Abstract

In mammals, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of AR antagonists like vinclozolin and procymidone or chemicals like di(2-ethylhexyl) phthalate (DEHP) that inhibit fetal testicular testosterone production demasculinize the males such that they display reduced anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, undescended testes, a vaginal pouch, epididymal agenesis, and small to absent sex accessory glands as adults. In addition to DEHP, di-n-butyl (DBP) also has been shown to display antiandrogenic activity and induce malformations in male rats. In the current investigation, we examined several phthalate esters to determine if they altered sexual differentiation in an antiandrogenic manner. We hypothesized that the phthalate esters that altered testis function in the pubertal male rat would also alter testis function in the fetal male and produce malformations of androgen-dependent tissues. In this regard, we expected that benzyl butyl (BBP) and diethylhexyl (DEHP) phthalate would alter sexual differentiation, while dioctyl tere- (DOTP or DEHT), diethyl (DEP), and dimethyl (DMP) phthalate would not. We expected that the phthalate mixture diisononyl phthalate (DINP) would be weakly active due to the presence of some phthalates with a 6-7 ester group. DEHP, BBP, DINP, DEP, DMP, or DOTP were administered orally to the dam at 0.75 g/kg from gestational day (GD) 14 to postnatal day (PND) 3. None of the treatments induced overt maternal toxicity or reduced litter sizes. While only DEHP treatment reduced maternal weight gain during the entire dosing period by about 15 g, both DEHP and DINP reduced pregnancy weight gain to GD 21 by 24 g and 14 g, respectively. DEHP and BBP treatments reduced pup weight at birth (15%). Male (but not female) pups from the DEHP and BBP groups displayed shortened AGDs (about 30%) and reduced testis weights (about 35%). As infants, males in the DEHP, BBP, and DINP groups displayed femalelike areolas/nipples (87, 70, and 22% (p < 0.01), respectively, versus 0% in other groups). All three of the phthalate treatments that induced areolas also induced a significant incidence of reproductive malformations. The percentages of males with malformations were 82% (p < 0.0001) for DEHP, 84% (p < 0.0001) for BBP, and 7.7% (p < 0.04) in the DINP group. In summary, DEHP, BBP, and DINP all altered sexual differentiation, whereas DOTP, DEP, and DMP were ineffective at this dose. Whereas DEHP and BBP were of equivalent potency, DINP was about an order of magnitude less active.

Exposure to synthetic environmental chemicals (e.g., DDT and its metabolites, especially p,p`-DDE, alkylphenol ethoxylates, PCBs, and dioxins) produces reproductive problems in a variety of vertebrate species via endocrine mechanisms (Ankley and Giesy, 1998; Colborn and Clement, 1992; Giesy and Snyder, 1998; Gray, 1991, 1992; Monosson, 1997; Van Der Kraak et al., 1998). Naturally occurring environmental chemicals (e.g., phytoestrogens and estrogenic mycotoxins) induce infertility in domestic animal species (Adams, 1989; Ueno, 1985) and can alter human reproductive function (Cain, 1960). Concerns over these findings have been compounded by a series of publications suggesting that in utero exposure to environmental chemicals may have contributed to the reported decline in human sperm counts, the increased incidences of urogenital malformations (e.g., hypospadias, testicular cancer, and undescended testes) (Carlsen et al., 1992), and altered sex ratio over the last 40–50 years. Sharpe and Skakkebaek (1993) hypothesized that in utero exposure to environmental estrogens could be responsible for the increased incidences of these alterations. This hypothesis is biologically plausible because hormones play critical roles as regulators of development in vertebrates, and exposure to hormonally active toxicants during sexual differentiation is known to produce abnormal reproductive phenotypes in humans and other animals (Gray, 1992; Schardein, 1993).

During mammalian sex differentiation, the androgens, testosterone (T), and the T metabolite dihydrotestosterone (DHT), produced by the fetal/neonatal male during sexual differentiation, are critical determinants of the male phenotype (Wilson, 1978). Differentiation of the Wolffian structures (e.g., the epididymis, vas deferens and seminal vesicles) is T mediated, while masculinization of the prostate and external genitalia is controlled by the more potent androgen DHT. In the central nervous system of the rat, some sex dimorphisms result from the action of estradiol, locally produced by the conversion of T by aromatase, while others, like play behavior, appear to be dependent upon androgens themselves (Gray, 1992). It appears that both T and DHT play important roles in the development of the brain in many mammals, including nonhuman primates, and other tissues like the spinal cord and levator ani muscles (T dependent). These developmental events can be altered by exposure to chemicals that bind AR and act either as hormone agonists or antagonists. Androgenic substances, like Danazol or methyltestosterone, masculinize human fetal females (i.e., female pseudohermaphroditism) (Schardein, 1993). Progestins act both as androgen antagonists in male offspring, demasculinizing them such that they display ambiguous genitalia with hypospadias (Schardein, 1993), and androgen agonists in females, virilizing the external genitalia. Laboratory studies of chemicals that inhibit or mimic the action of androgens also produce predictable alterations of sex differentiation in rodents (reviewed by Gray et al., 1999a).

In the last few years, transgenerational studies on developmental reproductive toxicity of the phthalates have demonstrated that several of these produce malformations in male rat offspring after in utero and neonatal treatment. A NOAEL of 50 mg/kg/d has been identified for DBP (Mylchreest et al., 1998, 1999, 2000), but other phthalates have been less thoroughly studied. It has been shown that the plasticizer DEHP also alters sexual differentiation in male rats in an antiandrogenic manner much like the effects of DBP (Gray et al., 1999a).

Administration of DEHP to pregnant rats by gavage at 750 mg/kg/day during the period of sexual differentiation (gestational day [GD] 14 to postnatal day [PND 3]) markedly demasculinizes and feminizes the male offspring. Treated male offspring display femalelike anogenital distance (AGD) at birth, retained nipples, cleft phallus with hypospadias, undescended testes, blind vaginal pouch, epididymal agenesis, and small to absent sex accessory glands. Arcadi et al. (1998) observed testicular histological alterations and testis weight reductions when DEHP was administered at dosage levels in the drinking water at 32.5 μl/l or 325 μl/l (estimated as 3 and 30 mg/kg/day) to the dam during gestation and lactation. Poon et al. (1997) also observed testicular effects in the male rat at relatively low dosage levels (3.7 mg DEHP/kg/day was identified as a NOAEL and 37 mg/kg/day was the LOAEL) when they administered DEHP in the diet for 90 days to young male rats (weighing 105–130 g at the start of the study). DEHP treatment caused mild vacuolation in the Sertoli cells at 36.8 mg/kg/day (500 ppm in the diet considered the LOAEL) in 7/10 males and mild seminiferous tubular atrophy at 5000 ppm in 9/10 males. In addition, subtle effects were seen in the testis of males in both the NOAEL” dose group and the group below the NOAEL. Four of ten males in each low-dose group (5 ppm or 0.37 mg/kg/day and 50 ppm or 3.7 mg/kg/day, which was used as the NOAEL) displayed minimal Sertoli cell vacuolation. The incidence of minimal Sertoli cell vacuolation (8/20) is significantly greater than the incidence in the control group (0/10) p < 0.03, Fisher`s Exact Test) and is consistent with the observation of increasingly severe Sertoli cell lesions at 500 and 5000 ppm.

Using the data from Poon et al. (1997), the Organization for Economic Cooperation and Development (OECD) estimated margin of exposure (MOE) of 19 for DEHP for children mouthing toys containing this chemical, whereas for DINP the value was 75 (EC 11/98 Opinion on Phthalate Migration, found at http://europa.eu.int/comm/food/fs/sc/sct/out19_en.html). A survey of phthalates revealed that 32 of 63 toys sampled contained DEHP, and 44 of 63 contained DINP. As a consequence of the low MOE, the European commission proposed a ban of several phthalates in childcare articles and toys (found at http://europa.eu.int/comm/dg03/press/1999/IP99829.htm), including DINP, DEHP, DNOP, DIDP, DBP, and BBP (Annex 1 to Directive 76/769/EEC and Annex IV of Directive 88/378/EEC; also, Environment News Service, 10/26/99, Environmental Data Services Ltd., London. E-mail: envdaily@ends.co.uk). The European Commission did not use the data of Arcadi et al. (1998) for the calculation of an MOE, and they did not include exposures to DEHP via other routes, although they did acknowledge that mouthing of toys might not be the major source of DEHP (see above EC references). The data from Arcadi et al. (1998) were considered as supportive of the effects reported by Poon et al. (1997).

Although in utero DEHP treatment alters androgen-dependent tissues, it does not appear to act as an androgen receptor (AR) antagonist like the pesticide vinclozolin (Gray et al., 1994; Kelce et al., 1994). In vitro studies found that neither DEHP nor the primary metabolite MEHP compete with androgens for binding to the AR at concentrations up to 10 μM (companion paper by Parks et al., 2000). In contrast to their lack of ability to bind to the AR, DEHP inhibits fetal Leydig cell testicular testosterone synthesis, reducing fetal male testosterone concentration to a female level from GD 17 to PND 2 (Parks et al., 2000).

In the current study, we compare the ability of six phthalate esters administered at one dose level to alter development of the male reproductive system, including the external genitalia (cleft phallus, hypospadias, vaginal pouch, and anogenital distance; DHT dependent), areola and nipple retention (DHT dependent), development of the ventral prostate (DHT dependent), seminal vesicles (T dependent), epididymides (T dependent), levator ani plus bulbocavernosus muscles (T dependent), and gubernacula (T dependent). We hypothesized that these phthalates would display the same structure–activity relationship as described by Foster et al. (1980) for the effects of short-term treatment with phthalate esters on the testis of the peripubertal male rat. We expected that phthalate esters with ester side chains four to six carbons in length in the ortho configuration would be antiandrogenic in utero (DEHP and BBP), while DOTP (C6-para position; also known as diethylhexyl terephthalate-DEHT), DEP (C2-ortho position), and DMP (C1-ortho position) would be inactive. In addition, we anticipated that mixture DINP would be weakly positive as a consequence of the presence of low amounts of phthalate with the ester groups (C6-7 long in the ortho position) (Fig. 1).

MATERIALS AND METHODS

Animals.

Ninety-day-old nulliparous female Sprague-Dawley rats were mated overnight (matings were confirmed by the presence of a sperm-positive vaginal smear) on proestrus and shipped from Charles River Breeding Laboratory, Raleigh, NC, on GD 2. Upon arrival, females were housed individually in clear polycarbonate cages (20 × 25 × 47 cm) with heat-treated (to eliminate resins), laboratory-grade pine shavings as bedding (Northeastern Products, Warrensburg, NY). Animals were maintained on Purina Rat Chow (5001) and filtered tap (Durham County) water (checked monthly for bacteria and every 4 months for pesticides and heavy metals) ad libitum in a room with a 14:10-h photoperiod (L/D, lights off at 11:00 AM, EST) and temperature of 20–24°C with a relative humidity of 40–50%.

Maternal Dosing.

Laboratory-grade corn oil (CAS # 8001-30-7, Sigma lot # 107h1649) was the vehicle chosen to prepare all dosing solutions. In the first block, DEHP (CAS # 117-81-7, Sigma lot # 106h3487, 99% purity), BBP (CAS # 85-68-7, Aldrich lot # 08523jq, 98% purity), DEP (CAS # 84-66-2, Sigma lot # 48h3537, 99% purity), DINP (CAS # 68515-48-0, Aldrich lot # 03005TR, purity = technical), and DMP (CAS # 131-11-3, Aldrich lot # 08812jy, 99% purity) were administered by gavage at 0 or 750 mg/kg/day in 2.5 μl of corn oil/g body weight from GD 14 (sperm detected on GD 1) until PND 3 (postcoital day 23 = PND 1). Dams (for n see Table 1) were assigned to treatments in a manner that provided similar means and variances in body weight before dosing was initiated. The dose administered included daily adjustments based on individual maternal weight changes throughout the dosing period. A second block was conducted to repeat the positive effects seen with DINP and BBP and to examine, for the first time, the effects of DOTP. In this block, pregnant rats per group were dosed, as above, with the vehicle, DEHP, BBP, DINP, or DOTP (CAS # 6422-86-2, Aldrich lot # 09704tr, 98% purity) in the vehicle.

Neonatal and Infantile Data.

Body weight and AGD were measured in offspring on PND 2 (as per Gray et al., 1999a,b) and one randomly selected male was necropsied from each litter in order to measure paired testes weight and histology (semithin plastic sections; for methods see Parks et al., 2000). In the second block, a second randomly selected male was removed for histological examination of the testes (methods as per Parks et al., 2000) in the control, DEHP, and BBP groups (Parks et al, 2000) on PND 3.

At 9–10 days of age, the inguinal region of each male pup was examined for suspected hemorrhagic testes; at 13 days of age they were examined for the presence of areolas/nipples, described as dark focal areas lacking hair (called an areola) with and without a nipple bud (in a blinded manner). Testes from control and DEHP-treated male pups were placed in Bouin's fixative for 24 h, after which they were rinsed and stored in 70% alcohol, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by Dr. N. R. Veeramachaneni for histopathological lesions.

At PND 28 male pups were weaned and housed in groups of 2 or 3 with littermates. After weaning, all male pups were examined daily for the onset of puberty, as indicated by preputial separation, and the data were analyzed using litter means.

Mounting behavior and intromissions.

Prior to necropsy, a subset of adult male rats from the control (n = 3) and DEHP (n = 6) groups were examined for the display of male mating behavior. The males examined from the DEHP group included those with (n = 2) and without (n = 4) cleft phallus and hypospadias. Each male was paired with a sexually receptive female rat for 30 min during the dark phase of the animal's diurnal cycle or until he displayed approximately 10 mounts (with or without intromission). A larger number of animals was not observed because DEHP animals displayed normal mounting behavior.

Necropsy.

At 3–5 (Block 1) or 4–7 (Block 2) months of age, males were killed by decapitation within 15 s of removal from the home cage in a separate room to control for the stress on hormone levels (see Table 2 for the numbers of animals observed). The order of necropsy was generally balanced with respect to treatments. Blood was collected for determination of serum testosterone (T) levels and the males were necropsied. The ventral surface of each male was shaved and examined for abnormalities, including the number and location of retained nipples, cleft phallus, vaginal pouch, and hypospadias. The animals were examined internally for undescended testes (with and without cranial suspensory ligaments), atrophic testes, epididymal agenesis, prostatic and vesicular agenesis, and abnormalities of the gubernacular cord. Weights taken at necropsy included body, pituitary, adrenal, kidney, liver, ventral prostate, levator ani plus bulbocavernosus muscles, seminal vesicles (with the coagulating gland and their fluids), testes, and epididymides. One testis was used for testis spermatid head count (TSHC) and one cauda epididymis was used to determine total and cauda epididymal sperm reserves as previously described (Gray et al., 1999b) for some of the rats.

Reproductive organs weights were taken from almost every male from each litter (BBP = 45 pups/11 litters, CON = 77 pups/19 litters, DEHP = 41 pups/15 litters, DEP = 12 pups/3 litters, DINP = 52 pups/14 litters, DMP = 21 pups/4 litters, DOTP = 39 pups/8 litters); nonreproductive organs were not weighed in all males due to a lack of effects on these organs in the first block (BBP = 30 pups/10 litters, CON = 45 pups/17 litters, DEHP = 23 pups/13 litters, DEP = 12 pups/3 litters, DINP = 29 pups/13 litters, DMP = 21 pups/4 litters, DOTP = 6 pups/6 litters). Some control (n = 3) and DEHP (n = 4) treated males were examined only for malformations, as above, but organ weights were not taken because these males were perfuse-fixed for histological examination of the testes, and one of the DEHP animals in this group had no siblings.

Statistical analysis of the data.

Data were analyzed using a one-way ANOVA (treatments) model on PROC GLM from SAS available on the U.S. EPA IBM mainframe computer. Post hoc tests were conducted when the overall ANOVA was significant at the p < 0.05 level using the LSMEANS available on SAS. (The LSMEANS is a two-tailed t-test which is appropriate for a priori hypotheses derived from our previous work with DEHP and DBP). For statistical purposes, the numbers/group are the number of litters, not the number of pups. Because the data analysis for maternal, pup, and adult necropsy data as two blocks or as a single study with two blocks yielded identical results, the results are presented with the blocks pooled. AGD, growth, pubertal, and necropsy data were analyzed at each age by ANOVA using litter mean values rather than individual values. AGD and organ weight data were analyzed with body weight as a covariate. Analysis of the epididymal, seminal vesicle, and ventral prostate weight data did not include zero values when complete agenesis was displayed. In addition, sex accessory tissue weight data also were excluded if the glands appeared infected (being impacted and/or discolored). The percentage of infant males displaying areolas (on a litter mean and individual basis) and the numbers of nipples/areolas per male were analyzed for treatment effects. Retained nipple data collected in adult males at necropsy were similarly analyzed. Categorical data (numbers of pups affected versus total pups and numbers of litters affected out of the total numbers of litters) were analyzed using Fisher`s Exact Test or Chi Square Test, as appropriate.

RESULTS

Maternal and Litter Effects

None of the phthalate ester treatments caused maternal death or overt maternal toxicity. Two DEP- (n reduced from 5 to 3 litters) and one DMP-treated (n reduced from 5 to 4 litters) dams died from dosing errors. DEHP and DINP treatments modestly reduced maternal weight gain, but maternal body weights were not significantly reduced at any time (Table 1). By the end of dosing on PND 3, only DEHP-treated dams displayed a significant reduction in weight change from the start of dosing. All the dams were pregnant and delivered live pups. The litter from one BBP-treated dam did not survive to 2 days of age, and another did not have any male pups at weaning (n reduced from 13 to 11 litters). The numbers of live pups at birth were not reduced by any of the phthalate ester treatments (Table 1), whereas pup weights were significantly reduced in the DEHP and BBP groups (Table 1).

Alterations in Neonatal and Infant Male Offspring

AGD, with or without covariance adjustment for body weight, was significantly reduced in male, but not female, pups in the BBP and DEHP treatment groups by about 30% at 2 days of age (Fig. 2). In contrast, both sexes displayed similar reductions in body size at this age (about 15%). These two treatments also produced a significant reduction in paired testes weights (about 35%) examined in one male per litter at this time (Fig. 3). At 8–9 days of age, several males (seven from three litters) in the DEHP group displayed hemorrhagic testes, as indicated by a darkening of the inguinal region. This also was observed in one male from the DINP treatment group. (This animal was not necropsied at this time. Hence, the observation should be considered as suspected rather than confirmed hemorrhagic testis, as several male pups in this litter died during lactation and were not necropsied).

Histological examination of some of the testes from DEHP-treated animals revealed focal interstitial hemorrhage (Fig. 4A), phagocytosis of extravasated erythrocytes, and aggregation of cells in the interstitium (Fig. 4B) by PND 2 and 3. Examination of thin sections of cell aggregates in the interstitium confirmed this phagocytic activity by the presence of erythrophagosomes. Occasional band cells and atypical cells with meandering nuclei were also evident. By PND 9–10, although some testes had focal granulomata (indicating that the hemorrhage was limited to focal areas and contained) (Fig. 4C), others manifested extensive coagulative necrosis, a sequel of hemorrhage (Fig. 4D). The latter were the grossly visible hemorrhagic testes. A closer examination of the luminal contents of blood vessels elsewhere in animals with hemorrhagic testes revealed presence of reticulocytes, indicating severe loss of erythrocytes, and therefore premature release of reticulocytes into circulation. The infarcted areas of testis (resulting from hemorrhage and coagulative necrosis) were characterized by loss of seminiferous epithelial architecture and deposits of hemosiderin. The natural progression of this lesion would be fibrosis, resulting in complete atrophy or even complete disappearance of testicular tissue. Notably, differentiating germ stem cells were also affected by DEHP. In PND 2 and PND 3 DEHP-treated animals, multinucleated gonocytes containing as many as 3–5 nuclei and some undergoing degenerative changes were observed (Figs. 4A and 4B).

Perinatal BBP, DEHP, and DINP treatments significantly induced areolas in male offspring (Fig. 5), based on analysis of either litter means or individual values. Body weights in BBP and DEHP animals were not reduced as compared to controls, either at weaning (28 days of age) or later in life (Tables 1 and 2).

Pubertal Landmarks

The age at preputial separation (PPS) was not delayed by phthalate ester treatments except in extremely malformed males in the BBP and DEHP groups (Table 1). In incomplete PPS, males typically displayed a permanent thread of tissue (persistent frenulum), a condition resulting from failure of normal liquefaction of the ectodermal ingrowth between the glans penis and prepuce. It is apparent that such alterations in PPS are not related to puberty per se but rather reflect abnormal differentiation of the prepuce. For this reason, data from the malformed males were not included in the analysis of the age at puberty.

Male Rat Sexual Behavior

When DEHP and control male rats were paired with sexually receptive females to examine male mating behavior, 4/6 treated males displayed mounts with pelvic thrusts versus 2/3 controls. Although treated males with malformations of the penis were unable to attain intromission, treated males with normal external genitalia displayed normal intromissions. Hence, these data do not support the hypothesis that PEs alter sexual differentiation of CNS with respect to male rat sexual behavior. However, our data on the levator ani/bulbocavernosus muscles would suggest that differentiation of other components of the nervous system such as the spinal cord are likely abnormal in BBP- and DEHP-treated animals.

Necropsy Data

Malformations.

The numbers of animals and litters examined for malformations are given in Table 1. DEHP, BBP, and DINP treatments induced malformations in male offspring (Fig. 6). DEHP and BBP were of equivalent potency (82 vs 84%, respectively; p < 0.0001 vs control). Both were considerably more potent than was DINP, which induced a significant level (7.7%) of reproductive malformations on an individual animal basis (4/52 pups, p < 0.05 by Chi Square on an individual basis and p <0.06 on a litter basis for 3/14) versus control (0/19 litters and 0/80 individuals).

The variability from animal to animal in the types of malformations displayed and the severity of these lesions was quite remarkable. Some litters were clearly more or less affected (or unaffected) than others and it was not unusual to find litters in which some siblings were mildly affected, displaying a single malformation, while others were severely affected with 12 malformations. Within the affected treatment groups, affected animals often displayed very different constellations of malformations. Every reproductive tissue that we examined was impacted to some degree by DEHP and BBP treatments. Even the 4 affected (out of 52) DINP males displayed diverse malformations.

The numbers of permanent nipples ranged from 1–14 (12 being the number typically found in a female rat vs zero in a male). Most DEHP and BBP and 2/52 DINP males displayed permanent nipples. In BBP- and DEHP-treated males, clefting of the phallus was complete in many males, resulting in hypospadias, while on occasion the phallus was partially cleft with epispadias. Cleft phallus and hypospadias were often concurrent with a vaginal pouch. Some DEHP- and BBP-treated males displayed complete agenesis of the ventral prostate and unilateral (one horn) or complete agenesis of the seminal vesicles and coagulating glands. Although not systematically recorded, it was noted that DEHP and BBP males often displayed agenesis of the bulbourethral glands.

Several testicular malformations were seen. These likely resulted from several different mechanisms involving:

Small (classified as less than 1.2 grams, which was smaller than any control testis) and atrophic [classified as less than 750 mg (half control size)] testes were seen in DEHP, BBP, and DINP male offspring at necropsy (Fig. 7, DINP small and atrophic testes). Several males (about 9%) in the DEHP and BBP groups displayed complete unilateral testicular agenesis/atrophy. In one case (DEHP group), both testes were absent. This lesion was not present in fetal or 2- to 3-day-old males (Parks et al., 2000).

Flaccid, fluid-filled testes were seen in the DINP, DEHP, and BBP groups. In some cases the fluid-filled testes were atrophic, lacking sperm, whereas in other cases testis size was normal or enlarged but still devoid of sperm. Fluid-filled testes were typically associated with ipsilateral agenesis of the epididymis.

Undescended testes were displayed by DEHP- and BBP-treated males. Some of these testes were free floating, completely lacking a gubernaculum, while others were attached to a thin elongated gubernaculum (ranging from 10 to 50 mm in length vs a normal length of less than 5 mm). Some scrotal testes lacked any gubernacular tissue, indicating that abdominal pressure and muscle tone around the inguinal ring were sufficient to induce scrotal retention of the testes, which then supported spermatogenesis. For example, one male displayed retained nipples, a small ventral prostate, and bilateral agenesis of the gubernacular cords, while the testes and all other tissues were normal. In one case of bilateral agenesis of the gubernaculum, the left testis had descended into the right scrotal sac, while the right testis was in the abdominal cavity. In another animal, both testes had descended into the contralateral scrotal sacs.

Specific comments on DINP males.

As indicated above, DINP induced a significant level (7.7%) of reproductive malformations on an individual animal basis (4/52, p < 0.05 by Chi Square) versus control (0/19 litters and 0/80 individuals). In addition, another male from one of the above litters in the DINP group displayed bilateral testicular atrophy, but these testes were not fluid filled or flaccid (Fig. 7). The fourth affected male in the DINP group was from a third litter and displayed unilateral epididymal agenesis with hypospermatogenesis and scrotal fluid-filled testis devoid of spermatids (Fig. 8). Another male was suspected of displaying a hemorrhagic testis at 9 days of age, but as 4 of 7 males died in this litter before 13 days of age, this effect was not confirmed by necropsy. In the DINP group, 2/52 males (from 2/14 litters) displayed nipples (no. of nipples = 1 and 6 for each of the two males) (Fig. 9). These are considered to be malformations, as permanently retained nipples represent a gross morphological alteration not normally seen in males of this species.

Body and organ weights.

Body weight and non–reproductive organ weights were not affected by perinatal PE treatment (Table 2). In the BBP and DEHP-treated male offspring, every reproductive organ was significantly affected (testis weight and sperm production [data not shown], cauda and caput epididymal weights and caudal sperm numbers [data not shown], and seminal vesicle [plus coagulating gland with fluids], ventral prostate, glans penis, and levator ani plus bulbocavernosus muscle weights). As previously reported for DEHP (Gray et al., 1999a), serum testosterone levels in adult male offspring at necropsy were not reduced by perinatal PE administration (Table 2), in marked contrast to the reductions in testosterone levels during fetal life.

DISCUSSION

The current study demonstrates that 0.75 g BBP and DEHP/kg/day severely alter sexual differentiation in the male rat with about equivalent potency. Although the effects of low dosages of BBP on male sexual differentiation are controversial (Ashby et al., 1997; Sharpe et al., 1995), it is clear that at 0.75 mg/kg BBP profoundly disrupts fetal sexual differentiation. DINP was about 10- to 20-fold less potent than BBP or DEHP, but it did display antiandrogenic activity. Better estimates of relative potency and determination of NOAELs will require execution of transgenerational dose-response studies. Although not tested here, several other studies have shown that DBP causes similar effects when administered during sexual differentiation (Gray et al., 1999a; Mylchreest et al., 1998; 1999; Wine et al., 1997). The profile of malformations for DBP is nearly identical to that for DEHP and BBP.

Mechanisms of Antiandrogen Action

The severity of the effects on the T-dependent tissues resulting from exposure to DEHP and BBP (which include a high incidence of epididymal agenesis) differs from the profile of malformations seen with AR antagonists like flutamide (Imperato-McGinley, 1992), vinclozolin (Gray et al., 1994, 1999b), and procymidone (Ostby et al., 1999). When administered at a dosage level that produces an equivalent degree of hypospadias, the AR antagonists have much less of an effect on T-dependent tissues and the testis than do the PEs. It was the differences in the constellation of effects obtained with the PEs that led us (Gray et al., 1999a) and Mylchreest et al. (1999) to hypothesize that although the PEs produce antiandrogenic effects during fetal life, they were not likely to be AR antagonists. Indeed, the recent work of Parks et al. (2000) on DEHP and of Foster et al. (1999) on DBP support this hypothesis. Our studies with DEHP (Parks et al., 2000) demonstrate that this PE acts by inhibiting fetal rat T production, which in turn lowers testicular and whole-body T to female levels. Similar studies with DBP have shown that it also reduces fetal rat T levels during sexual differentiation (Foster et al., 1999). Furthermore, the PEs and their monoester metabolites (DBP and MBP, DEHP, and MEHP; Parks et al., 2000; Foster et al., 1999) do not appear act as AR ligands in vitro at concentrations up to 10 μM.

Histological evaluation of the fetal and neonatal testis from DEHP- (Parks et al., 2000) and DBP-treated (Foster et al., 1999) rats also supports the hypothesis that the fetal testis is directly affected by PEs during sexual differentiation. Examination of the testes of fetal and neonatal rats treated with DEHP revealed abnormal fetal Leydig cell (LC) morphology. When the testes were examined for 3β HSD staining by immunohistochemistry at GD 20 and at 2–3 days of age, fetal LCs of PE-treated males appeared to be increased in number in the interstitium of the testis. In addition, Mylchreest et al. (1999) observed LC adenomas in young adult male offspring after maternal treatment with DBP during pregnancy. Taken together, these studies indicate that the several PEs act in an antiandrogenic manner distinct from that previously seen with AR antagonists such as vinclozolin and procymidone. The fact that neither the age at puberty, except in malformed males, nor the level of serum T in adulthood are altered in most PE-treated males during adult life indicates that T production by adult LCs during peripubertal and adult life is not permanently affected by perinatal PE treatment, in contrast to the marked reductions in T levels and production seen in fetal LCs during gestation. Hence, the fact that the androgen-dependent tissues are reduced in size by perinatal BBP and DBP treatments is an indication of abnormal organization of these tissues; they have been permanently imprinted and demasculinized and do not have potential to respond normally to the activational effects of androgens after puberty.

Although most of the effects of PEs on sexual differentiation in the male rat can be attributed to antiandrogenicity, it is unclear if hemorrhagic testes would result from this mechanism of action. At 8 to 9 days of age, several PE-treated males displayed hemorrhagic testis. This effect was never seen in fetal or neonatal males but this condition is likely causally related and a precursor to the absence of testicular tissue that was displayed during adulthood by about 9% of the DEHP- and BBP-exposed male offspring. One treated male displayed complete bilateral agenesis/atrophy of the testes as an adult. The fact that this male did not display hypospadias, cleft phallus, vaginal pouch, or a female tract demonstrates that LC and Sertoli cell functions were normal for at least a portion of sexual differentiation. The fact that none of the PE-exposed male offspring displayed any Mullerian duct derivatives indicates that the Sertoli cells secreted sufficient antimullerian hormone to induce regression of the progenitors of the female reproductive tract. While it is clear that the PEs target the Sertoli cell in the pubertal and neonatal male rat, causing vacuolation and premature germ cell release, the Sertoli cells of DEHP-treated fetal male rats did not display such vacuolation. It remains to be determined if their function is altered. Alteration of Sertoli cell paracrine activity could result in the alterations of Leydig and germ cells caused by perinatal PE treatment.

Detection of Developmental Reproductive Toxicity of PEs

The data presented here for the PEs BBP, DEHP, and DINP and published data on DBP (Foster et al., 1999; Gray et al., 1999a; Mylchreest et al., 1998; 1999) demonstrate that these PEs are developmental reproductive toxicants. This conclusion is in marked contrast to the conclusions of Koop and Juberg, 1999, who reported that DEHP and DINP were not specific developmental or reproductive toxicants. The discrepancy in interpretation arises from the fact that their review focused on a preponderance of negative standard teratology/developmental toxicity studies (all of which failed to identify PE-induced reproductive malformations), but failed to include some of the more recent transgenerational studies, which are positive. Several very competent scientists, using standard teratologic techniques, have reported the PEs are not teratogenic. DBP (Ema et al., 1993; 1994), BBP (Ema et al., 1992), DEHP (Hellwig et al., 1997; Thomas et al., 1986), and DINP (Hellwig et al., 1997) were negative or only induced a low incidence of malformations at maternally toxic dosage levels. Dosage levels above those used in the current study (1–4 g/kg/day) (Ema et al., 1992, 1993, 1994; Hellwig et al., 1997; Thomas et al., 1986). It is apparent from a review of the standard teratology studies that this test is inadequate for risk assessment of endocrine-disrupting chemicals. Endocrine-disrupting chemicals like the PEs, vinclozolin, procymidone, p,p` DDE, and linuron are false negatives in this assay. The inadequacy of this protocol arises from several sources. In older teratology studies the exposure period in the rat (from GD 6 to GD 15) did not include the critical period of sexual differentiation (Gray et al., 1994, 1999a, b; Wolf et al., 2000). Even studies conducted under the new developmental toxicity guidelines with the exposure continued through GD 19 (which includes some, but not all, of the period of sexual differentiation) cannot identify many of the malformations in male pups. Most reproductive malformations, induced in utero, are latent and are not apparent until after puberty or later in life (hypospadias, retained nipples, agenesis of sex accessory glands, prostatitis, and reproductive senescence). For example, in contrast to the results seen here with BBP, which induced malformations in 84% of the male offspring, administration of 2% BBP in the diet from GD 16–20 was found to be without effect on the fetal rat (Ema et al., 1992).

The multigenerational study design is currently the only test protocol that provides an adequate exposure regime and an examination of the reproductive function of the offspring. However, multigenerational studies that were conducted prior to the new U.S. EPA guidelines occasionally failed to detect malformations produced by chemicals with antiandrogenic activity, as the assessment of the reproductive system of the F1 was not thorough enough (Hodge et al., 1968; Waterman et al., 2000). In this regard, the new U.S. EPA reproductive test guidelines are a considerable improvement, as they have included several end points sensitive to endocrine disruption and require that three F1 animals per sex per litter be examined for gross malformations at weaning. However, it is unclear if the examination of weanling animals for reproductive malformations is entirely adequate, because the reproductive tissues are immature and some of them are quite small at this time. One may be able to detect severe hypospadias at weaning, but it is likely that many effects such as epispadias or agenesis of the sex accessory glands would not be detected at weaning.

For the PEs, few published multigenerational studies exist, even though these are high-production-volume chemicals that have been used for several decades. Multigenerational or transgenerational studies on DBP have been conducted in three laboratories using rats (Gray et al., 1999a; Mylchreest et al., 1998, 1999, 2000; Wine et al., 1997). These studies report similar reproductive effects in the F1 males with LOAELs of 66–100 mg/kg/day. F1 females were affected at 250 mg DBP/kg/day (Gray et al., 1999a), while adverse effects on the P0 females and males also were noted at 500 and 1000 mg DBP/kg/day (Gray et al., 1999a). DBP, DEHP, and di-n-hexyl phthalate also produced adverse reproductive effects in the continuous breeding protocol, with DEHP affecting both male and female mice (Lamb et al., 1987).

In contrast to the effects of DINP seen in the current study, a two-generation study of DINP in the diet at doses up about 500 mg/kg/day reported no adverse reproductive effects including fertility, testis weight, or histology in the F1 generation (Nikiforov et al., 1996; Waterman et al., 2000). However, this study did not include all of the androgen-sensitive end points measured herein, or examine a sufficient number of F1 animals to detect the low (7.7%) incidence of malformations that we found. Among other end points, they did not measure permanent nipples, areolas in infants, preputial separation, testis or epididymal sperm counts, or weigh androgen-dependent organs like the levator ani. In addition, neither the numbers of testes and epididymides examined histologically nor the histological results were presented, so it is unclear how many tissues were examined or what lesions, if any, were observed. Recently, we found that administration of DINP from GD 14 to PND 3 at doses of 1 and 1.5 g/kg/day reduced AGD (at 1.5) and increased the incidence of areolas in a dose-related fashion (L. E. Gray, unpublished data), confirming the weak antiandrogenic activity of DINP reported herein. Not unexpectedly, developmental toxicity studies with DINP are largely negative. In one developmental toxicity study of DINP, doses up to 1000 mg/kg/day (GD 6–15) were not teratogenic or selective developmental toxicants (Waterman et al., 1999). Another study found that the prenatal toxicity of DINP varied from mixture to mixture (their DINP with the same CAS number as used herein did not cause reproductive malformations), with only a few reproductive malformations seen at 1000 mg/kg/day [gonads in abnormal position in two fetuses (Hellwig et al., 1997)]. Herein, using end points sensitive to androgen disruption during fetal life, we found that DINP was antiandrogenic, inducing areolas and malformations (4/52) in male offspring, effects that could not possibly be assessed in these two teratology studies.

Relative Potency of PEs (PE-TEFs)

Although the current study only used a single dose level of each PE (the doses are low compared to those used in most developmental toxicity and pubertal studies of testicular toxicity), we are still able to make a preliminary estimate of the relative potency of PEs. Until recently, risk assessments were conducted on a chemical-by-chemical basis. However, as mandated under the Food Quality Protection Act (FQPA) of 1996, risk assessors are beginning to consider the risk posed by combined exposure to chemicals that act via the same mechanism. Therefore, risk assessments for PE-induced reproductive toxicity should consider these chemicals as a group and include exposures from multiple sources. Once adequate dose-response studies have been conducted, phthalate ester toxic equivalence factors (PE-TEF) can be developed for reproductive toxicity induced in utero. We propose the following preliminary PE-TEFs as follows; DEHP-TEF = 1; BBP-TEF = 1; DBP-TEF = 0.5 (based on the work of Mylchreest et al., 1999; Gray et al., 1999a); DINP (for the CAS # and lot used herein only)-TEF = 0.05–0.1; DOTP-, DMP-, AND DEP-TEF = 0. These values are presented to stimulate discussion and research about how we should estimate cumulative and aggregate risk to PEs. The PE-TEFs presented here, obviously, will be refined upon completion of adequate dose-response evaluations of the transgenerational effects of PEs.

DEP, DMP, and DOTP did not alter male rat sexual differentiation in the current study. Hence, the SAR profile for developmental reproductive toxicity of the PEs resembles that seen in young male rats (Foster et al., 1980), implying that there may be some similarity in the mechanism of action of the PEs in utero and during puberty, although the effects are very different. It appears that PEs with monoester metabolites with an ester group 4–6 carbons long are developmental reproductive toxicants. In order for a phthalate diester to be metabolized to an active monoester, the ester groups must be in the ortho position. DOTP, which is isomeric with DEHP but reportedly not metabolized to MEHP, was inactive in the current study and failed to cause testicular lesions in young adult Sprague-Dawley rats (administered as 1% in the diet for 90 days, with treatment initiated at 8 weeks of age; Barber and Topping, 1995).

As the numbers of litters and pups in the DEP and DMP groups are smaller than the other groups, we pooled the data from these two PEs for statistical purposes, given that these are both short- chain esters and we anticipated no effect from either. When the data were examined in this manner, the values for the DEP plus DMP (7 litters and 32 males) male offspring did not differ from controls. Our confidence in the DEP and DMP data are high, as these PEs did not induce areolas as did DINP, which was active but relatively weak as compared to BBP and DEHP. Nevertheless, as high levels of MEP, the monoester of DEP, have been detected in human urine (Brock et al., 2000), we plan to reexamine this PE to substantiate the lack of effect reported here.

Species-Specific PE-Induced Testicular Toxicity?

Koop and Juberg (1999) dismissed the reproductive and developmental effects of the PEs as irrelevant due to a species-specific peroxisome proliferator–activated receptor α (PPARα) mechanism, with effects occurring only in a few strains of rats and mice. However, a thorough review of the literature indicates otherwise. First, the testicular toxicity of the PEs appears to be unrelated to the species-specific expression of PPARα, as evidenced by the display of testicular pathology in DEHP-treated PPARα knockout mice (Ward et al., 1998). Second, a broad range of vertebrate species display testicular toxicity after PE treatment during development. The PEs have been shown to cause testicular alterations in numerous mammalian species, as long as the exposure included in utero or pubertal development. Affected vertebrates include several strains of rat (Long Evans, Sprague-Dawley, and Wistar), mice (including PPARα knockouts; Ward et al., 1998), hamsters (severe seminiferous tubular atrophy in most males after MEHP-treatment; Gray et al., 1982), ferrets (Lake et al., 1976), guinea pigs (Gray et al., 1982), and rabbits (Higuchi et al., 1999). In addition to mammals, fish (Patyna et al., 1999) and frogs (Higuchi et al., 1999) also display adverse reproductive outcomes when exposed to PEs during development. In fact, to date there are no studies of DEHP or MEHP that display negative results when the dosing regime includes the perinatal or pubertal periods of life and a sufficient number of animals is adequately evaluated. A study of DEHP in the nonhuman primate, which dosed four adult male common marmosets/group with DEHP at 100, 500, and 2500 mg/kg for 13 weeks, did not observe any testicular effects (Kurata et al., 1998). However, fetal and prepubertal nonhuman primates have not been similarly evaluated. Even in the rat, a species sensitive to PE-induced testicular damage during fetal and pubertal life, the testis is much less sensitive to the effects of PEs during adult life.

PE Exposures in Humans and Rats as Related to the Current Study

Although the current focus is on PEs in toys, these chemicals are ubiquitous in the environment. Some groups have particularly high DEHP exposures, with serum levels of MEHP in the ppm range (i.e., dialysis patients, transfusions, and occupational exposures). In addition, it was recently reported that human urine contained surprisingly high (ppm levels) of phthalate monoesters from as yet unidentified sources (Blount et al., 2000). A study of the exposure of newborn infants to DEHP and MEHP resulting from transfusions found plasma levels of DEHP between 3.4 and 11.1 μg/ml, while MEHP ranged from 2.4 to 15.1 μg/ml (Sjoberg et al., 1985a). Plonait et al. (1993) observed similar levels of DEHP in the serum of newborn infants after transfusion (6.1–21.6 μg DEHP/ml serum). For comparison, in male rats the concentrations of DEHP and MEHP in the serum 3 h after a single oral dose of 2.8 g/kg, which induces testicular lesions, are only about 4-fold higher than the levels in infants on dialysis, being 8.8 ± 1.7 and 63.2 ± 8.7 μg/ml, respectively (Teirlynck and Belpaire, 1985). In another study, Sjoberg et al. (1985b) reported that the testicular damage caused by treatment with 1 g DEHP/kg for 14 days to 25-day-old rats (40- and 60-day-old rats were unaffected) was associated with concentrations of MEHP in plasma ranging from 48 to 152 μg/ml. As the dosing regime used by Sjoberg et al. (1985b) is similar to that employed in the current study (0.75 g/kg/day for 12 days), which induced malformations in more than 80% of the male offspring, we would predict that maternal serum levels of MEHP in the current study would be about 35–115 μg/ml, which would be about 10-fold higher than the levels seen in dialysis patients. Assuming that administered dose is proportional to serum MEHP levels in the rat, we also would expect serum MEHP levels in the rat at the LOAEL (37 mg/kg/day) and NOAEL would be equivalent to those seen in dialysis patients. It is evident that dose-response studies that examine the effects of exposure to low levels of PEs are required and that these studies also should examine target tissue levels of DEHP and MEHP so they can be linked to known human exposure scenarios. Future animal studies should also include a sufficiently broad range of androgen-sensitive responses and use a sufficient number of animals such that malformations like those seen here in the DINP group (7.7% malformed) can be statistically detected.

Conclusions

The PEs DEHP, DBP, BBP, and DINP alter male rat sexual differentiation in an antiandrogenic fashion. While the specific mechanism of action remains to be identified, PE-induced reductions in fetal T levels result in malformations of androgen-dependent tissues. The structure-activity relationship for these fetal effects resembles that for pubertal testicular toxicity, implying that similar cellular and molecular targets are involved and that this target remains susceptible until fully differentiated after puberty, when reproductive effects can no longer be easily induced. Given that PE-induced reproductive lesions are displayed by a wide range of vertebrate species and the role of androgens and steroid hormone synthesis are highly conserved throughout the class Mammalia, it is premature to conclude that PE-induced alterations of sexual differentiation would also not be induced in the human if the male was exposed to concentrations of active PE metabolites during critical stages of reproductive development. Until we are certain of the precise mechanism responsible for PE-induced alteration of testicular function in the rat, it is impossible to accurately define the critical window of susceptibility in humans.

TABLE 1

Maternal and Neonatal Pup Effects of Various Phthalate Esters (PE) after Perinatal Maternal Exposure (GD 14–PND 3) to 0.75 g PE/kg/day on the Sprague-Dawley Rat

 Control BBP DEHP DINP DEP DMP DOTP  
Note. Values are litter means ± standard errors. 
* Indicates value differs from control by p < 0.05. 
** Indicates p < 0.01. 
Numbers of dams assigned to this study Block 1/Block 2 9/10 5/8 7/9 6/8 5/0 5/0 0/8  
Numbers of dams died 0  
Numbers of dams with live pups at day 2 19 13 16 14 8  
Numbers of dams with live pups at weaning 19 11 16 14 8  
Maternal weight GD 14 Block 1 (g) 303 ± 5.2 302 ± 4.8 303 ± 7.1 305 ± 5.2 301 ± 4.3 302 ± 4.7 —  
Maternal weight GD 14 Block 2 (g) 310 ± 4.0 309 ± 5.1 310 ± 3.9 309 ± 4.8 — — 309 ± 4.8  
Maternal weight gain to GD 21 (g) 104 ± 3.7 110 ± 5.7 79.3** ± 4.4 89.6* ± 5.0 106 ± 7.1 102 ± 5.6 104 ± 5.6  
Maternal weight gain to PND 3 (g) 15.4 ± 3.6 7.5 ± 3.3 0.12** ± 3.0 10.5 ± 4.8 23.7 ± 4.9 22.5 ± 5.8 3.2 ± 4.8  
Number of Live pups Day 1 13.5 ± 0.4 12.8 ± 1.1 13.3 ± 0.4 12.4 ± 0.7 14.7 ± 0.3 13.5 ± 0.9 13.1 ± 0.6  
Litter mean pup weight at birth (g) 6.84 ± 0.06 5.78** ± 0.18 5.78** ± 0.22 6.93 ± 0.11 6.60 ± 0.13 6.59 ± 0.24 6.80 ± 0.14  
Litter mean male pup weight at weaning (g) (no. pups/litters) 83.2 ± 1.4 (82/19) 87.8 ± 3.1 (48/11) 82.7 ± 2.9 (58/16) 85.7 ± 2.8 (52/14) 84.6 ± 8.5 (14/3) 80.7 ± 2.1 (21/4) 82.3 ± 4.5 (40/8)  
Litter mean male age at puberty (PPS) 43.5 ± 0.2 (81/19) 44.8 ± 1.0 (34/10) 44.5 ± 0.9 (37/10) 42.9 ± 0.5 (49/14) 42.7 ± 1.1 (13/3) 44.4 ± 0.8 (21/4) 42.8 ± 0.4 (40/8)  
Incomplete PPS due to genital malformations (No. without PPS/no. males observed) 9/46** 19/56** 
 Control BBP DEHP DINP DEP DMP DOTP  
Note. Values are litter means ± standard errors. 
* Indicates value differs from control by p < 0.05. 
** Indicates p < 0.01. 
Numbers of dams assigned to this study Block 1/Block 2 9/10 5/8 7/9 6/8 5/0 5/0 0/8  
Numbers of dams died 0  
Numbers of dams with live pups at day 2 19 13 16 14 8  
Numbers of dams with live pups at weaning 19 11 16 14 8  
Maternal weight GD 14 Block 1 (g) 303 ± 5.2 302 ± 4.8 303 ± 7.1 305 ± 5.2 301 ± 4.3 302 ± 4.7 —  
Maternal weight GD 14 Block 2 (g) 310 ± 4.0 309 ± 5.1 310 ± 3.9 309 ± 4.8 — — 309 ± 4.8  
Maternal weight gain to GD 21 (g) 104 ± 3.7 110 ± 5.7 79.3** ± 4.4 89.6* ± 5.0 106 ± 7.1 102 ± 5.6 104 ± 5.6  
Maternal weight gain to PND 3 (g) 15.4 ± 3.6 7.5 ± 3.3 0.12** ± 3.0 10.5 ± 4.8 23.7 ± 4.9 22.5 ± 5.8 3.2 ± 4.8  
Number of Live pups Day 1 13.5 ± 0.4 12.8 ± 1.1 13.3 ± 0.4 12.4 ± 0.7 14.7 ± 0.3 13.5 ± 0.9 13.1 ± 0.6  
Litter mean pup weight at birth (g) 6.84 ± 0.06 5.78** ± 0.18 5.78** ± 0.22 6.93 ± 0.11 6.60 ± 0.13 6.59 ± 0.24 6.80 ± 0.14  
Litter mean male pup weight at weaning (g) (no. pups/litters) 83.2 ± 1.4 (82/19) 87.8 ± 3.1 (48/11) 82.7 ± 2.9 (58/16) 85.7 ± 2.8 (52/14) 84.6 ± 8.5 (14/3) 80.7 ± 2.1 (21/4) 82.3 ± 4.5 (40/8)  
Litter mean male age at puberty (PPS) 43.5 ± 0.2 (81/19) 44.8 ± 1.0 (34/10) 44.5 ± 0.9 (37/10) 42.9 ± 0.5 (49/14) 42.7 ± 1.1 (13/3) 44.4 ± 0.8 (21/4) 42.8 ± 0.4 (40/8)  
Incomplete PPS due to genital malformations (No. without PPS/no. males observed) 9/46** 19/56** 
TABLE 2

Effects of Various Phthalate Esters on Male SD rat Offspring Body and Organ Weights and Serum Testosterone Levels after Perinatal Maternal Exposure (GD 14–PND 3) to 0.75 g/kg/day

 Control BBP DEHP DINP DEP DMP DOTP  
Note. Weight data are litter mean values ± standard errors. 
* Indicates value differ from control by p < 0.01. 
No. litters examined for repro/nonrepro organ wts. 19/17 11/10 15/15 14/13 3/3 4/4 8/6  
No. litters examined for malformations 19 11 16 14 4/4  8  
No. males: organ wts. (repro/nonrepro) 77/45 45/30 41/23 52/29 12/12 21/21 39/6  
No. Males examined for malformations 80 45 45 52 12 21 39  
Testes (mg) 3508 ± 53 2718* ± 300 2689* ± 241 3511 ± 72 3403 ± 128 3523 ± 86 3584 ± 91  
LABC (mg) 1275 ± 22 843* ± 74 851* ± 69 1211 ± 27 1287 ± 34 1234 ± 39 1301 ± 42  
Seminal vesicle plus CG (mg) 1857 ± 45 1154* ± 136 1184* ± 215 1725 ± 46 1637 ± 99 1798 ± 101 2082 ± 89  
Ventral prostate (mg) 685 ± 21 398* ± 54 511* ± 59 678 ± 25 642 ± 9.0 677 ± 15 727 ± 31  
Glans penis (mg) 108 ± 1.3 88* ± 4.0 81* ± 4.9 106 ± 1.8 111 ± 4.8 105 ± 5.3 110 ± 1.8  
No. nipples per male 5.1* ± 0.9 6.3* ± 1.1 0.11 ± 0.09 0  
Paired epididymides (mg) 1293 ± 18 966* ± 67 945* ± 82 1269 ± 21 1229 ± 25 1251 ± 26 1343 ± 23  
Cauda epididymis (mg) 312 ± 5.3 182* ± 21 180* ± 29 307 ± 6.8 288 ± 11 295 ± 9.8 330 ± 7.7  
Caput-corpus epididymis (mg) 333 ± 5.3 245* ± 20 278* ± 18 321 ± 7.5 315 ± 4.8 318 ± 12 343 ± 6.5  
Body weight (g) 613 ± 17 607 ± 30 606 ± 16 609 ± 19 547 ± 13 547 ± 15 659 ± 18  
Paired kidney (mg) 3754 ± 83 3510 ± 146 3516 ± 124 3744 ± 100 3662 ± 233 3635 ± 116 3959 ± 112  
Liver (g) 20.1 ± 0.8 18.8 ± 0.9 18.5 ± 0.9 21.0 ± 0.9 18.9 ± 1.3 19.9 ± 0.8 22.3 ± 1.5  
Pituitary (mg) 14.9 ± 0.45 15.0 ± 0.53 15.0 ± 0.54 15.2 ± 0.51 14.2 ± 1.0 14.6 ± 0.39 14.0 ± 0.34  
Adrenals (mg) 58.0 ± 1.9 57.3 ± 1.9 56.7 ± 1.9 53.7 ± 1.8 50.2 ± 2.9 54.2 ± 3.4 59.2 ± 2.1  
Serum T (ng/ml) 1.15 ± .13 1.45 ± 0.24 1.33 ± 0.16 1.89 ± 0.51 1.79 ± 0.37 1.40 ± 0.20 0.79 ± 0.12 
 Control BBP DEHP DINP DEP DMP DOTP  
Note. Weight data are litter mean values ± standard errors. 
* Indicates value differ from control by p < 0.01. 
No. litters examined for repro/nonrepro organ wts. 19/17 11/10 15/15 14/13 3/3 4/4 8/6  
No. litters examined for malformations 19 11 16 14 4/4  8  
No. males: organ wts. (repro/nonrepro) 77/45 45/30 41/23 52/29 12/12 21/21 39/6  
No. Males examined for malformations 80 45 45 52 12 21 39  
Testes (mg) 3508 ± 53 2718* ± 300 2689* ± 241 3511 ± 72 3403 ± 128 3523 ± 86 3584 ± 91  
LABC (mg) 1275 ± 22 843* ± 74 851* ± 69 1211 ± 27 1287 ± 34 1234 ± 39 1301 ± 42  
Seminal vesicle plus CG (mg) 1857 ± 45 1154* ± 136 1184* ± 215 1725 ± 46 1637 ± 99 1798 ± 101 2082 ± 89  
Ventral prostate (mg) 685 ± 21 398* ± 54 511* ± 59 678 ± 25 642 ± 9.0 677 ± 15 727 ± 31  
Glans penis (mg) 108 ± 1.3 88* ± 4.0 81* ± 4.9 106 ± 1.8 111 ± 4.8 105 ± 5.3 110 ± 1.8  
No. nipples per male 5.1* ± 0.9 6.3* ± 1.1 0.11 ± 0.09 0  
Paired epididymides (mg) 1293 ± 18 966* ± 67 945* ± 82 1269 ± 21 1229 ± 25 1251 ± 26 1343 ± 23  
Cauda epididymis (mg) 312 ± 5.3 182* ± 21 180* ± 29 307 ± 6.8 288 ± 11 295 ± 9.8 330 ± 7.7  
Caput-corpus epididymis (mg) 333 ± 5.3 245* ± 20 278* ± 18 321 ± 7.5 315 ± 4.8 318 ± 12 343 ± 6.5  
Body weight (g) 613 ± 17 607 ± 30 606 ± 16 609 ± 19 547 ± 13 547 ± 15 659 ± 18  
Paired kidney (mg) 3754 ± 83 3510 ± 146 3516 ± 124 3744 ± 100 3662 ± 233 3635 ± 116 3959 ± 112  
Liver (g) 20.1 ± 0.8 18.8 ± 0.9 18.5 ± 0.9 21.0 ± 0.9 18.9 ± 1.3 19.9 ± 0.8 22.3 ± 1.5  
Pituitary (mg) 14.9 ± 0.45 15.0 ± 0.53 15.0 ± 0.54 15.2 ± 0.51 14.2 ± 1.0 14.6 ± 0.39 14.0 ± 0.34  
Adrenals (mg) 58.0 ± 1.9 57.3 ± 1.9 56.7 ± 1.9 53.7 ± 1.8 50.2 ± 2.9 54.2 ± 3.4 59.2 ± 2.1  
Serum T (ng/ml) 1.15 ± .13 1.45 ± 0.24 1.33 ± 0.16 1.89 ± 0.51 1.79 ± 0.37 1.40 ± 0.20 0.79 ± 0.12 
FIG. 1.

Structure of phthalate esters which did (upper panel) or did not (middle panel) alter sexual differentiation of the male rat. Structure of dibutyl phthalate (DBP), which also alter sexual differentiation of the rat and rabbit, and the presumptive active metabolites of DBP (MBP) and DEHP (MEHP).

FIG. 1.

Structure of phthalate esters which did (upper panel) or did not (middle panel) alter sexual differentiation of the male rat. Structure of dibutyl phthalate (DBP), which also alter sexual differentiation of the rat and rabbit, and the presumptive active metabolites of DBP (MBP) and DEHP (MEHP).

FIG. 2.

Maternal DEHP and BBP treatments (0.75 g/kg/day from day 14 of gestation to day 3 of lactation) reduced anogenital distance (mm) in male but not female rat pups. Both of these phthalate esters reduced the sexual dimorphism in this trait by about 50%; **p < 0.001 for litter-based values, including body weight as a covariate in the analysis of these data.

FIG. 2.

Maternal DEHP and BBP treatments (0.75 g/kg/day from day 14 of gestation to day 3 of lactation) reduced anogenital distance (mm) in male but not female rat pups. Both of these phthalate esters reduced the sexual dimorphism in this trait by about 50%; **p < 0.001 for litter-based values, including body weight as a covariate in the analysis of these data.

FIG. 3.

Maternal DEHP and BBP treatments (0.75 g/kg/day from day 14 of gestation to day 3 of lactation) reduced paired testes weights in male offspring (1/litter) at 2 days of age, including body weight as a covariate in the analysis of these data.

FIG. 3.

Maternal DEHP and BBP treatments (0.75 g/kg/day from day 14 of gestation to day 3 of lactation) reduced paired testes weights in male offspring (1/litter) at 2 days of age, including body weight as a covariate in the analysis of these data.

FIG. 4.

Photomicrographs of testicular sections of rats exposed to DEHP at 750 mg/kg/day between GD 14 and PND 3 showing the progression of the testicular lesions from PND 2 to PND 10. (A) Testis at PND 2 stained with toluidine blue. Hemorrhage is seen, as evidenced by extravasated erythrocytes in the interstitium. Note aggregation of cells predominantly appearing to be fibroblasts and histiocytes. Phagocytosis of erythrocytes and erythrophagosomes are seen (arrows). Also note the presence of multinucleated gonocytes in seminiferous tubules (large arrow). (B) Testis at PND 3 stained with toluidine blue. Note encapsulated clustering of cells in the interstitium (arrow heads). Phagocytosis (arrow) and hemosiderin resulting from degraded erythrocytes within the cells are evident. Also note multinucleated gonocyte in the seminiferous tubule (large arrow). (C and D) Testes at PND 10 stained with hematoxylin and eosin. Formation of focal granulomata in the interstitium (C) and coagulative necrosis of the entire testis (D). When hemorrhage was limited to focal areas of the interstitium, localized granulomatous reaction was observed with remaining areas of the testis appearing normal; if the hemorrhage was multifocal and extensive, coagulative necrosis involving both interstitial and seminiferous epithelial elements was observed.

FIG. 4.

Photomicrographs of testicular sections of rats exposed to DEHP at 750 mg/kg/day between GD 14 and PND 3 showing the progression of the testicular lesions from PND 2 to PND 10. (A) Testis at PND 2 stained with toluidine blue. Hemorrhage is seen, as evidenced by extravasated erythrocytes in the interstitium. Note aggregation of cells predominantly appearing to be fibroblasts and histiocytes. Phagocytosis of erythrocytes and erythrophagosomes are seen (arrows). Also note the presence of multinucleated gonocytes in seminiferous tubules (large arrow). (B) Testis at PND 3 stained with toluidine blue. Note encapsulated clustering of cells in the interstitium (arrow heads). Phagocytosis (arrow) and hemosiderin resulting from degraded erythrocytes within the cells are evident. Also note multinucleated gonocyte in the seminiferous tubule (large arrow). (C and D) Testes at PND 10 stained with hematoxylin and eosin. Formation of focal granulomata in the interstitium (C) and coagulative necrosis of the entire testis (D). When hemorrhage was limited to focal areas of the interstitium, localized granulomatous reaction was observed with remaining areas of the testis appearing normal; if the hemorrhage was multifocal and extensive, coagulative necrosis involving both interstitial and seminiferous epithelial elements was observed.

FIG. 5.

Maternal treatment with DEHP, BBP, or DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with areolas (with and without nipple buds); **p < 0.01 based on litter means analysis.

FIG. 5.

Maternal treatment with DEHP, BBP, or DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with areolas (with and without nipple buds); **p < 0.01 based on litter means analysis.

FIG. 6.

Maternal treatment with DEHP, BBP or DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with malformations of the androgen-dependent organs and testes on an individual (as shown here) or litter basis.

FIG. 6.

Maternal treatment with DEHP, BBP or DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with malformations of the androgen-dependent organs and testes on an individual (as shown here) or litter basis.

FIG. 7.

Maternal treatment with DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with reproductive malformations on an individual (4/52, as shown here). The lesions seen in one male from the DINP group are displayed here. This male displayed paired testicular and epididymal atrophy and the anterior portion of the right testis appeared malformed (top right panel). A control testis and epididymis is provided in the top left for comparison. Both left and right testes from this DINP-treated male display atrophic tubules that lack any evidence of spermatogenesis (middle right and left panels, respectively). In addition, the lumen of both the left and right caudae epididymidis from this male contain cellular debris and reduced numbers of sperm.

FIG. 7.

Maternal treatment with DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with reproductive malformations on an individual (4/52, as shown here). The lesions seen in one male from the DINP group are displayed here. This male displayed paired testicular and epididymal atrophy and the anterior portion of the right testis appeared malformed (top right panel). A control testis and epididymis is provided in the top left for comparison. Both left and right testes from this DINP-treated male display atrophic tubules that lack any evidence of spermatogenesis (middle right and left panels, respectively). In addition, the lumen of both the left and right caudae epididymidis from this male contain cellular debris and reduced numbers of sperm.

FIG. 8.

Maternal treatment with DINP (0.75 g/kg/day from day 14 of gestation to day 3 of lactation) significantly increased the incidence of male offspring with reproductive malformations on an individual (4/52, as shown here). A photomicrograph of an undiluted sample of fluid from cauda epididymidis of a control (upper panel with sperm) and DINP male (lower panel), resulting from caput/corpus epididymal agenesis and a fluid-filled testis, is shown here.

FIG. 8.

Maternal treatment with DINP (0.75 g/kg/day from day 14 of gestation to day 3 of lactation) significantly increased the incidence of male offspring with reproductive malformations on an individual (4/52, as shown here). A photomicrograph of an undiluted sample of fluid from cauda epididymidis of a control (upper panel with sperm) and DINP male (lower panel), resulting from caput/corpus epididymal agenesis and a fluid-filled testis, is shown here.

FIG. 9.

Maternal treatment with DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with reproductive malformations on an individual (4/52, as shown here). One of the six permanent nipples seen in one male from the DINP group is shown here.

FIG. 9.

Maternal treatment with DINP (0.75 g/kg/day from GD 14 to day 3 of lactation) significantly increased the incidence of male offspring with reproductive malformations on an individual (4/52, as shown here). One of the six permanent nipples seen in one male from the DINP group is shown here.

1
To whom correspondence should be addressed. Fax: (919) 541-4041. E-mail: gray.earl@epa.gov.
The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

We would like to thank Christy Lambright, Cynthia Wolf and Carol Moeller for their excellent technical assistance in this project.

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