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Regular Article |
a Center for Biomedical Research, Population Council, New York, New York 10021
b Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205
c Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
ABSTRACT
Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg-1 kg-1 day-1 DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg-1 kg-1 day-1 DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg-1 kg-1 day-1 DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17ß-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21–48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg-1 kg-1 day-1 and 10 mg-1 kg-1 day-1, respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg-1 kg-1 day-1 DEHP for 28 days (Postnatal Days 62–89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.
Leydig cells, male reproductive tract, testis, testosterone, toxicology
INTRODUCTION
There is increasing public concern that environmental toxicants have the potential to impair human fertility because adverse developmental and reproductive effects are observed in laboratory animals and wildlife after exposure [1]. Phthalate esters have attracted considerable attention due to their high production volume and use in a variety of polyvinyl chloride-based consumer products. As a constituent of infant toys, building and food packaging products, and biomedical devices, di(2-ethylhexyl)phthalate (DEHP) is the most abundant phthalate in the environment. Western industrialized countries alone produce 1–4 million tons of this compound annually [2]. The ability of this compound to leach readily into biologicals during hemodialysis, extracorporeal membrane oxygenation therapy, and exchange blood transfusions is another potential hazard [3]. Exposure of the general human population to DEHP is estimated at 30 µg-1 kg-1 day-1, primarily from residues in food [4]. Occupational and clinical exposure (e.g., i.v. exposure via blood transfusions) may increase this level severalfold to between 0.5 and 5 mg [3, 5]. The use in previous studies of high DEHP dosage levels that were outside the range of this compound's presence in the environment [6] and the lack of epidemiologic data warrant further investigation at lower levels of exposure.
Environmental toxicants are classified as endocrine disruptors if they interfere with regulation of cellular function by endogenous steroids through inhibition of receptor-binding and/or transcriptional activation, or if they affect steroid biosynthesis and/or degradation. The reproductive toxicity of DEHP is attributable to the action of its primary metabolite, mono(2-ethylhexyl)phthalate (MEHP), in Sertoli and germ cells [7–10], although disruption of Leydig cell structure and function has also been reported [2, 11, 12]. However, it is not clear whether changes in androgen biosynthesis contribute to DEHP modulation of Sertoli cell function.
The male primary steroid hormone, testosterone (T), is produced almost exclusively by Leydig cells in the testis. Postnatally, the process of Leydig cell differentiation involves transformation through three stages, designated progenitor, immature, and adult Leydig cells at 21, 35, and 56 days of age, respectively [13]. Development is accompanied by a progressive increase in the capacity for T production. LH, secreted by the pituitary, is the primary regulator of Leydig cell function. During mammalian development, T promotes acquisition of the male phenotype and maintains fertility in adulthood. Testosterone is also the precursor molecule for the highly potent androgen dihydrotestosterone in some androgen-dependent tissues (e.g., the prostate) and undergoes aromatization to estradiol in the brain [14, 15]. Because inhibition of T production has adverse consequences for male fertility, experiments were conducted to investigate the ability of DEHP to impair Leydig cell androgen biosynthesis. Male rats were exposed to this compound at different stages of development. The results show that DEHP affects Leydig cell T biosynthesis through changes in steroidogenic enzyme activity and serum LH concentrations.
MATERIALS AND METHODS
Chemicals
DEHP (lot no. 107H345, >99% purity), sodium bicarbonate (NaHCO3), EDTA, soy bean trypsin inhibitor, BSA, bovine lipoprotein, Hepes, heparin, Dulbecco modified Eagle medium nutrient mixture:Ham F-12 (DMEM/F-12, 1:1 mixture without phenol red), albumin, Percoll, 22R-hydroxycholesterol, pregnenolone, 17
-hydroxyprogesterone, androst-4-ene-3,17-dione, etiocholan-3ß-ol-17-one, NAD, nitro blue tetrazolium, and gentamycin were purchased from Sigma Chemical Company (St. Louis, MO). Dulbecco PBS, Medium 199, and 10x-strength Hanks balanced salt solution were obtained from Life Technologies (New York, NY), and collagenase, dispase, and DNAse from Boehringer Mannheim GmbH (Mannheim, Germany). Ovine LH was provided by the National Hormone and Pituitary Program (National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], Rockville, MD).
Experimental Design
Long-Evans rats (Charles River, Wilmington, MA), were used for this study because a substantial toxicological data base has been generated from this strain in studies of endocrine disruptors and male reproduction [16]. Male rats were exposed to DEHP by maternal gavage during gestation or lactation, or directly at developmental stages thereafter (Fig. 1). Control rats were fed only the corn oil vehicle. Feed and water were provided ad libitum, and animals were kept on a 12L:12D cycle. Timed-pregnant rats were housed individually; pups (after weaning), prepubertal, and young adult rats were kept in groups of three through the entire duration of experiments. Allocation of animals to dose groups was done by body weight randomization to ensure equal weight distribution between groups.
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A recent study of di(n-butyl) phthalate, a closely related compound to DEHP, that involved exposure of pregnant dams during late gestation (Gestation Days [GD] 12–21) determined the no-observed-adverse-effect-level and lowest-observed-adverse-effect-level to be 50 mg-1 kg-1 day-1 and 100 mg-1 kg-1 day-1, respectively [2]. The GD 12–21 time period is considered the major period for male sexual differentiation in the rat [17]. Furthermore, a previous study found no signs of toxicity after exposure of pregnant dams to DEHP doses less than or equal to 200 mg-1 kg-1 day-1 from GD 6 to 15 [18]. Based on these observations, initial experiments were conducted to determine whether gestational and lactational exposure of male rats to DEHP can affect Leydig cell androgen biosynthesis in the prepubertal, pubertal, and adult postnatal period. Pregnant (GD 12–21) and nursing (Postnatal Days [PND] 1–21) dams (n = 7) were gavaged with 100 mg-1 kg-1 day-1 (Fig. 1a). The dams were timed pregnant from the supplier while the day after birth was designated PND 1. Male offspring were subsequently and randomly obtained from every dam in each group and analyzed at 21, 35, and 90 days of age.
The effects of DEHP may be influenced by the developmental stage at which animals are exposed [2, 16]. To examine the possibility of age-dependent differential sensitivity of male rats to DEHP with regard to Leydig cell function, weanling prepubertal rats were gavaged with 0, 1, 10, 100, or 200 mg-1 kg-1 day-1 for two differing 14-day periods, PND 21–34 or 35–48, or for a longer period of 28 days, from PND 21 to 48 (Fig. 1b). In addition, young adult rats were gavaged with 0, 1, 10, 100, or 200 mg-1 kg-1 day-1 DEHP for 28 days (PND 62–89) (Fig. 1b). The time periods for exposure were chosen to approximate the prepubertal, pubertal, and adult periods of male reproductive tract development. Leydig cell T production was measured at the end of DEHP treatment in all cases, i.e., at PND 21, 35, 49, and 90.
Feed intake and body weights were measured for all control and DEHP-exposed groups. Animals were killed within 24 h of the last DEHP administration, using a protocol approved by the Institutional Animal Care and Use Committee of Rockefeller University (protocol 91200-R2). At death, the body weight, and weights of the testis and seminal vesicles (with coagulating glands) were obtained. Immediately following decapitation, trunk blood was collected for measurement of serum LH and T concentrations. Testicular interstitial fluid (IF) was obtained using the method of Turner et al. [19]. Briefly, the caudal pole of the testis was punctured with a 23-gauge needle, taking care to avoid damage to blood vessels and seminiferous tubules. Testes were then centrifuged at 50 x g for 15 min. IF samples were stored at -20°C until T levels were measured by RIA.
Hormone Measurements
Serum LH levels were measured using 125I rat LH (Covance Laboratories Inc., Vienna, VA), and materials obtained from the National Hormone and Pituitary Program, namely, rat antibody NIDDK-anti-rLH-S11, and LH reference standards (NIDDK-rLF-RP-3). The secondary immunoglobulin-G (IgG) antiserum was supplied by ICN Pharmaceuticals (Costa Mesa, CA). The lower limit of detection for this assay is 0.12 ng/ml, and LH values are expressed in relation to the RP-3 standards. The intraassay and interassay coefficients of variation were 5% and 10%, respectively [20]. The concentrations of T in the serum and IF, and the amounts of T produced by Leydig cells, were measured with a tritium-based RIA as previously described, with a 7–8% interassay variation [21].
Leydig Cell T Production
Purified Leydig cells were obtained from rats of different ages by collagenase digestion of the testis followed by Percoll density centrifugation of the cell suspension, according to the method of Klinefelter et al. [22]. Prior to loading onto a 55% continuous Percoll density gradient, Leydig cells isolated from 49- and 90-day-old rats were subjected to centrifugal elutriation to remove germ cell and sperm contaminants. The fraction of Leydig cells remaining in the elutriator chamber at a flow rate greater than 16 ml/min and rotor speed of 2000 rpm was collected. This step was omitted when Leydig cells were isolated from 21- and 35-day-old rats due to the absence of sperm in the testis of young rats. Progenitor Leydig cells (PND 21) were harvested from the Percoll gradient at a band between 1.062 and 1.070 g/ml, immature Leydig cells (PND 35) between 1.070 and 1.088, and Leydig cells obtained on PND 49 and 90 at 1.070 and greater. Cell yields were estimated with a hemocytometer, and purity was assessed by histochemical staining for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), using 0.4 mM etiocholan-3ß-ol-17-one as the enzyme substrate [23]. Progenitor Leydig cell fractions were typically 90% enriched for cells that stain weakly for the marker enzyme 3ß-HSD, and immature and adult Leydig cells were 95–97% enriched for cells that stained intensely. The culture medium was composed of DMEM/F-12, containing 0.1% BSA and 0.5 mg/ml bovine lipoprotein, and was buffered with 14 mM NaHCO3 [24]. Leydig cells were incubated at a density of 0.5–1 x 106 cells in microcentrifuge tubes at a temperature of 34°C for 3 h without (basal) or with a maximally stimulating dose of ovine LH (100 ng/ml). Testosterone production values were normalized to ng/106 cells.
Enzyme Assay
Androgen biosynthesis in Leydig cells involves conversion of cholesterol through a number of intermediate steroids into T, and these reactions are catalyzed by four enzymes: cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3ß-HSD, cytochrome P450 17-hydroxylase/17,20 lyase (P45017), and 17ß-hydroxysteroid dehyrogenase (17ß-HSD). Enzyme activity was measured by incubation of Leydig cells in the presence of saturating concentrations (20 µM) of steroid substrates: 22R-hydroxycholesterol (P450scc), pregnenolone (3ß-HSD), progesterone (P45017), or androstenedione (17ß-HSD). 11-Dehydroxycorticosterone was used as an internal control. The capacity of Leydig cells to carry out specific steroidogenic reactions was calculated by summing all possible five 3-ketosteroid products (pregnenolone, 17-hydroxyprogesterone, progesterone, androstenedione, and T) after 120 min of incubation. For example, to measure P450scc activity, 22R-hydroxycholesterol was added to the incubation medium, and pregnenolone, 17-hydroxyprogesterone, progesterone, and T were measured and summed. The steroid intermediates were measured by using an HPLC/UV (240-nm wavelength) detection system (Shimadzu SCL-10AVP; Shimadzu, Tokyo, Japan). Analysis and quantitation of steroids were performed as previously described [25].
Testicular Histology
The testicular interstitium was examined for focal proliferative lesions that may indicate Leydig cell hyperplasia and also for seminiferous tubule damage and germ cell degeneration. Tissue was fixed by immersion in Bouin fluid and embedded in paraffin. Sections (5–6 µm in thickness) were stained with hematoxylin and eosin and evaluated by light microscopy.
Statistics
There were at least seven animals in each experimental group. Assays of samples from the same study were conducted in triplicate. All data were analyzed by one-way ANOVA with multiple comparisons performed by the Duncan multiple range test to identify differences between groups. Differences were considered to be significant if P was less than or equal to 0.05.
RESULTS
Gestational Exposure
Gavage of pregnant dams with 100 mg-1 kg-1 day-1 from GD 12 to 21 did not affect body weights (g) that were similar in control and DEHP-treated dams (n = 7) at the beginning of the experiment (283 ± 5 versus 284 ± 5) and also at the end (384 ± 6 versus 376 ± 9). Male offspring were analyzed at 21, 35, and 90 days of age. Prenatal exposure to DEHP did not affect body weight (g) in male rats at 21 (36.6 ± 1 versus 39.3 ± 1.2), 35 (119.5 ± 3 versus 120 ± 3.8), and 90 days (431 ± 8 versus 445 ± 9). The weights (g) of the testes were also similar in control and DEHP-exposed rats on PND 21 (0.19 ± 0.05 versus 0.2 ± 0.06), PND 35 (1.03 ± 0.04 versus 1.08 ± 0.04), and PND 90 (3.4 ± 0.06 versus 3.7 ± 0.08). The weights of the seminal vesicles (g) were not affected by DEHP exposure. For example, the wet weights (g) of this organ measured 1.42 ± 0.07 in control and 1.41 ± 0.03 in DEHP-exposed rats at 90 days. However, prenatal exposure of male rats to DEHP adversely affected Leydig cell androgen biosynthesis. At 21 and 35 days, serum T levels were significantly lower in DEHP-exposed rats, compared to control (P < 0.05) (Fig. 2a). Depressed serum T levels were associated with reduced serum LH concentrations in these animals (Fig. 2b). Leydig cell T production (basal and LH-stimulated) after DEHP-exposure were lower at PND 21 but compared favorably with control at PND 35 (Table 1). However, all measurements were similar in control and DEHP-exposed rats at 90 days (Table 1 and Fig. 2).
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Lactational Exposure
Gavage of nursing dams with 100 mg-1 kg-1 day-1 DEHP from the PND 1 to PND 21 did not affect body weight (g), measuring 312 ± 8 versus 328 ± 7 in control and DEHP-treated rats (n = 7) at the beginning of the experiment, and 337 ± 10 versus 335 ± 8 at the end, respectively. Male offspring were analyzed immediately, i.e., at the end of exposure on PND 21, and also at 35 and 90 days of age. DEHP exposure did not affect body weight (g) in male rats (PND 21, 46 ± 2 versus 49 ± 3; PND 35, 121 ± 7 versus 134 ± 6; PND 90, 475 ± 19 versus 483 ± 16). At 21 days of age, serum T concentrations were slightly reduced in DEHP-exposed rats (control 2.01 ± 0.06; DEHP, 1.75 ± 0.06 ng/ml, P < 0.05), but there were no differences in the serum levels of LH (control, 0.28 ± 0.05; DEHP, 0.30 ± 0.06 ng/ml). Other measurements, including body weights, serum T and LH levels, and the weights of the seminal vesicles (with coagulating glands) were comparable in control and DEHP-exposed rats at 35 and 90 days of age (data not shown).
Direct Exposure of Prepubertal and Young Adult Rats
Dosing of growing rats with DEHP did not cause any signs of toxicity as determined by the rate of body weight gain. At 270 g, the greatest weight attained by rats that were gavaged directly with DEHP in the present study, the highest dose of DEHP (200 mg-1 kg-1 day-1) represents an intake of 54 mg. This level of intake is proportionately reduced for younger and smaller rats. The maximally tolerated dose of DEHP in rats, estimated from a 90-day toxicity study by the National Toxicology Program, is 6000 mg-1 kg-1 feed-1 [26]. In the present study, there were no differences in feed intake between groups, averaging 15 ± 3 g per day. Thus, using NTP data, a rat weighing 270 g will ingest 90 mg DEHP per day and, expectedly, suffer no toxicity over a 90-day period. The 90 mg per rat is far greater than the highest dose fed to DEHP-treated rats in this study.
Prepubertal rats (n = 10) were gavaged with 0, 1, 10, 100, or 200 mg/kg/day DEHP for 14 days at two different periods (PND 21–34 or 35–48). Exposure of rats to DEHP for 14 days, regardless of the exposure period, did not affect the rate of body weight gain, serum hormone (T and LH) levels, and testis and seminal vesicle weights (Tables 2 and 3). However, Leydig cell T production was reduced by DEHP exposure at doses equal to or greater than 10 mg-1 kg-1 day-1 (Figs. 3 and 4). Reduced androgen biosynthesis was associated with inhibition of steroidogenic enzyme activity at the dose of 10 mg-1 kg-1 day-1 and greater (Table 4).
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Gavage of prepubertal rats for 28 days (PND 21–48, n = 10) with 0, 1, 10, 100, or 200 mg-1/kg-1/day-1 DEHP did not affect body weights. This exposure paradigm, however, caused a dose-dependent increase in the serum concentrations of LH and T. Testicular interstitial fluid T levels were also elevated in DEHP-treated rats (Table 5), and Leydig cells from rats treated with the higher DEHP doses exhibited increased capacity for T production (Fig. 5).
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Unlike prepubertal rats, treatment of young adult rats with DEHP for 28 days (PND 62–89, n = 10) with 0, 1, 10, 100, or 200 mg-1 kg-1 day-1 affected neither serum hormone levels (LH, T) nor Leydig cell T production (data not shown).
Testicular Histology
Histological evaluation of the testis presented no evidence of Leydig cell hyperplasia, seminiferous tubule damage, germ cell degeneration, or delayed spermiation in all experimental groups (data not shown).
DISCUSSION
Data obtained in this study show that DEHP modulates Leydig cell function and exhibits differential effects on steroidogenesis. For example, exposure of male rats to DEHP during early reproductive tract development adversely affects Leydig cell androgen biosynthesis. The data also show that prenatal exposure to DEHP suppressed pituitary function because, compared to control, serum LH levels were lower at 21 and 35 days of age in male rats exposed to this compound in utero (Fig. 2). Similarly, reduced serum T levels in 21-day-old nursing male rats exposed to DEHP via maternal gavage failed to stimulate a rise in serum LH concentrations. On the other hand, there were no apparent changes in reproductive parameters measured in young adult rats exposed to 100 mg-1 kg-1 day-1 DEHP for 28 days (PND 62–89). These observations suggest that growing rats are more susceptible to the effects of DEHP and/or its metabolites than adults.
DEHP-induced inhibition of Leydig cell T production was associated with two factors: 1) a decrease in pituitary LH secretion and 2) reduced steroidogenic enzyme activity. Both factors compromise the primary function of Leydig cells: T biosynthesis and secretion. LH stimulates the process of differentiation in developing Leydig cells and maintains steroidogenic enzyme gene expression and cell volume in mature cells [27]. Thus, attenuation of pituitary LH secretion during development adversely affects acquisition of steroidogenic capacity, thereby decreasing androgen biosynthesis by individual Leydig cells [28]. It is therefore not surprising that male offspring that were exposed to DEHP in utero or during nursing exhibited decreases in serum T levels that were pronounced in the immediate postexposure period. While there were no decreases in serum LH levels, Leydig cell steroidogenesis was reduced by DEHP treatment of prepubertal rats for 14 days (PND 21–34 or 35–48) (Figs. 3 and 4), suggesting that DEHP and/or its metabolites have direct effects on Leydig cell function. Reduced steroidogenesis is presumably due to a decrease in the activity of T biosynthetic enzymes, although there were no apparent decreases in serum T levels (Tables 3 and 4). It could be that steroidogenic enzyme inhibition was not profound enough to affect Leydig cell T production at the time of serum T measurement or that compensatory mechanisms were activated in vivo to counteract the effect of decreased enzyme activity. It is also possible that enzyme assays conducted in vitro stimulate higher than normal levels of steroidogenesis because they provide more substrate than is ordinarily available to Leydig cells in vivo. Thus, in vitro enzyme assays have greater sensitivity in detecting changes in steroidogenic capacity compared to measurements of serum T levels.
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Interestingly, exposure of prepubertal rats to DEHP for 28 days (PND 21–48) enhanced Leydig cell androgen biosynthetic capacity and increased serum T levels, in association with elevated LH concentrations (Fig. 5 and Table 5). This finding contrasts with the observed decrease in androgen biosynthesis after rats were gavaged with DEHP for 14 days (Figs. 3 and 4). The finding of higher serum T levels and increased androgen biosynthesis after a 28-day exposure of growing rats to DEHP is probably due to a compensatory mechanism. Normally, when androgen biosynthesis is sufficiently depressed, the lowered serum T concentrations act via negative feedback mechanisms to induce increased LH output from the pituitary. LH stimulates Leydig cells to secrete more T, which in the course of time, and acting via the same pathway, restores pituitary LH secretion to normal levels. In this manner, the negative feedback mechanism serves as a homeostatic control for the hypothalamo-pituitary-gonadal axis [29]. However, the simultaneous occurrence of higher than normal levels of LH and T suggests a disruption of this mechanism. Oishi and Hiraga [30] also reported increased gonadal steroidogenesis after DEHP treatment of mice. Similarly, chronic DEHP administration to male Fischer 344 rats caused hypertrophy of gonadotropes in the anterior pituitary and a higher incidence of pituitary tumors compared to control rats [31]. The mechanisms responsible for these observations are not presently clear. However, persistently high levels of T production by Leydig cells may have, at least, two consequences. First, long-term suppression of steroidogenesis was found to prevent age-related deficits in Leydig cell androgen biosynthesis [32]. It is therefore possible that enhanced steroidogenesis for prolonged periods will cause premature deterioration of Leydig cell capacity for androgen biosynthesis. Second, Leydig cell hyperplasia has been associated with chronic LH stimulation and also with damage to seminiferous tubules, germ cell loss, and disruption of spermatogenesis [33, 34]. Testicular lesions were absent in the present study, but serum LH concentrations were elevated in prepubertal rats exposed to DEHP for 28 days at doses that were equal to or greater than 10 mg-1 kg-1 day-1 (Table 5). A potential consequence of DEHP-induced chronic LH stimulation could therefore be increased Leydig cell proliferative activity.
A major issue regarding endocrine disruptors in the environment is whether their effects are permanent or reversible. It has been hypothesized that the earlier in development a lesion is inflicted, the greater the likelihood of its persistence into adulthood [35, 36]. It has also been proposed that compounds that bind cellular DNA cause irreversible and delayed effects that become apparent only later in life [37]. DEHP is not known to bioaccumulate in body tissues and is eliminated from the body within 3–4 days after last administration [6]. The present data show that male offspring that were exposed to DEHP in utero and during the nursing period showed reduced androgen status only in the neonatal and peripubertal periods; these animals exhibited serum T levels that were similar to controls evaluated at 90 days of age. This observation suggests that the inhibitory effects of DEHP on Leydig cells are reversible and/or that a new population of Leydig cells had developed after exposure to DEHP was terminated, restoring Leydig cell function over time. However, behavioral deficits may become apparent in adulthood if the brain is not exposed to adequate androgen levels early in development [15].
Previous studies using high doses of DEHP (0.5 to 6 g-1 kg-1 day-1) demonstrated varying degrees of reproductive anomalies, including testicular atrophy and damage [7–12]. The present study found no cellular or morphological changes in the testis as evaluated by light microscopy, and there were no DEHP-induced changes in the weights of the seminal vesicles (with coagulating glands). It is possible that the changes in the serum levels of T, namely, a decrease after gestational exposure to DEHP and an increase following peripubertal exposure for 28 days, although statistically significant, were not intense enough or occurred for too short a period of time to cause detectable changes in testicular and seminal vesicle weights. Variations in the severity of DEHP-induced lesions is probably related to the window and duration of exposure and the dosages used in different studies.
In conclusion, data from the present study show that the effects of DEHP on androgen biosynthesis are more pronounced early in development than at later stages. The results also indicate that the lowest dose of DEHP affecting steroidogenesis and causing changes in serum LH and T levels is 10 mg-1 kg-1 day-1, representing the lowest-observed-effect-level; 1 mg-1 kg-1 day-1 constitutes the no-observed-effect-level, because there were no detectable effects at this dose. However, it remains to be determined that these exposure levels can impact T production in human Leydig cells. It has been suggested that species-specific differences in the pharmacokinetics of phthalates may protect humans against the toxic effects seen in rats because hydrolysis of DEHP to its monoester metabolite, MEHP, occurs at much lower rates in nonhuman primates than in rodents [38]. Additional studies are therefore required to better define the sensitivity of humans to phthalates and their metabolites in comparison to rodents that have been more extensively studied. Data from such investigations, along with observations from rodent studies, will facilitate the process of risk assessment for humans regarding chronic and concomitant exposure to environmentally relevant levels of DEHP and other phthalates.
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ACKNOWLEDGMENTS
The authors are grateful to Evan Read for help with manuscript preparation, and to Drs. Louise Parks and Li You for comments on the manuscript. The reagents for LH RIA were generously provided by Dr. A.F. Parlow and the National Hormone and Pituitary Program (NIDDK, Rockville, MD).
FOOTNOTES
First decision: 22 March 2001.
1 This work was supported in part by the National Institute of Environmental Health Sciences (ES 10233). Preliminary data were presented at the 33rd Annual Meeting of the Society for the Study of Reproduction, 15–18 July 2000, University of Wisconsin, Madison, WI. Although this study was funded in part and the data presented herein were approved for publication by the U.S. Environmental Protection Agency, this paper does not necessarily reflect the views and policies of the Agency. 
2 Correspondence: Benson T. Akingbemi, Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; benson{at}popcbr.rockefeller.edu
Accepted: May 31, 2001.
Received: January 11, 2001.
REFERENCES
-Dihydrotestosterone formation is necessary for embryogenesis of the rat prostate. Endocrinology 1988; 122:1159-1164[Abstract]This article has been cited by other articles:
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 |
|
  M. Culty, R. Thuillier, W. Li, Y. Wang, D. B. Martinez-Arguelles, C. G. Benjamin, K. M. Triantafilou, B. R. Zirkin, and V. Papadopoulos In Utero Exposure to Di-(2-ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat Biol Reprod, June 1, 2008; 78(6): 1018 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lin, R.-S. Ge, G.-R. Chen, G.-X. Hu, L. Dong, Q.-Q. Lian, D. O. Hardy, C. M. Sottas, X.-K. Li, and M. P. Hardy Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero PNAS, May 20, 2008; 105(20): 7218 - 7222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. G. Rosenberg, H. Chen, J. Folmer, J. Liu, V. Papadopoulos, and B. R. Zirkin Gestational Exposure to Atrazine: Effects on the Postnatal Development of Male Offspring J Androl, May 1, 2008; 29(3): 304 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Onorato, P. W. Brown, and P. L. Morris Mono-(2-ethylhexyl) Phthalate Increases Spermatocyte Mitochondrial Peroxiredoxin 3 and Cyclooxygenase 2 J Androl, May 1, 2008; 29(3): 293 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
IN MEMORIAM Matthew P. Hardy, Ph.D. 1957-2007 Biol Reprod, March 1, 2008; 78(3): 563 - 564. [Full Text] [PDF]
|
|
|
|
 |
|
 I Svechnikova, K Svechnikov, and O Soder The influence of di-(2-ethylhexyl) phthalate on steroidogenesis by the ovarian granulosa cells of immature female rats J. Endocrinol., September 1, 2007; 194(3): 603 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-S. Ge, G.-R. Chen, Q. Dong, B. Akingbemi, C. M. Sottas, M. Santos, S. C. Sealfon, D. J. Bernard, and M. P. Hardy Biphasic Effects of Postnatal Exposure to Diethylhexylphthalate on the Timing of Puberty in Male Rats J Androl, July 1, 2007; 28(4): 513 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Plummer, R. M. Sharpe, N. Hallmark, I. K. Mahood, and C. Elcombe Time-Dependent and Compartment-Specific Effects of In Utero Exposure to Di(n-butyl) Phthalate on Gene/Protein Expression in the Fetal Rat Testis as Revealed by Transcription Profiling and Laser Capture Microdissection Toxicol. Sci., June 1, 2007; 97(2): 520 - 532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Martin, R. J. Brennan, W. Hu, E. Ayanoglu, C. Lau, H. Ren, C. R. Wood, J. C. Corton, R. J. Kavlock, and D. J. Dix Toxicogenomic Study of Triazole Fungicides and Perfluoroalkyl Acids in Rat Livers Predicts Toxicity and Categorizes Chemicals Based on Mechanisms of Toxicity Toxicol. Sci., June 1, 2007; 97(2): 595 - 613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Supornsilchai, O. Soder, and K. Svechnikov Stimulation of the pituitary-adrenal axis and of adrenocortical steroidogenesis ex vivo by administration of di-2-ethylhexyl phthalate to prepubertal male rats J. Endocrinol., January 1, 2007; 192(1): 33 - 39. [Abstract] [Full Text] [PDF]
|
|
|
|
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S. A. Lahousse, D. G. Wallace, D. Liu, K. W. Gaido, and K. J. Johnson Testicular Gene Expression Profiling following Prepubertal Rat Mono-(2-ethylhexyl) Phthalate Exposure Suggests a Common Initial Genetic Response at Fetal and Prepubertal Ages Toxicol. Sci., October 1, 2006; 93(2): 369 - 381. [Abstract] [Full Text] [PDF]
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M. Ma, T. Kondo, S. Ban, T. Umemura, N. Kurahashi, M. Takeda, and R. Kishi Exposure of Prepubertal Female Rats to Inhaled Di(2-ethylhexyl)phthalate Affects the Onset of Puberty and Postpubertal Reproductive Functions Toxicol. Sci., September 1, 2006; 93(1): 164 - 171. [Abstract] [Full Text] [PDF]
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 P R Dalsenter, G M Santana, S W Grande, A J. Andrade, and S L Araujo Phthalate affect the reproductive function and sexual behavior of male Wistar rats Human and Experimental Toxicology, June 1, 2006; 25(6): 297 - 303. [Abstract] [PDF]
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S. W. Grande, A. J. M. Andrade, C. E. Talsness, K. Grote, and I. Chahoud A Dose-Response Study Following In Utero and Lactational Exposure to Di(2-ethylhexyl)phthalate: Effects on Female Rat Reproductive Development Toxicol. Sci., May 1, 2006; 91(1): 247 - 254. [Abstract] [Full Text] [PDF]
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 R. M. David Proposed Mode of Action for In Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract Toxicol Pathol, April 1, 2006; 34(3): 209 - 219. [Abstract] [Full Text] [PDF]
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K Svechnikov, V Supornsilchai, M-L Strand, A Wahlgren, D Seidlova-Wuttke, W Wuttke, and O Soder Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary-gonadal axis and Leydig cell steroidogenesis J. Endocrinol., October 1, 2005; 187(1): 117 - 124. [Abstract] [Full Text] [PDF]
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N. Bhattacharya, J. M. Dufour, M.-N. Vo, J. Okita, R. Okita, and K. H. Kim Differential Effects of Phthalates on the Testis and the Liver Biol Reprod, March 1, 2005; 72(3): 745 - 754. [Abstract] [Full Text] [PDF]
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 S. Bagel-Boithias, V. Sautou-Miranda, D. Bourdeaux, V. Tramier, A. Boyer, and J. Chopineau Leaching of diethylhexyl phthalate from multilayer tubing into etoposide infusion solutions Am. J. Health Syst. Pharm., January 15, 2005; 62(2): 182 - 188. [Abstract] [Full Text] [PDF]
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J. C. Corton and P. J. Lapinskas Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract? Toxicol. Sci., January 1, 2005; 83(1): 4 - 17. [Abstract] [Full Text] [PDF]
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S. M. Duty, A. M. Calafat, M. J. Silva, J. W. Brock, L. Ryan, Z. Chen, J. Overstreet, and R. Hauser The Relationship Between Environmental Exposure to Phthalates and Computer-Aided Sperm Analysis Motion Parameters J Androl, March 1, 2004; 25(2): 293 - 302. [Abstract] [Full Text] [PDF]
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 C. J. Thompson, S. M. Ross, and K. W. Gaido Di(n-Butyl) Phthalate Impairs Cholesterol Transport and Steroidogenesis in the Fetal Rat Testis through a Rapid and Reversible Mechanism Endocrinology, March 1, 2004; 145(3): 1227 - 1237. [Abstract] [Full Text] [PDF]
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B. T. Akingbemi, R. Ge, G. R. Klinefelter, B. R. Zirkin, and M. P. Hardy Phthalate-induced Leydig cell hyperplasia is associated with multiple endocrine disturbances PNAS, January 20, 2004; 101(3): 775 - 780. [Abstract] [Full Text] [PDF]
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A. S. Cupp, M. Uzumcu, H. Suzuki, K. Dirks, B. Phillips, and M. K. Skinner Effect of Transient Embryonic In Vivo Exposure to the Endocrine Disruptor Methoxychlor on Embryonic and Postnatal Testis Development J Androl, September 1, 2003; 24(5): 736 - 745. [Abstract] [Full Text] [PDF]
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