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,#
From the * Population Council and The Rockefeller
University, New York, New York; Department of
Pathology, Wenzhou Medical College, Wenzhou, Zhejiang, China;
Department of Urology, Huaxi Medical School,
Sichuan University, Chengdu, Sichuan, China;
Department of Anatomy, Physiology &
Pharmacology, College of Veterinary Medicine, Auburn University, Alabama; ||
Department of Neurology, Mount Sinai School of
Medicine, New York, New York; and ¶ Department of
Pharmacology and Therapeutics, McGill University, Montréal,
Canada.
| Correspondence to: Dr Matthew P Hardy, The Population Council, 1230 York Ave, New York, NY 10021 (e-mail: m-hardy{at}popcbr.rockefeller.edu) |
| Received for publication September 27, 2006; accepted for publication January 29, 2007. |
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Abstract |
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Key words: Androgen, puberty, steroidogenesis, testis, endocrine disruptor, toxicology
Previous animal studies of phthalates were generally conducted with high
doses and short exposure periods. Typically the doses of DEHP that were
analyzed (
500 mg/kg body weight) are at least 100 times higher than the
estimated human daily DEHP exposure and were associated with decreased
testosterone (T) production and lower sperm counts
(Sjoberg et al, 1986a;
Sjoberg et al, 1986b;
Foster et al, 2001).
Translation of the results of controlled high-dose acute studies in rodents to
the human exposure risk scenario has been difficult. Acute exposure paradigms
do not approximate real-life situations for human populations who may be
subjected to prolonged low-level exposures. We previously observed that, in
striking contrast to the high-dose exposures, elevations in T levels occur
when rats are treated orally with 10 and 100 mg/kg/d DEHP for 28 days during
puberty (Akingbemi et al, 2001;
Akingbemi et al, 2004a).
Similar results were obtained in rats treated by inhalation with a low dose of
DEHP comparable with 10 mg/kg/d oral treatment
(Kurahashi et al, 2005) and in
boars exposed intramuscularly to DEHP at 50 mg/kg twice per week
(Ljungvall et al, 2005) during
puberty. Therefore, the present study was designed to investigate
dose-dependent effects of DEHP on pubertal timing and to determine whether
Leydig cell steroidogenesis is affected by phthalate in a dose-dependent
manner.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Earlier we reported on dose-dependent effects of DEHP (10 to 200 mg/kg) (Akingbemi et al, 2001; Akingbemi et al, 2004b). Accordingly, 10 mg/kg/d DEHP was selected based on our previously determined lowest observed effect level (LOEL) in pubertal rats (Akingbemi et al, 2001; Akingbemi et al, 2004a). To expand the dose range further and address the possibility that different phthalate-mediated effects could be seen at the high and low ends of the dose-response range, 500 or 750 mg/kg/d DEHP was selected based on the previously observed LOEL for androgen inhibition (Foster et al, 2001).
Cohorts of 40 male rats from mixed-size litters were randomly allocated by body weight into 4 groups (n = 10/group). Rats were gavaged with DEHP (10, 500, or 750 mg/kg) or the corn oil vehicle (control) daily from postnatal day (PND) 21 (ie, at weaning) to 48. This age interval was used because the prepubertal period is a time of active reproductive tract development, and hormonally active chemicals are known to exhibit greater potency during sexual differentiation in rodents and humans than at later times. Body weight and preputial separation (an index of pubertal onset) were recorded. The observers were blinded to treatment condition to avoid bias. At the end of treatment, animals were killed and blood was collected for determination of serum hormone (luteinizing hormone [LH] and T) concentrations. The weights of androgen-dependent tissues such as seminal vesicles and prostates were also measured in selected experiments, and all experiments were repeated at least twice; final sample sizes are presented in "Results."
Preputial Separation Assay
To assess the effect of chronic DEHP exposure on male preputial separation,
prepubertal Long-Evans male rats were assigned to different groups as above.
Preputial separation, an easily scored external sign of sexual development in
male rats, can be used as an index of change in peripubertal androgen
secretion (Korenbrot et al,
1977). The separation of the prepuce from the glans penis, termed
preputial separation, has been shown to be androgen dependent and to occur
around the time of puberty in rats. The average age of preputial separation is
39 to 45 days, just preceding the appearance of mature sperm in the caput
epididymis. In pilot experiments, preputial separation was found to occur
before day 51 and prior to the increase in circulating androgen levels. The
time course of the accumulative frequency of rats with preputial separation
was calculated.
Short-Term Exposure to DEHP In Vivo on Immature Leydig Cell Function
Previously we observed that after gavage with 0, 1, 10, 100, or 200 mg/kg/d
DEHP for 14 days from PND 21 to 34, the rate of body weight gain, serum
hormone (T and LH) levels, and individual testes and seminal vesicle weights
were unaffected in pubertal rats (Akingbemi
et al, 2001). Similarly, 500 mg/kg/d administered over a more
chronic setting of 28 (PND 21 to 49) days did not affect these parameters. To
address whether compensatory changes in androgen synthesis and feedback
suppression of pituitary function were associated with 28-day exposure, the
higher dose, 500 mg/kg/d, was also evaluated after a shorter 14-day exposure
during the first half (PND 21 to 34) of the 28-day exposure setting. Leydig
cell T production was examined in addition to the parameters that were
measured after 28-day exposure.
Leydig Cell Purification
Purified Leydig cells were obtained from the testes of 35-day-old rats by
collagenase digestion, followed by Percoll density centrifugation as described
previously (Ge and Hardy,
1998). In an initial purification step, Leydig cells from
49-day-old rats were sedimented in solutions of bovine serum albumin (BSA)
(Salva et al, 2001). After
centrifugation through a 55% continuous Percoll gradient, Leydig cells from
35-day-old rats were harvested at densities between 1.070 and 1.088 g/mL,
whereas cells from 49-day-old rats were harvested at densities corresponding
to 1.070 g/mL (ie, to the bottom of the tube). Cell yields were estimated
with a hemocytometer, and purity was assessed by histochemical staining for 3
ß-hydroxysteroid dehydrogenase using 0.4 mM etiocholan-3ß-ol-17-one
as the enzyme substrate (Payne et al,
1980). Leydig cell preparations were 95% to 97% enriched for cells
that stained intensely for this marker enzyme.
Ex Vivo T Production by Leydig Cells After DEHP Exposure In Vivo
To measure the rates of T production in spent media, aliquots of 0.2
x 106 Leydig cells obtained from 35-day-old rats were
incubated in microcentrifuge tubes in 1 mL of culture medium. The culture
medium consisted of Dulbecco modified Eagle medium/F-12 buffered with 14 mM
NaHCO3 containing 0.1% BSA and 0.5 mg/mL bovine lipoprotein
(Sigma-Aldrich, St Louis, Mo). The cells were incubated at 34°C for 3
hours using the maximally stimulating dose of 100 ng/mL ovine LH
(NIDDK-oLH-26; provided by the National Hormone and Pituitary Program,
National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda,
Md) or 5 µM 22(R)-hydroxycholesterol. Leydig cell T production values were
expressed as ng/106 cells.
Treatment With MEHP In Vitro
MEHP is the primary metabolite formed from DEHP in vivo and is the mediator
of its effects in cells (Huber et al,
1996). DEHP is a protoxin, metabolized in the gastrointestinal
tract to MEHP which is 10 times more potent than the parent compound
(Huber et al, 1996).
Therefore, incubation of purified Leydig cells with MEHP was used to assess
the consequences of phthalate exposure in vitro. A range of MEHP
concentrations were tested (10–9–10–2
M) in cultures of Leydig cells maintained at 34°C for 18 hours. MEHP was
added alone or in combination with 0.5 ng/mL ovine LH (provided by the
National Hormone and Pituitary Program, NIDDK). The cells were incubated in a
concentration of 0.5 ng/mL (low concentration) LH to maintain Leydig cell
viability, and this dose elicits a 25% to 50% increase in T production
relative to control.
RNA Analysis by Reverse Transcriptase Polymerase Chain Reaction
Pituitary LH ß subunit (Lhb) and androgen receptor
(Ar) mRNA expression levels were quantified by real-time reverse
transcriptase polymerase chain reaction (PCR) to assess whether the action of
DEHP on T production is direct or indirect. Total RNA was isolated from
pituitary glands using Trizol following the manufacturer's instructions
(Invitrogen, Carlsbad, Calif). The cDNA synthesis step and real-time PCR were
performed using a thermocycler (ABI7900HT; Applied Biosystems, Foster City,
Calif) as previously described (Yuen et
al, 2002). The RNA levels for the house keeping genes ribosomal
protein S11 (Rps11), tubulin (Tuba), and ß-actin
(Actb) were also assayed in all samples to select one that could be
used as an internal control. Lhb and Ar mRNA measurements
were normalized using a robust global normalization algorithm. All control
crossing threshold (Ct) values were corrected by the median difference in all
samples from Actb. All samples were then normalized by the difference
from the median Ct of the 3 corrected control gene Ct levels in each sample,
with the value converted to a nominal copy number per cell by assuming 2500
Actb mRNA molecules per cell and an amplification efficiency of 93%
for all reactions. The primer sequences used for the assays were: Ar
sense: 5'-TATGGT GAGCGTGGACTTTC-3', antisense:
5'-GCCCATGCCAG AGAAGTAGT-3'; Lhb sense:
5'-CAGTGTGCACCTACC GTGAG-3', antisense:
5'-GGGGAAGGTCACAGGTCAT T-3'; Rps11 sense:
5'-CGAGGGCACCTACATAGACA-3', antisense:
5'-GAGATAGTCCCGGCGGATGA-3'; Actb sense:
5'-GCCTCAACACCTCAAACCAC-3', antisense:
5'-CCACAGCTGAGAGGGAAATC-3'; Tuba sense: 5'-AGC
GCCCAACCTACACTAAC-3', antisense: 5'-GGGAAGTG
GATGCGAGGGTA-3'.
Hormone Assays
Serum LH concentrations were measured using 125I rat LH (Covance
Laboratories, Vienna, Va) and materials obtained from the National Hormone and
Pituitary Program (rat antibody [NIDDK-anti-rLH-S11] and LH reference
standards [NIDDK-rLF-RP-3]). The secondary immunoglobulin G antiserum was
supplied by MP Biochemicals (Solon, Ohio). The lower limit of detection for
this assay is 0.12 ng/mL, and LH values were expressed in relation to the
standards. The intra-assay and interassay coefficients of variation were 5%
and 10%, respectively. Steroid hormone (T) concentrations were measured by a
previously described tritium-based radioimmunoassay validated for use with rat
antiserum (Akingbemi et al,
2001; Akingbemi et al,
2004a). The assays were performed in triplicate, and the intra-
and interassay coefficients of variation were 10% and 15%, respectively.
Statistics
Data are presented as mean ± SEM. For the in vivo studies, the data
represent the averages from 2 separate experiments and the data for the in
vitro studies were combined from at least 3 separate experiments. Data were
analyzed by 1-way analysis of variance (ANOVA) with Dunnett's multiple
comparison test to identify differences among groups (control, 10, 500, and
750 mg/kg DEHP). The time course of the accumulative frequency of rats with
preputial separation was fitted by sigmoidal curvilinear regression. The
average age at which 50% of the males attained complete preputial separation
was calculated. Data were analyzed by 2-way ANOVA with PND and drug dose as
independent measures. Differences were considered significant at P
.05.
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Results |
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High-Dose Short-Term Exposure to DEHP Inhibits Leydig Steroidogenesis
When the 500 mg/kg/d dose was tested for 14 days as shown above, decreases
were seen in testis weights and serum T levels. The 500 mg/kg dose of DEHP
also decreased T production by Leydig cells in vitro under
substrate-saturating conditions (5 µM) of 22(R)-hydroxycholesterol
(Table 2), suggesting that
inhibition occurs at the step of cholesterol side-chain cleavage enzyme
activity and/or later in the T biosynthetic pathway.
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Biphasic Effect of MEHP on T Production In Vitro
The above in vivo experiment suggests a direct action of DEHP on Leydig
cells. Since DEHP is protoxin and MEHP is the primary metabolite formed from
DEHP in vivo with 10 times more potency compared with the parent compound
(Huber et al, 1996), a wide
range of MEHP concentrations was tested on Leydig cells incubated in vitro.
The cells were incubated in the presence and absence of LH (0.5 ng /mL). At
100 µM and 1 mM, MEHP increased LH-stimulated T production, whereas a
higher concentration, 10 mM, was inhibitory
(Figure 3). Cell viability as
assessed by Trypan blue exclusion staining was not affected at any of the
doses tested (unpublished observations). In the absence of LH, 1 mM MEHP
increased T production, although it was 10 times less potent than when
combined with LH. This indicated that phthalate effects at low doses might
have a direct stimulatory effect on T production.
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Discussion |
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It was established previously that during days 21 to 49 of pubertal development, oral exposures to 10 mg/kg/d DEHP caused Leydig cell hyperplasia and persistently elevated T levels (Akingbemi et al, 2004a). This observation was confirmed independently in 2 other studies: rats treated by inhalation with a low dose of DEHP (comparable to an oral dose of 10 mg/kg/d) (Kurahashi et al, 2005) and boars that received DEHP at 50 mg/kg twice a week intramuscularly during puberty had elevated serum T levels (Ljungvall et al, 2005). In boar testes, Leydig cell hyperplasia results from low-dose DEHP exposure, similar to what has been observed in rats (Ljungvall et al, 2005). The present observations extend these earlier findings and show that DEHP-mediated increases in androgen levels are sufficient to advance the timing of preputial separation. Preputial separation has been shown to be androgen dependent, and we have established that increases in T to adult levels advanced the timing of preputial separation from 41 days to 30 days using T-releasing implants (Hardy, unpublished data). Human exposure to DEHP is uncontrolled and is undoubtedly composed of both low- and high-dose levels. In this scenario, distinct low- and high-dose effects may cancel each other, which could explain why, to date, there is a failure to show conclusively that DEHP has antiandrogenic effects in human male infants (Kaiser, 2005).
Pituitary Lhb and Ar mRNA levels are sensitive to regulation by androgen in circulation. Androgen suppresses Lhb expression (Fallest et al, 1995) and increases Ar expression (Okada et al, 2003). In the present study, we did not observe any effect of phthalate exposure on Lhb or Ar mRNA levels in either low-(10 mg/kg) or high-dose (500–750 mg/kg) conditions that either increased or suppressed serum T levels. This indicates that effects of DEHP on serum T levels are not mediated through pituitary gland regulations of Lhb mRNA. An apparent trend toward reduced Lhb mRNA levels at 10 mg/kg was associated with the increased T (and androgen negative feedback) levels at this dose. Therefore, a direct action of DEHP on Leydig cells has been purposed in the present study. Accordingly, a direct action of DEHP was assessed using the DEHP metabolite, MEHP. Direct exposure of Leydig cells to a wide range of MEHP concentrations in vitro clearly demonstrated that this metabolite induced a biphasic effect on T production that was similar to the trend obtained after exposure to DEHP in vivo. Low concentrations (100 µM) of MEHP stimulated T production and high concentrations (>10 mM) were suppressive. The MEHP concentration that was associated with higher rates of T, 100 µM, equates to a serum level of 30 µg/mL, 10 times lower than a 10 mg/kg/d exposure regimen (Akingbemi et al, 2004a). These results indicate that chronic direct stimulation of Leydig cells by MEHP increases T production. The stimulatory effect of low-dose exposure may take time to develop, as 10 mg/kg/d DEHP did not increase serum T levels at PND 35 in rats treated from PND 21 for 14 days (Akingbemi et al, 2001; Akingbemi et al, 2004a). This latency of the effect on plasma concentration of T was also seen in boars exposed to 50 mg/kg/d DEHP (Ljungvall et al, 2005). The direct stimulatory effect of phthalate on T production may contribute to the increase in circulating androgen levels that ensue from low-dose exposures, along with the previously reported Leydig cell hyperplasia (Akingbemi et al, 2004a; Ljungvall et al, 2005).
The male reproductive toxicity associated with gestational, prenatal, or
pubertal exposure to DEHP and other phthalates has largely been documented at
doses of 500 mg/kg/d (Gray et al,
2000; Foster et al,
2001; Wilson et al,
2004; Borch et al,
2005). Of concern is that the inhibitory effects of phthalate
exposures on pubertal development were achieved at doses 750 mg/kg/d
(Table 1). Interpretation of
the reproductive toxicity of phthalate exposure at doses this high may be
confounded by systemic toxicity (Table
1). The 500 mg/kg/d dose of DEHP appears to be intermediate, as it
did not affect pubertal development or serum T concentrations
(Table 1) subsequent to a
28-day treatment period. However, testis weights and T levels were
significantly reduced by day 14 within the 28-day treatment period (PND 21 to
35), indicating that pubertal rats are more susceptible to inhibition by DEHP,
compared to adults.
In conclusion, data from the present study showed that DEHP has a biphasic effect on Leydig cell function, with low-dose exposures increasing T production and advancing the onset of puberty. In contrast, high doses of DEHP are antiandrogenic and delay the onset of puberty. Importantly, these high doses may also be outside the range of real environmental exposure levels and therefore results obtained under such conditions should be considered in this light.
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Acknowledgments |
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Footnotes |
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The data were presented orally in preliminary form at the 39th Annual Meeting of the Society for the Study of Reproduction, Omaha, Neb, July 29–August 1, 2006.
# These authors contributed equally to this article. 
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