showed a clear negative effect in male peproductive system at different stages of theirelopment. Animals sacrificed on PND14 present

n Mylchreest et al. (1999), DBP produced a complendocrine phenotype initially similar to that produy a high dose of the potent androgen receptor (AR

agonist flutamide, even if different sensitivities of cain androgen-dependent tissues were found. Howray et al. (1998)suggested that the antiandrogenic

ects of DBP observed in vivo did not appear to beiated directly at the level of androgen receptor, sBP proved to be negative in both competitive bindnd transcriptional activation assays with the ARonclusion, DBP seems to produce antiandrogeniects on male sexual differentiation, such as thoseuced by AR antagonists, without interacting direith the AR.Mylchreest et al. (1999)eventually sugested that DBP may exert its antiandrogenic acty indirectly interfering with androgen signalling paays during sexual differentiation, probably by actirectly on the fetal testes. In fact, exposure during

136 V. Salazar et al. / Toxicology 205 (2004) 131–137

pre-natal period, when imprinting of hormonal signalsis critical for normal development of the testis, mayhave disrupted regulation of Leydig cell growth anddifferentiation. Since AR expression is modulated byandrogen levels (Bentvelsen et al., 1994, 1995) andLeydig cells produce androgens, either of these mech-anisms may have been involved in the pathogenesis ofour findings. In more recent studies (Mylchreest et al.,2002), it was suggested that Leydig cell proliferation islikely a compensatory mechanism to increase testicularsteroidogenesis triggered by testosterone insufficiency.Shultz et al. (2001)carried out a study whose resultsled to the identification of several possible mechanismsby which DBP can induce its antiandrogenic effects onthe developing male reproductive tract without directlyinteracting with the AR. Its results suggested that theantiandrogenic affects of DBP are due to decreasedtestosterone synthesis, that the DBP-induced Leydigcell hyperplasia is mediated by an enhanced expres-sion of cell survival proteins (such as TRPM-2 andBcl-2) and that gonocyte degeneration may be affectedby downregulation of c-kit. These findings seem to bein accordance with unpublished data recently obtainedby our group in which testosterone plasmatic levels in12-week-old DBP-exposed animals were significantlylower as compared with control rats.

In very recent studies, more information about pos-sible mechanisms of action has been obtained.Wilsonet al. (2004)demonstrated that DBP significantly re-duced ex vivo testosterone production in neonatal an-i antr an-t in-d ultso dt fromt d int oft

BP,r ingsi akeq note ica to1 -t dn -like

chemical in the developing female rat but it only alteredandrogen-dependent processes in males during devel-opment. This article affirmed that DBO did not pro-duce any of the classical effects of estrogen exposurein females, such as delayed puberty, abnormal estrouscycles, increased uterine weight, or reduced fertility.However, data obtained in our study did show indeedan evident delay in both vaginal opening (2 days delay)and in occurrence of first estrous (2 days delay as well)in female offspring exposed to DBP during pregnancyand lactation.

On the other hand,Yu et al. (2003)studied the estro-genic activity of certain environmental endocrine dis-rupters, DBP among them, in vitro in the breast cancercell line T47D. Its results indicated that, compared withcontrol cells, DBP markedly enhanced the proliferationof T47D cells in a time-dependent and dose-dependentfashion. This might suggest that these chemicals possescertain estrogenic activity and that they may exert theiraction through estrogenic receptors.

In conclusion, it seems clear now that the develop-ment of the reproductive tract of male offspring maybe more susceptible than other organ systems to DBPtoxicity after maternal exposure. Future studies shouldaim to a better understanding of DBP’s mechanisms ofpathogenesis through antiandrogenic metabolic path-ways. The contradictory estrogenic properties of DBPshould be also deeply studied in order to shed lighton the involvement of estrogenic receptors and itsmetabolic pathways.

A

eang

R

A s: re-

B 994.at is

B Lin-F.H.,n byl tract

mals and that it was accompanied by a significeduction in the insl3 gene expression when quified by real-time RT-PCR. Testicular atrophyuced by DBP was in part explained by the resbtained byKobayashi et al. (2003)that suggeste

hat the suppression of spermatogenesis resultinghe changes in the expression of genes involvehe inhibin/activin–follistatin system may be onehe mechanisms of DBP pathogenesis.

Concerning the potential estrogenic activity of Desults are quite contradictory. On one hand, findn the studies previously mentioned seemed to muite clear that its effects are antiandrogenic andstrogenic.Jorgensen et al. (2000)evaluated estrogenctivity for DBP and its potency resulted 10(3)-0(6)-fold lower than that of 17�-estradiol. In addi

ion, Mylchreest et al. (1999)reported that DBP diot elicit the responses expected of an estrogen

cknowledgement

This work has been possible through a Europrant EUK1-CT-2002-00128 EURISKED.

eferences

utian, J., 1973. Toxicity and health threats of phthalate esterview of the literature. Environ. Health Perspect. 4, 3–26.

entvelsen, F.M., McPhaul, M.J., Wilson, J.D., George, F.W., 1The androgen receptor of the urogenital tract of the fetal rregulated by androgen. Mol. Cell. Endocrinol. 105, 21–26.

entvelsen, F.M., Brinkmann, A.O., van der Schoot, P., van derden, J.E., van der Kwast, T.H., Boersma, W.J., Schroder,Nijman, J.M., 1995. Developmental pattern and regulatioandrogens of androgen receptor expression in the urogenitaof the rat. Mol. Cell. Endocrinol. 113, 245–253.

V. Salazar et al. / Toxicology 205 (2004) 131–137 137

Ema, M., Amano, H., Itami, T., Kawasaki, H., 1993. Teratogenic eval-uation of di-n-butyl phthalate in rats. Toxicol. Lett. 69, 197–203.

Ema, M., Amano, H., Ogawa, Y., 1994. Characterization of the de-velopmental toxicity of di-n-butyl phthalate in rats. Toxicology86, 163–174.

Ema, M., Harazono, A., Miyawaki, E., Ogawa, Y., 1997. Develop-mental effects of di-n-butyl phthalate after a single administrationin rats. J. Appl. Toxicol. 17, 223–229.

Ema, M., Miyawaki, E., Kawashima, K., 1998. Further evaluation ofdevelopmental toxicity of di-n-butyl phthalate following admin-istration during late pregnancy in rats. Toxicol. Lett. 98, 87–93.

Ema, M., Miyawaki, E., Kawashima, K., 2000a. Critical period foradverse effects on development of reproductive system in maleoffspring of rats given di-n-butyl phthalate during late pregnancy.Toxicol. Lett. 111, 271–278.

Ema, M., Miyawaki, E., Kawashima, K., 2000b. Effects of dibutylphthalate on reproductive function in pregnant and pseudopreg-nant rats. Reprod. Toxicol. 14, 13–19.

Ema, M., Miyawaki, E., 2001a. Adverse effects on development ofthe reproductive system in male offspring of rats given monobutylphthalate, a metabolite of dibutyl phthalate, during late preg-nancy. Reprod. Toxicol. 15, 189–194.

Ema, M., Miyawaki, E., 2001b. Effects of monobutyl phthalate onreproductive function in pregnant and pseudopregnant rats. Re-prod. Toxicol. 15, 261–267.

Ema, M., 2002. Antiandrogenic effects of dibutyl phthalate and itsmetabolite, monobutyl phthalate, in rats. Congenit. Anom. Kyoto42, 297–308.

Gray Jr., L.E., Ostby, J.S., Mylchreest, E., Foster, P.M.D., Kelce,W.R., 1998. Dibutyl phtalate (DBP) induces antiandrogenic butnot estrogenic in vivo effects in Long Evans hooded rats. Toxicol.Sci. 42 (Suppl. 1), 176.

Harris, C.A., Henttu, P., Parker, M.G., Sumpter, J.P., 1997. The es-trogenic activity of phthalate esters in vitro. Environ. Health Per-spect. 105, 802–811.

J J.P.,lud-iron.

J 000.ls ofrspect.

J n sex–41.

K wa,eptortatinon of

L J.R.,the

Marsman, D., 1995. NTP technical report on the toxicity studiesof Dibutyl Phthalate (CAS No. 84-74-2) Administered in Feedto F344/N Rats and B6C3F1. Mice Toxic. Rep. Ser. 30, G1–G5.

Marx, J.L., 1972. Phthalic acid esters: biological impact uncertain.Science 178, 46–47.

Mayer, F.L., Stalling, D.L., Johnson, J.L., 1972. Phthalate esters asenvironmental contaminants. Nature 238 (5364), 411–413.

Mylchreest, E., Cattley, R.C., Foster, P.M., 1998. Male reproductivetract malformations in rats following gestational and lactationalexposure to di(n-butyl) phthalate: an antiandrogenic mechanism?Toxicol. Sci. 43, 47–60.

Mylchreest, E., Sar, M., Cattley, R.C., Foster, P.M., 1999. Disruptionof androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different fromflutamide. Toxicol. Appl. Pharmacol. 156, 81–95.

Mylchreest, E., Sar, M., Wallace, D.G., Foster, P.M., 2002. Fetaltestosterone insufficiency and abnormal proliferation of Leydigcells and gonocytes in rats exposed to di(n-butyl) phthalate. Re-prod. Toxicol. 16, 19–28.

Saillenfait, A.M., Payan, J.P., Fabry, J.P., Beydon, D., Langonne,I., Gallissot, F., Sabate, J.P., 1998. Assessment of the develop-mental toxicity, metabolism, and placental transfer of di-n-butylphthalate administered to pregnant rats. Toxicol. Sci. 45, 212–224.

Shiota, K., Chou, M.J., Nishimura, H., 1980. Embryotoxic effects ofdi-2-ethylhexyl phthalate (DEHP) and di-n-buty phthalate (DBP)in mice. Environ. Res. 22, 245–253.

Shultz, V.D., Phillips, S., Sar, M., Foster, P.M., Gaido, K.W., 2001.Altered gene profiles in fetal rat testes after in utero exposure todi(n-butyl) phthalate. Toxicol. Sci. 64, 233–242.

Sonnenschein, C., Soto, A.M., Fernandez, M.F., Olea, N., Olea-Serrano, M.F., Ruiz-Lopez, M.D., 1995. Development of amarker of estrogenic exposure in human serum. Clin. Chem. 41,1888–1895.

S ,as a

men-13–

S E.D.,ride

W , G.,sionstal rat

W E.,-

ealth

Y nvi-

obling, S., Reynolds, T., White, R., Parker, M.G., Sumpter,1995. A variety of environmentally persistent chemicals, incing some phthalate plasticizers, are weakly estrogenic. EnvHealth Perspect. 103, 582–587.

orgensen, M., Vendelbo, B., Skakkebaek, N.E., Leffers, H., 2Assaying estrogenicity by quantitating the expression leveendogenous estrogen-regulated genes. Environ. Health Pe108, 403–412.

ost, A., Vigier, B., Prepin, J., Perchellet, J.P., 1973. Studies odifferentiation in mammals. Recent Prog. Horm. Res. 29, 1

obayashi, T., Niimi, S., Kawanishi, T., Fukuoka, M., HayakaT., 2003. Changes in peroxisome proliferator-activated recgamma-regulated gene expression and inhibin/activin-follissystem gene expression in rat testis after an administratidi-n-butyl phthalate. Toxicol. Lett. 138, 215–225.

amb 4th, J.C., Chapin, R.E., Teague, J., Lawton, A.D., Reel,1987. Reproductive effects of four phthalic acid esters inmouse. Toxicol. Appl. Pharmacol. 88, 255–269.

oto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F.Olea, N., Serrano, F.O., 1995. The E-SCREEN assaytool to identify estrogens: an update on estrogenic environtal pollutants. Environ. Health Perspect. 103 (Suppl. 7), 1122.

pencer, J.R., Torrado, T., Sanchez, R.S., Vaughan Jr.,Imperato-McGinley, J., 1991. Effects of flutamide and finasteon rat testicular descent. Endocrinology 129, 741–748.

ilson, V.S., Lambright, C., Furr, J., Ostby, J., Wood, C., HeldGray Jr., L.E., 2004. Phthalate ester-induced gubernacular leare associated with reduced insl3 gene expression in the fetestis. Toxicol. Lett. 146, 207–215.

ine, R.N., Li, L.H., Barnes, L.H., Gulati, D.K., Chapin, R.1997. Reproductive toxicity of di-n-butylphthalate in a continuous breeding protocol in Sprague–Dawley rats. Environ. HPerspect. 105, 102–107.

u, Z., Zhang, L., Wu, D., 2003. Estrogenic activity of some eronmental chemicals. Wei Sheng Yan Jiu 32, 10–12.