Effects of Dibutyl Phthalate in Male Rabbits following in Utero, Adolescent, or Postpubertal Exposure

Ty T. Higuchi*, Jennifer S. Palmer*, L. Earl Gray, Jr.{dagger} and D. N. Rao Veeramachaneni*,1

* Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523-1683; and {dagger} National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

Received November 11, 2002; accepted January 15, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated sequelae in male rabbits following exposure to dibutyl phthalate (DBP) at a dose level known to adversely affect testicular function in rodents without causing systemic toxicity. Because rabbits have a relatively long phase of reproductive development simulating better than rodents the reproductive development of humans, and because the use of rabbits facilitates multiple evaluations of mating ability and seminal quality, we used this animal model. Rabbits were exposed to 0 or 400 mg DBP/kg/day in utero (gestation days [GD] 15–29) or during adolescence (postnatal weeks [PNW] 4–12), and male offspring were examined at 6, 12, and 25 weeks of age. Another group was exposed after puberty (for 12 weeks) and examined at the conclusion of exposure. The most pronounced reproductive effects were in male rabbits exposed in utero. Male offspring in this group exhibited reduction in numbers of ejaculated sperm (down 43%; p < 0.01), in weights of testes (at 12 weeks, down 23%; p < 0.05) and in accessory sex glands (at 12 and 25 weeks, down 36%; p < 0.01 and down 27%; p < 0.05, respectively). Serum testosterone levels were down (at 6 weeks, 32%; p < 0.05); a slight increase in histological alterations of the testis (p < 0.05) and a doubling in the percentage (from 16 to 30%, p < 0.01) of abnormal sperm; and 1/17 males manifesting hypospadias, hypoplastic prostate, and cryptorchid testes with carcinoma in situ-like cells. In the DBP group exposed during adolescence, basal serum testosterone levels were reduced at 6 weeks (p < 0.01) while at 12 weeks, testosterone production in vivo failed to respond normally to a GnRH challenge (p < 0.01). In addition, weight of accessory sex glands was reduced at 12 weeks but not at 25 weeks after a recovery period; there was a slight increase in the percentage of abnormal sperm in the ejaculate; and 1/11 males was unilaterally cryptorchid. In both of these DBP-treated groups, daily sperm production, epididymal sperm counts, mating ability, and weights of body and nonreproductive organs were unaffected. Thus, DBP induces lesions in the reproductive system of the rabbit, with the intrauterine period being the most sensitive stage of life.

Key Words: DBP; reproductive toxicity; abnormal male sexual differentiation; ejaculated sperm counts; atypical germ cells; semen quality; testosterone.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phthalates, used as plasticizers to produce polymeric materials and a variety of consumer products, are among the highest-volume chemicals produced with an estimated 2–8 million tons per annum (WHO, 1992Go). Environmental contamination or exposure occurs as a result of the manufacture, use, or disposal by leaching out of plastic products or directly through one of the numerous consumer products (cosmetics, pharmaceuticals, etc.) (Autian, 1973;GoJaeger and Rubin, 1972;GoPerwack et al., 1981Go). One of the phthalates, dibutyl phthalate (DBP), has been found in the sediment, water, air, biota, and even in stored foods (Aurela et al., 1999;GoBove et al., 1978;GoGiam et al., 1980;GoPetersen and Breindahl, 2000Go). In a recent study, urine samples from a human reference population of 289 adults contained 7 to 294 ng/ml monobutyl phthalate reflecting exposure to DBP (Blount et al., 2000Go). Furthermore, serum concentrations of DBP ranging from 15 to 276 mg/l were detected in 28 of 41 (68%) of females (6 months to 8 years of age) diagnosed with premature thelarche (a physical change characteristic of puberty before age 8) versus none in controls (Colon et al., 2000Go).

DBP is known to be a developmental and reproductive toxicant. In rodents, teratogenic response to DBP was observed only at maternally toxic doses (~1 g DBP/kg/day) from gestation day (GD) 6 to 15 (Ema et al., 1993,Go1994Go). When treatment was extended to include the critical window of reproductive development (GD 12–21; GD 14–PND 3; or GD 16–19) marked effects on development of the male reproductive system were observed in rats at 250–750 mg DBP/kg/day. The sequelae included: reduced anogenital distance, retained thoracic nipples, cryptorchidism, hypospadias, malformed epididymis and vas deferens, hemorrhagic testes, and testicular atrophy (Gray et al., 1999,Go2001;GoMylchreest et al., 1998,Go1999,Go2000,Go2002Go). Exposure after weaning, delayed puberty in the male rat, and reduced weights of testis and other reproductive organs (Gray et al., 1999Go). Postpubertal exposure (2 g DBP/kg/day for 4 to 9 days) also caused a decrease in weights of testes and severe atrophy of seminiferous epithelium in rats (Cater et al., 1977;GoFoster et al., 1980;GoGray et al., 1982Go).

Although considerable attention has been given to the study of teratological and reproductive toxicity of DBP in rodents, very little is known regarding its effects in species such as rabbits, which have a relatively long infantile (quiescent) period of reproductive development. For example, during late gestation to postnatal week (PNW) 6, the seminiferous epithelium contains only gonocytes, pre-spermatogonia, and Sertoli cells (Gondos et al., 1973Go). From PNW 5 to 7 Leydig and Sertoli cells undergo differentiation, and the onset of spermatogenesis begins around PNW 7–8 when the first adult spermatogonia (types A and B) are formed (Gondos et al., 1973Go, 1976Go). Spermatocytes are observed at PNW 8, spermatids appear by PNW 12, coinciding with the formation of the tubular lumen, and mature spermatozoa appear in the lumen by PNW 13–14 (Gondos et al., 1973Go). This adolescent period of reproductive development in rabbits, relative to lifespan, is longer than that of rodents and better approximates the comparable phase of human reproductive development. Furthermore, use of rabbit facilitates multiple evaluations of endocrine profiles, mating ability, and semen quality. Use of this species belonging to an order of mammals (lagomorpha) different from that of rodents (rodentia), also addresses the issue that phthalate-induced effects on reproductive development and function may only be rodent-specific. Herein, we report sequelae in male rabbits following exposure to DBP in utero, during adolescence, or after puberty at a dose level known to adversely affect testicular function in rodents without causing systemic toxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental design.
Three exposures were performed: in utero (GD 15–29), adolescent (PNW 4–12), and postpubertal (12 weeks of exposure) (Fig. 1Go). In utero exposure was begun on GD 15 to ensure that major organogenesis was completed before the start of the treatment and that the window of exposure corresponded with the onset of gonadal differentiation (Gondos and Conner, 1973Go) and development of the male reproductive tract. Adolescent exposure encompassed the infantile period of testicular differentiation—differentiation of Leydig cells and Sertoli cells, onset of spermatocytogenesis, and initiation of puberty (Gondos et al., 1973,Go1976Go). Postpubertal exposure encompassed at least six cycles of the seminiferous epithelium, which has been suggested to be optimal to evaluate the effects of xenobiotics on gametogenesis in adult rabbits (Amann, 1982Go).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 1. Overview of experimental design. Arrows indicate hormonal analyses; arrows with a sphere indicate evaluations before and after a challenge with gonadotropin-releasing hormone. GD: gestation day; PNW: postnatal week.

 
Rabbit husbandry.
Six-month-old, specific-pathogen-free, Dutch-Belted rabbits were obtained from Myrtle’s Rabbitry (Thompson Station, TN) and individually housed in standard stainless steel cages in the CSU animal care facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. Animals were treated according to the guidelines of the institutional Animal Care and Use Committee. The room was maintained at a 12-h light-dark cycle at approximately 19–21°C and 40% humidity. Rabbits were fed certified rabbit ration (#7009, Harlan, Teklad, Madison, WI, provided water ad libitum, and bedded with kiln-dried aspen shavings ~2 inches below the stainless-steel floor.

After acclimatization for 3–4 weeks, rabbit does were artificially inseminated with 20 x 106 spermatozoa, pooled from semen collected from 10 bucks using the procedures previously described (Veeramachaneni, et al., 2001aGo). Rabbits were palpated for pregnancy 14 days after insemination and pregnant does were randomly assigned to treatment groups for appropriate exposures. On GD 28, pregnant does were provided with nesting boxes. At PNW 3, female pups were culled, and at PNW 6 male pups were weaned and individually housed.

Dosing.
Dibutyl phthalate (DBP; CAS# 84–74–2, lot # 03912 ES, 99.8% pure) was obtained from Aldrich Chemical Co. (Milwaukee, WI). DBP was administered orally in a mixture of deionized water:Karo light corn syrup (60:40). For in utero exposure, rabbit does were dosed with vehicle only (control; n = 5) or 400 mg DBP/kg/day (n = 8) from GD 15 to 29. This resulted in 12 control male pups (representing 5 litters) and 17 DBP-exposed male pups (representing 8 litters). In adolescent exposure, rabbit pups (n = 11 representing 4 litters) were individually administered 400 mg DBP/kg/day from PNW 4 to 12. Of these experimental animals, one pup from a litter was randomly assigned to a 25-week group (6 each for in utero and adolescent exposures) and the remaining to a 12-week group (11 for in utero and 5 for adolescent exposures). Because of limited resources, it was not possible to monitor or evaluate all of the male rabbits from each litter until 25 weeks. Likewise, in order to minimize costs and use of animals, control rabbits from in utero exposure (6 each for 12- and 25-week age groups) were utilized as controls for the adolescent exposure experiment. Given the observations seen herein, follow-up studies are being designed to replicate and expand understanding of the adverse effects of DBP on the male reproductive system.

For postpubertal exposure, 6–8-month-old bucks (n = 12) were trained to ejaculate into an artificial vagina. Three semen samples were collected on alternate days and analyzed prior to the beginning of treatment. Rabbits were ranked based on body weight and before-treatment seminal parameters and alternately assigned to 0 (n = 6) or 400 mg DBP/kg/day (n = 6), ensuring representative distribution between the two treatment groups. Treatment continued for 12 weeks.

The chosen dose of DBP was based on previous reports that adverse effects on male reproductive development occur between 100 and 500 mg DBP/kg/day without maternal or systemic toxicity (Foster et al., 2001;Mylchreest et al., 1998,Go1999,Go2000Go).

General procedures.
For in utero and adolescent exposure groups, body weights were recorded at 6, 12, and 25 weeks of life. Testicular descent was monitored weekly from 6 weeks of life by scrotal palpation until descent was complete, or 12 weeks of age. At 6, 12, and 25 weeks, 4 to 5 ml of blood was collected by jugular puncture, and serum separated and stored at –20°C for hormone assays.

For postpubertal exposure, body weights were recorded before treatment and weekly thereafter. Serum samples were collected before and at the conclusion of treatment and were stored as described above.

Evaluation of mating ability and semen characteristics.
Mating ability and semen characteristics were evaluated according to procedures previously described (Veeramachaneni et al., 2001aGo). Briefly, male rabbits were trained to ejaculate into an artificial vagina using a female teaser, and one seminal ejaculate was collected every third day between 22 and 24 weeks of age (n = 6 ejaculates/animal) for in utero and adolescent exposure groups. For postpubertal exposure, three ejaculates (per animal) were collected before the beginning of the treatment and three after the end of treatment.

For each episode when an ejaculate was collected, mating ability was subjectively evaluated by monitoring the outcome and recording: (1) sexual interest, (2) status of penile erection, (3) number of mounts to accomplish ejaculation, and (4) time taken to ejaculate after the introduction of a female teaser. Once the teaser was introduced, a maximum period of 180 s was allowed for evaluation.

For each sample, the ejaculate volume (after removing the gel) was recorded (to the nearest 0.05 µl) and an aliquot (50 µl) of semen was fixed in 950 µl of buffered formal saline and stored at 4°C until evaluation. Sperm concentration was determined by hemocytometer and total sperm per ejaculate calculated by multiplying the value of concentration of sperm by the corresponding ejaculate volume. Morphologic features of sperm were evaluated in a treatment-blinded manner, using a light microscope equipped with differential interference contrast optics. Two hundred sperm/ejaculate were evaluated in wet smears for abnormalities of the acrosome, head, mid-, and principal pieces, retention of cytoplasmic droplets, and presence of residual cytoplasm using criteria previously established for rabbits (Veeramachaneni et al., 2001aGo).

Tissue sampling.
Rabbits were euthanized by CO2 asphyxiation at 12 or 25 weeks of age for in utero and adolescent exposures and at the end of a 12-week postpubertal exposure (10–12 months of age). Liver, kidney, thyroid, testes, epididymides, accessory sex glands, and hypothalamus were evaluated for any gross abnormalities, removed, and weighed. Testes and epididymides were weighed individually. In the 12-week group, both left and right testes, and in the 25-week group, only left testes were sliced into two pieces; one piece was fixed in Bouin’s fixative and the other in 4% glutaraldehyde in 0.1 M sodium cacodylate and processed for light and transmission electron microscopy (Veeramachaneni et al., 1993Go). The right testis was decapsulated, weighed, frozen, and stored at –80°C to determine the daily sperm production (DSP). The right epididymis was dissected from the testis, cut into two segments (caput-corpus and cauda), weighed, frozen, and stored at –80°C to determine the epididymal sperm reserves (ESR). The hypothalamus was removed by incisions rostral to the preoptic area, caudal to the mamillary bodies, lateral to the hypothalamic sulci, and dorsal to the anterior commissure. The hypothalamus was bisected through the infundibulum; one-half was weighed, placed in formic acid:methanol (1:9, v/v), and stored at –20°C until assayed for gonadotropin-releasing hormone (GnRH; Nett and Adams, 1977Go).

Daily sperm production and epididymal sperm reserves.
Daily sperm production and epididymal sperm reserves were determined by procedures described for rabbit (Amann and Lambiase, 1969Go) with minor modifications. Briefly, testicular parenchyma was thawed, minced on a watch glass, and homogenized for 1 min in a semi-micro Waring blender using 50 ml buffer (0.145 M NaCl containing 4 mM NaN3 and 0.05% [v/v] Triton X-100). The number of homogenization-resistant elongated spermatids was determined without further dilution, using a hemocytometer. DSP was calculated by using a time divisor of 5.35 days (Amann et al., 1974Go) and expressed per weight of testicular parenchyma. Capita-corpora and caudae epididymidis were thawed, minced on a watch glass, homogenized for 3 min using 50 and 125 ml of buffer, and counted using a hemocytometer without further dilution. ESR was expressed per epididymal segment.

Histopathology of the testis.
Seminiferous epithelium and interstitium were evaluated using 5-µm-thick sections stained with hematoxylin and eosin (light microscopy) and 60–80-nm sections stained with uranyl acetate and lead citrate (transmission electron microscopy). Using a Nikon FXA light microscope, one hundred randomly selected, essentially round (<1.5 x width) seminiferous tubules from each animal were classified into one of eight different grades, using criteria established for evaluating bovine testis (Veeramachaneni et al., 1986Go), with minor modifications for rabbits. Briefly, grade 0, normal intact seminiferous epithelium; grade 1, seminiferous epithelium with pyknotic cells and desquamation or focal vacuolation; grade 2, seminiferous epithelium intermediate between grades 1 and 3; grade 3, seminiferous epithelium with pre-meiotic germ cells and Sertoli cells; grade 4, Sertoli cells only; grade 5, no seminiferous epithelium, leaving only the basement membrane; grade 6, seminiferous tubule with sperm stasis, sperm granuloma, or mineralization; and grade 7, fibrotic seminiferous tubule. A weight between 0 and 1, based on relative degree of loss of germ cells, was assigned to each grade: 0, 1/4, 2/4, 3/4 to grades 0, 1, 2, 3, respectively, and 4/4 to grades 4 through 7, to indicate relative degree of germinal epithelial loss (DGEL). DGEL was calculated by multiplying the percentage of tubules in each grade by the respective assigned weight and summing products.

In 3 DBP-treated rabbits, one from each exposure group, unique lesions were encountered at light microscopic level. Corresponding tissue sections from these animals, along with six appropriate control sections, were examined using a JEOL 1200EX transmission electron microscope. Evaluation was limited to characterization of subtle lesions and abnormal cells because quantitative analysis is impractical with electron microscopy.

Hormone measurements.
Hypothalamic content of GnRH was determined at 12 and 25 weeks in in utero and adolescent exposures and after 12 weeks of postpubertal exposure. Each hypothalamus was homogenized in its storage medium and GnRH was extracted and assayed using established procedures (Moss et al., 1980;GoNett and Adams, 1977Go). Samples were analyzed in one batch, and the intra-assay coefficient of variation was 3.2%. Concentrations of testosterone were determined at 6 weeks for in utero and adolescent exposures.

To assess the ability of the pituitary-gonadal axis to respond to GnRH input, GnRH-challenge tests were conducted at 12 and 25 weeks on in utero and adolescent exposure groups; rabbits in the 25-week age group were not subjected to GnRH challenge at 12 weeks. In postpubertal exposure, concentrations of testosterone were determined before treatment and GnRH-challenge tests were conducted after 12 weeks of postpubertal exposure. Each time, a baseline blood sample was taken, 10 µg GnRH was injected (im), and two samples were collected at 30- and 120-min postinjection. Each sample was extracted and assayed for testosterone using a validated RIA (Berndtson et al., 1974Go). Intra-assay and inter-assay coefficients of variation were 5.6 and 7.5%, respectively.

Statistical analyses.
Data were analyzed using Statview (version 5.0, SAS Institute, Inc., Cary, NC). Treatment was fixed for each experiment and all parameters were random. Difference between means was determined by t-test. The level of significance was set at p < 0.05. This model was used to evaluate body weights, hormone concentrations, and necropsy data. In postpubertal exposure, data for repeated measures on the same animal (endocrine parameters, sexual capacity, and semen characteristics) were analyzed by comparing before- and after-treatment values. Percentage values were transformed using arcsine of the square root of the percentage/100 to account for any inequalities in variance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There was no effect of DBP treatment on maternal survival or maintenance of pregnancy to full term when administered at 400 mg/kg/day between GD 15 and 29.

Gross Morphological Abnormalities
Descent of testes and differentiation of reproductive tract.
Testicular descent occurred between PNW 6 and 9 in all (12/12) controls and in 16 out of 17 pups exposed to DBP in utero. The one DBP-exposed rabbit in which testes failed to descend even by 12 weeks also had a poorly developed penis and malformed prepuce (giving an appearance of feminized external genitalia), hypospadias, hypoplastic seminal vesicle and prostate, and agenesis of bulbourethral gland (Fig. 2Go). However, the epididymides did not manifest agenesis or segmental aplasia. One out of 11 pups exposed to DBP during adolescence was unilaterally cryptorchid.



View larger version (132K):
[in this window]
[in a new window]
 
FIG. 2. Accessory sex glands and external genitalia of a 12-week-old cryptorchid rabbit exposed to DBP in utero (A, C) compared to an age-matched control (B, D). Note hypoplastic seminal vesicle (SV) and prostate (P) and missing bulbourethral gland (asterisks) (A) in the DBP-exposed rabbit. The external genitalia appear feminized with poorly developed penis and malformed prepuce (C).

 
Body and organ weights.
In utero exposure to DBP had no adverse effects on F1-male body weights at 6, 12, or 25 weeks of age (Table 1Go). At 12 weeks of age, weights of paired testes (p < 0.05) and accessory sex glands (p < 0.01) were lower in DBP-treated pups compared to controls. At 25 weeks, weight of accessory sex glands (p < 0.01), but not that of testes (p > 0.1), remained lower in DBP-treated rabbits. There were no differences (p > 0.1) in weights of paired epididymides at 12 or 25 weeks.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Effects of in Utero, Adolescent or Postpubertal Exposures to 400 mg DBP/kg/day on Organ Weights
 
Similar to in utero exposure, exposure to DBP during adolescence had no effect on weight gain from PNW 4 to 12 (data not presented) or body weight at 6, 12, or 25 weeks of age. There were no significant differences (p > 0.1) in weights of paired testes or epididymides, but the weight of accessory sex glands was lower (p < 0.05) in DBP-treated pups at 12 weeks. This decrease in the weight of accessory sex glands was not apparent at 25 weeks.

Postpubertal exposure to DBP also had no effect on body weight throughout the dosing period (data not presented). After 12 weeks of exposure, weights of reproductive and visceral organs did not differ (p > 0.1) between DBP-treated and control rabbits (Table 1Go) but there was a significant increase (p < 0.05) in the weight of the thyroid gland in DBP-treated rabbits.

Mating ability.
None of the three windows of exposure to DBP had any significant effect on mating ability (data not presented). Failure to ejaculate during 180 s in the presence of a teaser female occurred only once in in utero exposure groups and once in postpubertal DBP exposure groups. These incidences of failure accounted for only 3% (1/36) for in utero and 6% (1/18) for postpubertal exposure of the total number of times mating ability was evaluated. In both instances of failure to ejaculate, the rabbits expressed no sexual interest and achieved no erection.

Production of spermatozoa.
There were no differences (p > 0.1) in DSP and the number of spermatozoa in the caput-corpus or cauda epididymides between control and DBP rabbits in any of the three treatment regimens (Table 2Go). However, ejaculate volume (p < 0.01), sperm concentration (p < 0.05), and total sperm per ejaculate (p < 0.01) were lower in rabbits exposed to DBP in utero compared to controls. No decline in semen parameters was observed in adolescent or postpubertal exposure groups.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Effects of in Utero, Adolescent or Postpubertal Exposures to 400 mg DBP/kg/day on Sperm Production and Semen Characteristics
 
Semen quality, as indicated by sperm morphology, was poorer (p < 0.01) in in utero and adolescent exposure groups than in controls. DBP-treated rabbits in these two groups had almost twice as many morphologically abnormal sperm (30.1% in in utero exposure and 25.7% in adolescent exposure) as controls (16.6%; Table 2Go). A majority of the abnormal sperm (16 and 18%) had acrosomal and nuclear defects (Fig. 3Go). These included acrosomal dysplasia and vesiculation and malformed nuclei. A significant reduction (5%; p < 0.01) in the number of morphologically normal sperm was also observed following DBP treatment in the postpubertal group. In contrast, control rabbits in this exposure group had a 2% increase in the number of morphologically normal sperm (Table 2Go).



View larger version (72K):
[in this window]
[in a new window]
 
FIG. 3. Sperm acrosomal and nuclear defects in rabbits exposed to DBP in utero, during adolescence, or after puberty. Differential interference contrast (A, B), and electron microscopy (C) revealed that these defects included dysplasia and vesiculation of acrosomes and incomplete condensation and vacuolation of sperm nuclei. That these defects originated during spermiogenesis, and not during epididymal transit, is evident from histological examination of testicular sections (D). Arrows: acrosomal vesiculation, Arrowheads: incomplete nuclear condensation. Original magnification (A) x930; (B, D) x1030; (C) x5000.

 
Histopathology of the testis.
Histopathological changes representative of grades 1 and 2 were observed in all rabbits from each treatment group (Table 3Go, Figs. 4A and 4BGoGo). However, the frequency of these changes was higher in DBP-treated rabbits compared to controls. The incidence of grade 4 tubules with total germinal epithelial loss was low, only occurring in a few isolated DBP-treated rabbits from in utero and adolescent exposure groups (Fig. 4CGo). Grades 5–7 tubules were not observed. Reflective of the increased incidence of grades 1 and 2 in DBP-treated rabbits, the DGEL was significantly higher (p < 0.05) in all DBP groups than in controls (Table 3Go). Accordingly, desquamated premature germ cells were frequently observed in the lumina of seminiferous tubules (Fig. 4DGo).


View this table:
[in this window]
[in a new window]
 
TABLE 3 Histopathological Changes in the Seminiferous Epithelium after in Utero, Adolescent or Postpubertal Exposures to 400 mg DBP/kg/day
 


View larger version (153K):
[in this window]
[in a new window]
 
FIG. 4. Pathological changes of the seminiferous epithelium after in utero, adolescent, or postpubertal exposure to DBP. Grades 1 and 2 with focal vacuolation of seminiferous epithelium were less frequent in control rabbits (A) than in DBP-treated rabbits (B). Grade 4 tubules containing predominantly Sertoli cells and a few germ cells were observed in rabbits exposed to DBP in utero or during adolescence (C). Immature germ cells resulting from premature desquamation (arrows) were seen in the lumina of seminiferous tubules in DBP-treated rabbits (D). Numbers indicate grades of pathological change. Original magnification: x100; H&E staining.

 
Atypical germ cells resembling gonocytes and pre-spermatogonia were also encountered in the undescended testes of one 12-week-old, bilaterally cryptorchid rabbit from the in utero exposure group (1/17) and one 25-week-old unilaterally cryptorchid rabbit from the adolescent exposure group (1/11). Gonocytes should not be present beyond the first few weeks of life and their presence at 12 and 25 weeks of age indicates an abnormality (Veeramachaneni, 2000aGo). This germ cell atypia was further characterized using transmission electron microscopy, which confirmed abnormal cells resembling gonocytes and pre-spermatogonia (Fig. 5Go). The cellular atypia ranged from alterations in the nucleolonema to more advanced changes characterized by unusual membranous profiles, irregular nuclear contours, chromatin clumps, and swollen mitochondria. These morphological features are similar to carcinoma in situ (CIS) reported in humans (Nielsen et al., 1974;GoSkakkebaek, 1972Go).



View larger version (198K):
[in this window]
[in a new window]
 
FIG. 5. Transmission electron micrographs of atypical gonocytes (A, B), an unaffected pre-spermatogonium (C), and two atypical germ cells (D) in the undescended testis of a 25-week-old unilaterally cryptorchid rabbit exposed to DBP from PNW 4 to 12. Cellular atypia ranged from alterations in the nucleolonema (white arrow; A, D) to more advanced changes characterized by irregular nuclear contours, chromatin clumps, swollen mitochondria (black arrow; compare with panel C), unusual membranous profiles and lipid droplets (B). Note presence of two nucleoli (A) and a meandering nucleolus (D). Original magnification A, B, D: x3500; C: x4000.

 
Interestingly, atypical germ cells also were identified in the seminiferous epithelium of one rabbit (1/6) in the postpubertal exposure group (Fig. 6Go). These cells had irregular nuclear contours, perinuclear inclusions, and occasional pale staining of the cytoplasm. Electron microscopic evaluation confirmed light microscopic observations and further revealed pars amorpha in nucleoli, chromatin clumping, and swollen mitochondria.



View larger version (143K):
[in this window]
[in a new window]
 
FIG. 6. Photomicrographs of atypical germ cells from a male rabbit exposed to DBP after puberty. Light microscopic examination of H&E-stained sections (A) revealed that these atypical germ cells (white arrows) had abnormal nuclei, perinuclear cytoplasmic inclusions, and pale cytoplasm compared to a normal germ cell (arrowhead). In addition, upon electron microscopic examination (B), abnormal nucleoli, irregular nuclear contours, chromatin clumping and mitochondrial swelling (black arrow) were observed. Asterisks indicate Sertoli cell nuclei. Original magnification A: x1125; B: x3500.

 
GnRH and testosterone.
There were no changes in the hypothalamic GnRH content (p > 0.1) in in utero or postpubertal DBP exposures (Table 4Go), but it was significantly higher (p < 0.01) immediately following the adolescent exposure at 12 weeks. This increase in GnRH content was not observed at 25 weeks of age.


View this table:
[in this window]
[in a new window]
 
TABLE 4 Effects of in Utero, Adolescent or Postpubertal Exposures to 400 mg DBP/kg/day on Reproductive Hormones
 
Serum testosterone was significantly lower in DBP-treated rabbits at 6 weeks for in utero (5.62 ± 0.87 ng/ml; p < 0.05) and adolescent (5.03 ± 0.61; p < 0.01) exposures compared to controls (8.31 ± 0.84). This decrease was not apparent in subsequent measurements at 12 and 25 weeks. There was no change (p > 0.1) in serum concentration of testosterone at the end of treatment in postpubertal exposure compared to before-treatment values.

Injection of GnRH resulted in an increase in serum concentration of testosterone, 30 or 120 min later, in all rabbits exposed in utero at 12 or 25 weeks (Table 4Go). The magnitude of change from baseline concentration in DBP-treated rabbits was not different from that of controls except at 120 min post-GnRH in the 12-week group, in which it was significantly higher (p < 0.05). This was probably because the baseline concentration in this exposure group was 67% lower than that of control. In adolescent exposure groups, the concentration as well as the magnitude of change in testosterone at 30 and 120 min post-GnRH was significantly lower (p < 0.05) in DBP-treated rabbits at 12 weeks; this effect was not apparent at 25 weeks. In postpubertal exposure, the response of serum testosterone to exogenous GnRH was similar between controls and DBP-treated rabbits.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DBP caused developmental and/or reproductive toxicity in male Dutch-Belted rabbits exposed in utero, during adolescence, or after puberty. The most pronounced effects were observed in rabbits exposed in utero. This supports earlier findings in rodents that the male reproductive system is more susceptible to DBP (Mylchreest et al., 1998, Go1999, Go2000;Go; Wine et al., 1997Go) or diethylhexyl phthalate (DEHP; Gray et al., 2000;GoParks et al., 2000Go) in developing animals than in adult animals.

Rats exposed to >500 mg DBP/kg/day from GD 12 to 21 had a high incidence of testicular maldescent and impaired differentiation of Wolffian duct (epididymis and seminal vesicle) and urogenital sinus/genital tubercle (prostate and external genitalia) (Mylchreest et al., 1999,Go2000Go). Reflective of these perturbations in androgen-dependent phenomena, the weights of the testes, epididymides, seminal vesicles, and prostate were also reduced at PND 100, long after cessation of treatment (Mylchreest et al., 1999Go). Similarly, we observed cryptorchidism and malformations of reproductive tract (1/17) and decreased weights of the testes (12 weeks) and accessory glands (12 and 25 weeks) in rabbits exposed to DBP from GD 15 to 29. One of the 11 rabbits exposed during adolescence also had testicular maldescent and decreased weights of accessory sex glands at 12 weeks. The low incidences of the reproductive tract malformations observed in rabbits compared to those observed in rodent studies may be due to a lower dose (400 mg DBP/kg/day in rabbit vs. >500 mg DBP/kg/day in rodents; Mylchreest et al., 2000Go) or species differences in metabolism of DBP (Foster et al., 1983Go). Despite the low incidence, these findings confirm the previously reported adverse effects of DBP on the developing male reproductive system.

In rodents, in utero exposure to DBP also caused Leydig cell hyperplasia, testicular atrophy with widespread germ cell loss, multinuclear gonocytes, and decreased number of homogenization-resistant spermatids (Mylchreest et al, 1998,Go2002;GoWine et al., 1997Go). Similarly, rodents treated with 1000 mg DEHP/kg/day from PND 6 to 11 had decreased proliferation of Sertoli cells immediately after the final dose, which eventually recovered to normal levels by PNW 6. In spite of the recovery in number of Sertoli cells, a decrease in the number of testicular spermatids was observed at PNW 19 (Dostal et al., 1988Go). It was not clear how these perturbations in rat testis would have ultimately manifested in its final product—the mature spermatozoa.

By using the rabbit as an animal model, we were able to overcome the inherent limitations of use of rats in which collection and evaluation of seminal ejaculates are not easily accomplished and some aspects of sexual differentiation are neonatal rather than prenatal. We observed deteriorated seminal parameters in rabbits exposed to DBP in utero (25 weeks after the cessation of treatment). In utero exposure caused a reduction in ejaculate volume whereas adolescent and postpubertal exposures did not, indicating a permanent effect on function of accessory sex glands of rabbits exposed to DBP in utero. Sperm concentration was also lower following in utero exposure but not after adolescent or postpubertal exposure to DBP. Considering that both daily sperm production and epididymal sperm reserves were unaffected following in utero exposure, the lower sperm concentration in this group suggests impairment in epididymal transit and/or emission of sperm during ejaculation.

Irrespective of the window of exposure, DBP treatment lowered the number of morphologically normal sperm in the ejaculates, although the effect was more robust after in utero exposure. The majority of the abnormal sperm, regardless of window of exposure, included acrosomal and nuclear defects. Morphogenesis of these defects was observed in later stages of seminiferous epithelial cycle indicating that they originated during spermiogenesis and not during epididymal transit. Furthermore, the fact that these defects continued to occur 25 weeks and 13 weeks after the cessation of treatment (for in utero and adolescent exposures, respectively) indicated that these effects are lasting.

In addition, we observed atypical germ cells resembling carcinoma in situ (CIS) in the undescended testes of one rabbit exposed to DBP in utero and another treated during adolescence. CIS is believed to arise from transformation of gonocytes or pre-spermatogonia, and it is generally accepted that testicular germ cell tumors, except for spermatocytic seminoma, develop from CIS (Diekmann and Skakkebaek, 1999; Looijenga and Oosterhuis, 1999Go). It has been suggested that CIS cells also occur in the infantile testis adjacent to teratomas (yolk sac tumors). Since transformation of germ cells in the rabbit testis to CIS-like cells seems to be associated with the type of chemical insult and not with the abdominal location of the testis per se (Veeramachaneni et al., 2001bGo), it is logical to conclude that DBP induces unique testicular lesions in the rabbit. CIS-like cells have been observed directly adjacent to a spontaneously occurring seminoma-like tumor in this species (Veeramachaneni and VandeWoude, 1999Go). In our studies with Dutch-Belted rabbits we have never observed abdominal testes, malformed accessory sex glands, or feminized external genitalia in an untreated male. To date, out of 114 control male rabbits (in our 6 historical studies) we have encountered only one animal with a testis located in the inguinal canal. We have seen CIS-like cells in the testes of other treated male offspring, which also were associated with cryptorchidism (Veeramachaneni, 2000a,Gob;GoVeeramachaneni et al., 2001bGo).

We also observed atypical germ cells in one rabbit exposed to DBP after puberty. It is assumed that these atypical cells were derived from a more differentiated phenotype than a gonocyte (Looijenga and Oosterhuis, 1999Go), since exposure began well beyond infantile and pubertal development when gonocytes are not expected to be present. As with the CIS-like cells observed in in utero and adolescent exposures, the fate of atypical germ cells observed in postpubertal exposure is not known. It is interesting to note that spermatocytic seminoma (a form of testicular cancer that occurs in older men) develops from transformed B spermatogonia but not from CIS (Dekker et al., 1992Go). Detailed morphometric analysis of the incidence of the atypical germ cells observed in DBP-treated rabbits and studies investigating whether these cells ultimately develop into cancerous lesions are warranted.

All exposures in the current study caused germinal epithelial loss in DBP-treated rabbits as indicated by a slight but significant increase in DGEL immediately following or several months after cessation of treatment. These observations are consistent with the reports of increased germ cell loss in rodents without recovery as adults (Mylchreest et al., 1998,Go1999Go). Likewise, acute exposure of postpubertal rodents to high doses of DBP (2000–2400 mg DBP/kg/day) has been reported to cause severe testicular atrophy and immediate reduction in testis weight (Cater et al., 1977;GoFoster et al., 1980;GoGray et al., 1982Go).

Phthalate-mediated male reproductive toxicity in the developing testis is thought to occur by a complex interaction between Sertoli, Leydig, and germ cells (Mylchreest et al., 2002Go). In the current study, in utero or adolescent exposure to DBP decreased concentrations of testosterone at 6weeks. In rats, in utero phthalate treatment alters Leydig cell differentiation such that the cells proliferate, forming large clusters, rather than differentiating. The resultant immature fetal Leydig cells fail to synthesize normal testosterone and insl-3 (Wilson, Gray et al., in preparation). During puberty, phthalates appear to inhibit FSH-mediated processes in Sertoli cells, and increase the exfoliation of germ cells and incidence of multinuclear gonocytes (Li et al., 1998, Go2000Go). Furthermore DBP decreases the testicular testosterone concentrations by inhibiting 3ß-hydroxysteroid dehydrogenase, a key enzyme in the steroidogenic pathway (Mylchreest et al., 2002Go); however, it is unclear if this represents a direct effect of phthalate monoesters on this enzyme.

In utero exposure to DBP did not alter the hypothalamic GnRH content or the basal or GnRH-stimulated concentrations of testosterone either at 12 or 25 weeks. It should be noted, however, that although rabbits exposed to DBP during adolescence had basal levels of testosterone similar to controls at 12 weeks, GnRH-stimulated testosterone production was significantly decreased 30 or 120 min post-stimulation. Furthermore, the hypothalamic GnRH content was higher in DBP-treated rabbits at 12 weeks. Without measuring the concentrations of gonadotropins, we were unable to ascertain whether this effect was mediated by lowered pituitary responsiveness to GnRH or the inability of the testis to respond to gonadotropin. This effect was not permanent, and the ability to respond to GnRH-stimulation returned to normal at 25 weeks. Comparable effects were found in Leydig cell steroidogenic function of rats exposed to DEHP (Akingbemi et al., 2001Go). Thus, similar to observations made in rodents, it appears that in utero exposure to DBP directly affects the endocrine milieu of the developing rabbit testis, probably by acting on differentiating Leydig cells.

It is uncertain if the phthalate-induced testicular toxicity in postpubertal rodents was a result of changes in gonadotropins and gonadal hormones or a direct effect on the testis. A majority of the studies using rats indicates testicular toxicity during this life stage is a result of compromised Sertoli cell function, since metabolic and morphological changes are observed immediately after treatment (Creasy et al., 1983,Go1987;GoFoster et al., 1980;GoGray and Gangolli, 1986;GoHeindel and Chapin, 1989;GoHeindel and Powell, 1992Go). Because hypothalamic GnRH content and concentrations of testosterone were not affected following DBP treatment in postpubertal rabbits, we assume DBP-induced testicular changes occurred without altering the hormones necessary to drive reproductive or metabolic processes, supporting the hypothesis of a direct effect on Sertoli cells or germ cells.

Mating ability was not affected to any significant extent in any exposure window. Similarly, mating ability was not affected in rats exposed to 80 or 385 mg DBP/kg/day from GD 0 to PND 21 (Wine et al., 1997Go). Since sexual function in rabbits is known to be sensitive to developmental exposure of a variety of xenobiotics (Veeramachaneni et al., 2000a,Gob,GoVeeramachaneni et al., 2001aGo), lack of any significant detrimental effect on mating ability following DBP exposure indicates that this chemical does not affect sexual function.

The observations that in utero, adolescent, or postpubertal exposure to DBP decreased the percentage of morphologically normal sperm in the ejaculate and increased germinal epithelial loss, and that exposure to DBP in utero or during adolescence decreased the number of sperm in the ejaculate by 23 and 20% should not be overlooked. Human males have low reproductive efficiency, only ejaculating twice the number of normal sperm where fertility might be expected to decline (Amann, 1982Go). Therefore, the human testis would be more vulnerable to toxicological insult, assuming similar responses occur. Several epidemiological studies have attempted to correlate exposure to phthalate-containing materials with decline in semen quality and increased risk of testicular cancer (Murature et al., 1987;GoHardell et al., 1997;GoHansen, 1999Go). A weak negative correlation between sperm density and phthalate concentration was found in a random population of college students (Murature et al., 1987Go), while a 7-fold increase in risk for developing testicular cancer was determined in workers occupationally exposed to phthalate-containing materials (Hardell et al., 1997Go). However, another study using a larger population found no increased risk for testicular cancer in these workers (Hansen, 1999Go). More recently, Duty et al., (2002) Go reported that urinary DEP levels were associated with increased DNA damage in human sperm.

Collectively, these results indicate that exposure to DBP during development affects the male rabbit, a nonrodent species, and that males exposed to DBP in utero or during adolescence are more sensitive to toxicological injury to the reproductive system than adults.


    ACKNOWLEDGMENTS
 
We are grateful to Colleen Kane, Shannon Lee, Ginger Sammonds, and Carol Moeller for technical assistance, Xiaoming Sha and Terry Nett for expert help with RIA, and Gary Klinefelter and Heywood Sawyer for reviewing the manuscript. This study was supported by the CSU-CVMBS Research Council, NIH Training Grant HD07031, and U.S. EPA STAR grant R829429.


    NOTES
 
The research described in this article has been reviewed by the National Health 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 do mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed. Fax: (970) 491-3557. E-mail: rao{at}colostate.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Akingbemi, B. T., Youker R. T., Sottas, C. M., Ge, R., Katz, E., Klinefelter, G. R., Zirkin, B. R., and Hardy, M. P. (2001). Modulation of rat Leydig cell steroidogenic function by di(2-ethylhexyl)phthalate. Biol. Reprod. 65, 1252–1259.[Abstract/Free Full Text]

Amann, R. P. (1982). Use of animal models for detecting specific alterations in reproduction. Fundam. Appl. Toxicol. 2, 13–26.[Medline]

Amann, R. P., Kavanaugh, J. F., Griel, L. C., Jr., and Voglmayr, J. K. (1974). Sperm production of Holstein bulls determined from testicular spermatid reserves, after cannulation of rete testis or vas deferens, and by daily ejaculation. J. Dairy Sci. 57, 93–99.[ISI][Medline]

Amann, R. P., and Lambiase, J. T., Jr. (1969). The male rabbit: III. Determination of daily sperm production by means of testicular homogenates. J. Anim. Sci. 28, 369–374.[ISI][Medline]

Aurela, B., Kulmala, H., and Soderhjelm, L. (1999). Phthalates in paper and board packaging and their migration into Tenax and sugar. Food Addit. Contam. 16, 571–577.[CrossRef][ISI][Medline]

Autian, J. (1973). Toxicity and health threats of phthalate esters: Review of the literature. Environ. Health Perspect. 4, 3–26.[Medline]

Berndtson, W. E., Pickett, B. W., and Nett, T. M. (1974). Reproductive physiology of the stallion: IV. Seasonal changes in the testosterone concentration of peripheral plasma. J. Reprod. Fertil. 39, 115–118.[CrossRef][Medline]

Blount, B. C., Silva, M. J., Caudill, S. P., Needham, L. L., Pirkle, J. L., Sampson, E. J., Lucier, G. W., Jackson, R. J., and Brock, J. W. (2000). Levels of seven urinary phthalate metabolites in a human reference population. Environ. Health Perspect. 108, 979–982.

Bove, J. L., Dalven, P., and Kukreja, V. P. (1978). Airborne di-butyl and di-(2-ethylhexyl)-phthalate at three New York City air sampling stations. Int. J. Environ. Anal. Chem. 5, 189–194.[ISI]

Cater, B. R., Cook, M. W., Gangolli, S. D., and Grasso, P. (1977). Studies on dibutyl phthalate-induced testicular atrophy in the rat: Effect on zinc metabolism. Toxicol. Appl. Pharmacol. 41, 609–618.[ISI][Medline]

Colon, I., Caro, D., Bourdony, C. J., and Rosario, O. (2000). Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development. Environ. Health Perspect. 108, 895–900.[ISI][Medline]

Creasy, D. M., Beech, L. M., Gray, T. J., and Butler, W. H. (1987). The ultrastructural effects of di-n-pentyl phthalate on the testis of the mature rat. Exp. Mol. Pathol. 46, 357–371.[CrossRef][ISI][Medline]

Creasy, D. M., Foster, J. R., and Foster, P. M. (1983). The morphological development of di-n-pentyl phthalate induced testicular atrophy in the rat. J. Pathol. 139, 309–321.[ISI][Medline]

Dekker, I., Rozeboom, T., Delemarre, J., Dam, A., and Oosterhuis, J.W. (1992). Placental-like alkaline phosphatase and DNA flow cytometry in spermatocytic seminoma. Cancer 69, 993–996.[ISI][Medline]

Dieckmann, K. P., and Skakkebaek, N. E. (1999). Carcinoma in situ of the testis: Review of biological and clinical features. Int. J. Cancer 83, 815–822.[CrossRef][ISI][Medline]

Dostal, L. A., Chapin, R. E., Stefanski, S. A., Harris, M. W., and Schwetz, B. A. (1988). Testicular toxicity and reduced Sertoli cell numbers in neonatal rats by di(2-ethylhexyl)phthalate and the recovery of fertility as adults. Toxicol. Appl. Pharmacol. 95, 104–121.[ISI][Medline]

Duty, S. M., Singh N. P., Silva, M. J., Barr, D. B., Brock, J. W., Ryan, L., Herrick, R. F., Christiani, D. C., and Hauser, R. (2002). The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay. Environ. Health Perspect. DOI: 10.1289/ehp.5756. Accessed 6 December 2002.

Ema, M., Amano, H., Itami, T., and Kawasaki, H. (1993). Teratogenic evaluation of di-n-butyl phthalate in rats. Toxicol. Lett. 69, 197–203.[CrossRef][ISI][Medline]

Ema, M., Amano, H., and Ogawa, Y. (1994). Characterization of the developmental toxicity of di-n-butyl phthalate in rats. Toxicology 86, 163–174.[CrossRef][ISI][Medline]

Foster, P. M., Cook, M. W., Thomas, L. V., Walters, D. G., and Gangolli, S. D. (1983). Differences in urinary metabolic profile from di-n-butyl phthalate-treated rats and hamsters. A possible explanation for species differences in susceptibility to testicular atrophy. Drug Metab. Dispos. 11, 59–61.[Abstract]

Foster, P. M., Thomas, L. V., Cook, M. W., and Gangolli, S. D. (1980). Study of the testicular effects and changes in zinc excretion produced by some n-alkyl phthalates in the rat. Toxicol. Appl. Pharmacol. 54, 392–398.[CrossRef][ISI][Medline]

Giam, C. S., Atlas, E., and Chang, H. S. (1980). Phthalate esters, PCB and DDT residues in the Gulf of Mexico atmosphere. Atmos. Environ. 14, 65–69.[CrossRef][ISI]

Gondos, B., and Conner, L. A. (1973). Ultrastructure of developing germ cells in the fetal rabbit testis. Am. J. Anat. 136, 23–42.[ISI][Medline]

Gondos, B., Renston, R. H., and Conner, L. A. (1973). Ultrastructure of germ cells and Sertoli cells in the postnatal rabbit testis. Am. J. Anat. 136, 427–439.[ISI][Medline]

Gondos, B., Renston, R. H., and Goldstein, D. A. (1976). Postnatal differentiation of Leydig cells in the rabbit testis. Am. J. Anat. 145, 167–181.[ISI][Medline]

Gray, L. E., Jr., Ostby, J., Furr, J., Price, M., Veeramachaneni, D. N. R., and Parks, L. (2000). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol. Sci. 58, 350–365.[Abstract/Free Full Text]

Gray, L. E., Jr., Ostby, J., Furr, J., Wolf, C. J., Lambright, C., Parks, L., Veeramachaneni, D. N., Wilson, V., Price, M., Hotchkiss, A., Orlando, E., and Guillette, L. (2001). Effects of environmental antiandrogens on reproductive development in experimental animals Hum. Reprod. Update 7, 248–264.[Abstract/Free Full Text]

Gray, L. E., Jr., Wolf, C., Lambright, C., Mann, P., Price, M., Cooper, R. L., and Ostby, J. (1999). Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p‘-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15, 94–118.[ISI][Medline]

Gray, T. J., and Gangolli, S. D. (1986). Aspects of the testicular toxicity of phthalate esters. Environ. Health Perspect. 65, 229–235.[ISI][Medline]

Gray, T. J., Rowland, I. R., Foster, P. M., and Gangolli, S. D. (1982). Species differences in the testicular toxicity of phthalate esters. Toxicol. Lett. 11, 141–147.[CrossRef][ISI][Medline]

Hansen, J. (1999). Risk for testicular cancer after occupational exposure to plastics. Int. J. Cancer 82, 911–912.[CrossRef][ISI][Medline]

Hardell, L., Ohlson, C. G., and Fredrikson, M. (1997). Occupational exposure to polyvinyl chloride as a risk factor for testicular cancer evaluated in a case-control study. Int. J. Cancer 73, 828–830.[CrossRef][ISI][Medline]

Heindel, J. J., and Chapin, R. E. (1989). Inhibition of FSH-stimulated cAMP accumulation by mono(2-ethylhexyl) phthalate in primary rat Sertoli cell cultures. Toxicol. Appl. Pharmacol. 97, 377–385.[CrossRef][ISI][Medline]

Heindel, J. J., and Powell, C. J. (1992). Phthalate ester effects on rat Sertoli cell function in vitro: Effects of phthalate side chain and age of animal. Toxicol. Appl. Pharmacol. 115, 116–123.[CrossRef][ISI][Medline]

Jaeger, R. J., and Rubin, R. J. (1972). Migration of a phthalate ester plasticizer from polyvinyl chloride blood bags into stored human blood and its localization in human tissues. N. Engl. J. Med. 287, 1114–1118.[ISI][Medline]

Li, L. H., Jester, W. F., Jr., Laslett, A. L., and Orth, J. M. (2000). A single dose of Di-(2-ethylhexyl) phthalate in neonatal rats alters gonocytes, reduces sertoli cell proliferation, and decreases cyclin D2 expression. Toxicol. Appl. Pharmacol. 166, 222–229.[CrossRef][ISI][Medline]

Li, L. H., Jester, W. F., Jr., and Orth, J. M. (1998). Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured Sertoli cells and gonocytes from neonatal rats. Toxicol. Appl. Pharmacol. 153, 258–265.[CrossRef][ISI][Medline]

Looijenga, L. H. J., and Oosterhuis, J. W. (1999). Pathogenesis of testicular germ cell tumours. Rev. Reprod. 4, 90–100.[Abstract/Free Full Text]

Moss, G. E., Adams, T. E., Niswender, G. D., and Nett, T. M. (1980). Effects of parturition and suckling on concentrations of pituitary gonadotropins, hypothalamic GnRH and pituitary responsiveness to GnRH in ewes. J. Anim. Sci. 50, 496–502.[ISI][Medline]

Murature, D. A., Tang, S. Y., Steinhardt, G., and Dougherty, R. C. (1987). Phthalate esters and semen quality parameters. Biomed. Environ. Mass Spectrom. 14, 473–477.[ISI][Medline]

Mylchreest, E., Cattley, R. C., and Foster, P. M. (1998). Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: An antiandrogenic mechanism? Toxicol. Sci. 43, 47–60.[Abstract]

Mylchreest, E., Sar, M., Cattley, R. C., and Foster, P. M. (1999). Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol. Appl. Pharmacol. 156, 81–95.[CrossRef][ISI][Medline]

Mylchreest, E., Sar, M., Wallace, D. G., and Foster, P. M. (2002). Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate. Reprod. Toxicol. 16, 19–28.[CrossRef][ISI][Medline]

Mylchreest, E., Wallace, D. G., Cattley, R. C., and Foster, P. M. (2000). Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to Di(n-butyl) phthalate during late gestation. Toxicol. Sci. 55, 143–151.[Abstract/Free Full Text]

Nett, T. M., and Adams, T. E. (1977). Further studies on the radioimmunoassay of gonadotropin-releasing hormone: Effect of radioiodination, antiserum, and unextracted serum on levels of immunoreactivity in serum. Endocrinology 101, 1135–1144.[ISI][Medline]

Nielsen, H., Nielsen, M., and Skakkebaek, N. E. (1974). The fine structure of possible carcinoma in-situ in the seminiferous tubules in the testes of four infertile men. Acta Pathol. Microbiol. Scand. (A) 82, 235–248.

Parks, L. G., Ostby, J. S., Lambright, C. R., Abbott, B. D., Klinefelter, G. R., Barlow, N. J., and Gray, L. E., Jr. (2000). The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicol. Sci. 58, 339–349.[Abstract/Free Full Text]

Perwack, J., Goyer, M., and Schimke G. (1981). An exposure and risk assessment for phthalate esters, di(2-ethylhexyl) phthalate, di-n-butyl phthalate, dimethyl phthalate, diethyl phthalate, di-n-octyl phthalate, butyl benzyl phthalate. EPA-440/4–81–020. NTIS No. PB85–211936. U.S. Environmental Protection Agency, Office of Water Regulations and Standards, Washington, DC.

Petersen, J. H., and Breindahl, T. (2000). Plasticizers in total diet samples, baby food, and infant formulae. Food Addit. Contam. 17, 133–141.[CrossRef][ISI][Medline]

Skakkebaek, N. E. (1972). Possible carcinoma-in situ of the testis. Lancet 2,516–517.[CrossRef][ISI][Medline]

Veeramachaneni, D. N. R. (2000a). Deteriorating trends in male reproduction: Idiopathic or environmental? Anim. Reprod. Sci. 60–61, 121–130.[CrossRef][ISI]

Veeramachaneni, D. N. R., Higuchi, T. T., Palmer, J. S., and Kane, C. M. (2000b). Dibromoacetic acid, a disinfection by-product in drinking water, impairs sexual function and fertility in male rabbits. Biol. Reprod. 62, 354. (Abstract).

Veeramachaneni, D. N. R., Moeller, C. L., Pickett, B. W., Shiner, K. A., and Sawyer, H. R. (1993). On processing and evaluation of equine seminal samples for cytopathology and fertility assessment: The utility of electron microscopy. J. Equine Vet. Sci. 13, 207–215.[ISI]

Veeramachaneni, D. N. R., Ott, R. S., Heath, E. H., McEntee, K., Bolt, D. J., and Hixon, J. E. (1986). Pathophysiology of small testes in beef bulls: Relationship between scrotal circumference, histopathologic features of testes, and epididymides, seminal characteristics, and endocrine profiles. Am. J. Vet. Res. 47, 1988–1999.[ISI][Medline]

Veeramachaneni, D. N. R., Palmer, J. S., and Amann, R. P. (2001a). Long-term effects on male reproduction of early exposure to common chemical contaminants in drinking water. Hum. Reprod. 16, 979–987.[Abstract/Free Full Text]

Veeramachaneni, D. N. R., Palmer, J. S., Tessari, J. D., and Awoniyi, C. A. (2001b). Cellular manifestations of undescended testes depends upon the etiology of cryptorchidism but not the abdominal location per se. J. Androl. Suppl. P5–6–059. (Abstract)

Veeramachaneni, D. N. R., and Vandewoude, S. (1999). Interstitial cell tumour and germ cell tumour with carcinoma in situ in rabbit testes. Int. J. Androl. 22, 97–101.[CrossRef][ISI][Medline]

WHO (1992). Diethyhexyl phthalate. Bulletin 131, Environmental Health Criteria. World Health Organization, Geneva.

Wine, R. N., Li, L. H., Barnes, L. H., Gulati, D. K., and Chapin, R. E. (1997). Reproductive toxicity of di-n-butylphthalate in a continuous breeding protocol in Sprague-Dawley rats. Environ. Health Perspect. 105, 102–107.[ISI][Medline]