1 Department of Biological Sciences, University of Missouri-Columbia, Columbia, Missouri, 2 Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri, 3 Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois and 4 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
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Abstract |
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Key words: endocrine disruption/fetus/oral contraceptive/prostate/testes
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Introduction |
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In 1993, the American College of Obstetrics and Gynecology concluded that oral contraceptive use during early pregnancy or shortly before pregnancy is not associated with increased risk of fetal malformation (American College of Obstetrics and Gynecology, 1993). Even when malformations are limited specifically to those of the genitourinary tract, where there are tissues known to be sensitive to reproductive steroidal hormones, the association between OC use in early pregnancy and malformations is still generally considered absent (Kallen et al., 1991
; Raman-Wilms et al., 1995
; Martinez-Frias et al., 1998
). However, there have also been reports to the contrary (Li et al., 1995
; Li, 1998
). Despite the general lack of positive epidemiological data linking OC use during pregnancy with malformations at birth, concern remains because prenatal exposure to exogenous sex hormones is known to cause adverse effects other than externally observable malformations in both humans and animals (Gray, 1992
; Newbold, 1995
). In addition, OC used in the USA contain 17
-ethinyl oestradiol (EE), a synthetic oestrogen with an oestrogenic potency very similar to that of diethylstilbestrol (DES), another synthetic oestrogen known to be a reproductive teratogen.
DES is a non-steroidal synthetic oestrogen that was given to more than 3 million women in the United States during the 1950s and 1960s, primarily to prevent miscarriage and premature births. Use for this purpose of DES was banned in the USA in 1971 after the chance discovery that DES- exposed daughters were at an increased risk of developing clear-cell adenocarcinoma of the vagina and cervix (Herbst, 1981; Mittendorf, 1995
). Further follow-up of DES-exposed daughters found they were also at increased risk for developing a variety of reproductive tract abnormalities. These ranged from vaginal epithelial cell changes, cervicovaginal, uterine and Fallopian tube structural anomalies, increased incidence of infertility, ectopic pregnancies, premature births, and possibly increased incidence of immune and behavioural disorders, such as depression (Bibbo et al., 1977
; Herbst, 1981
; Stillman, 1982
; Blair et al., 1992
; Hines, 1992
; Giusti et al., 1995
; Newbold, 1995
). Damage to developing reproductive organs in women was typically found if exposure to DES occurred prior to week 18 of pregnancy (Herbst et al., 1979
).
In males, in-utero DES exposure has also been associated with a variety of reproductive abnormalities, including increased incidence of epididymal cysts, cryptorchidism, hypospadias, and possibly sperm abnormalities (Bibbo et al., 1977; Stillman, 1982
; Giusti et al., 1995
). However, studies of DES effects in men have been limited to cohorts of only a few hundred exposed males, so the effects of DES on human males are less certain than in females. Of particular importance is whether exposure began prior to the end of the first trimester and continued into the second trimester of pregnancy, since it is during this time that the greatest damage to reproductive organs would occur (Wilcox et al., 1995
). It is unknown whether DES-exposed daughters and sons are at increased risk for other hormonally mediated cancers, such as breast or prostate cancer, as they are just approaching the age when these types of cancers are more likely to occur.
DES and EE have similar potencies as assessed by their effect on uterine growth, a well-established oestrogen bioassay, when administered subcutaneously to prepubertal rats (Branham et al., 1988). However, in a separate set of experiments on immature rats, the relative potencies of DES and EE varied depending upon whether administration was oral or subcutaneous, with these oestrogenic chemicals being more similar during oral administration (Reel et al., 1996
). In any event, both DES and EE are more potent stimulators of uterine growth than 17ß-oestradiol, the primary endogenous oestrogen (Branham et al., 1988
; Reel et al., 1996
). In addition, EE has been shown recently to cause similar incidence of uterine adenocarcinomas as DES in adult mice following neonatal exposure. An estimated 3% of DES administered to pregnant mice reaches the fetus, primarily concentrating in the reproductive tract (Shah and McLachlan, 1976
). While metabolism of EE by the maternal liver does occur, unconjugated EE reaches the fetus (Slikker et al., 1982
), although uptake of EE into fetal tissues after administration to pregnant mice has not been reported.
One basis for the high oestrogenic activity of both DES and EE relative to oestradiol is that DES and EE show little binding to oestrogen-binding plasma proteins, alpha-fetoprotein in rodents and sex hormone binding globulin in humans (Akpoviroro and Fotherby, 1980; Branham et al., 1988
). These plasma oestrogen-binding proteins modulate the levels of free, bioavailable oestrogen in serum (Nagel et al., 1998
).
Despite the similarity in oestrogenic potency of DES and EE, it appears that lessons learned from DES have not been applied to the evaluation of risks resulting from fetal exposure to EE (American College of Obstetrics and Gynecology, 1993). One reason for this is that the high-dose exposures of DES (1.5150 mg/day) (Bern et al., 1987
) are regarded as irrelevant to the low doses of EE (3550 µg/day) found in oral contraceptives. Importantly, even the very high doses of DES that were once used did not result in external malformations in most offspring, which is why millions of women were administered DES over two decades. This clearly shows that external malformations are not an expected outcome of fetal exposure to oestrogenic chemicals, except in a very small proportion of cases, even with very high doses. These findings also clearly demonstrate that the absence of malformations at birth does not preclude reproductive dysfunction later in life due to fetal exposure to oestrogenic chemicals (Gill et al., 1976
).
Following the initial observation of increased vaginal adenocarcinoma in DES-exposed daughters, a re-evaluation of the animal literature on DES suggested that reproductive disorders in exposed offspring should have been expected. The animal literature regarding the adverse effects of developmental exposure to DES extends back at least to the early 1960s (Dunn and Green, 1963; Takasugi and Bern, 1964
), but these findings were ignored, as many physicians and scientists apparently believed that the animal data were irrelevant to human health. Subsequent experiments with mice and rats have resulted in findings that were highly consistent with findings in humans (Newbold, 1995
; Swan and vom Saal, 2001
). The literature on DES has contributed to a general awareness regarding the high level of conservation of the hormonal mechanisms mediating differentiation of the reproductive organs in mammals (Kavlock and Ankley, 1996
; National Research Council, 1999
).
While there is now an abundance of data concerning the consequences of developmental exposure to DES, there have been relatively few studies evaluating the long-term effects resulting from prenatal exposure to EE. A MEDLINE January 2001 search for diethylstilbestrol/prenatal and ethinyl oestradiol/prenatal found 488 and 14 references respectively. Prenatal exposure to EE has been shown to disrupt reproductive function in both male and female mice at higher than clinically relevant doses, while effects at doses within the clinical range are unexplored. In male mice, fetal exposure to EE results in an increased incidence of cryptorchidism (Walker et al., 1990; Yasuda et al., 1985b
), epididymal azoospermia (Yasuda et al., 1988
), atrophy of seminiferous tubules, and altered Sertoli and Leydig cell differentiation (Yasuda et al., 1985a
, 1986a
, Yasuda et al., b
). Female mice exposed to EE prenatally develop follicular cell hyperplasia (Yasuda et al., 1987
), ovarian hypoplasia, and increased degeneration of primordial follicles (Yasuda et al., 1985b
). However, these histological and functional responses to prenatal EE have only been studied at maternal doses of 20 µg/kg/day and above, with alterations typically occurring at 200 µg/kg/day. The clinically relevant dose for EE in most OC is ~0.5 µg/kg/day; this is based on use of an OC with 35 µg EE by a woman who weighs 68 kg.
Since there is little information as to whether exposure to clinically-relevant doses of EE during prenatal life can disrupt the development of reproductive organs in experimental animals, the goal of the current study was to investigate the effects on adult male reproductive organs following in-utero exposure to doses of EE that were at or below the clinically-relevant dose range for EE found in oral contraceptives.
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Materials and methods |
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Treatment of pregnant females with 17-ethinyl oestradiol
Virgin females (aged 34 months) were time-mated by being placed daily with a stud male for 4 h beginning 2 h before the end of the dark phase of the light:dark cycle. Mating was verified by the presence of a seminal plug (day 0 of pregnancy). Plug-positive mice were either left unhandled or were given (orally) an average dose of 0, 0.002, 0.02, 0.2, 2, 20 or 200 µg/kg EE dissolved in 30 µl of tocopherol-stripped corn oil (ICN, Aurora, OH, USA) once daily during gestational days 017. Dams were weighed every 3 days throughout gestation and allowed to deliver normally on gestation day 19. The corn oil was administered by electronic micropipette (Rainen Instruments, Woburn, MA, USA) because mice readily consume corn oil that is pipetted into their mouth, and this procedure is not as stressful as gavage (force-feeding by stomach tube). No plug-positive mice dosed with 20 or 200 µg/kg of EE remained pregnant, and only offspring from the 0.002, 0.02, 0.2 and 2 µg/kg groups were generated. Each group consisted of 1012 dams.
Reproductive tissue collection in males
Total litter size ranged from 6 to 18 [mean 10.9 ± 2.2 (SD)]. The number of males in each litter ranged from 2 to 10 (mean 5.5 ± 1.7). Female offspring were removed at weaning, and males remained in litter groups of two to five males per cage. No more than five males were kept for any litter. For males killed when 50 days old, a randomly selected male was individually housed at weaning. For males killed when 5 months old, a randomly selected male was individually housed, beginning at 4 months of age. The reason that males were individually housed is that when male mice are housed in groups, a non-linear hierarchy is often observed where there is one dominant male and the remaining males are subordinate; this can have marked effects on reproductive organs and behaviour in CF-1 mice. However, individual housing for 1 month eliminates the prior effects of subordination in CF-1 mice (unpublished observation). Following weaning when one male per litter was individually housed, the remaining males from the same litter continued to be housed together. Similarly, when another male per litter was removed at 4 months of age, the remaining animals continued to be housed together until used for additional, separate experiments. At both 50 days and 5 months of age, animals were weighed and euthanized. The coagulating glands, seminal vesicles, prostate and one testis and epididymis were then removed and weighed. Fluid was removed from the seminal vesicles and coagulating glands by blotting prior to being weighed. Prostate and testes were immediately frozen in liquid nitrogen, and then stored at 70°C.
Daily sperm production
Daily sperm production (DSP) was determined using a frozen right or left testis from control and treated males by a procedure that has been previously described (Robb et al., 1978; Cooke et al., 1991
; Joyce et al., 1993
). Briefly, after being removed and weighed, the tissues were placed in liquid nitrogen, and subsequently kept at -70°C until being examined. Testes were then homogenized for 3 min in 25 ml of physiological saline containing 0.05% (v/v) Triton X-100 (Sigma, St Louis, MO, USA) using a semimicro Waring container (PGC # 77-8549) on a Waring blender (Fisher # 14-509-10). Step 1416 spermatids (stage IIVIII) survive this homogenization, and their nuclei can then be counted using a haemocytometer (Fisher # 02-671-10). To count the spermatids, a 200 µl sample of homogenate was diluted with 300 µl of saline and 500 µl of 4% Trypan blue, which stains spermatids and facilitates counting (Cooke et al., 1991
). Sample aliquots of 5.5 µl were then placed on the haemocytometer and counted twice at 100x magnification under a microscope to determine the average number of spermatids per sample. These values were used to obtain the total number of spermatids per testis, and this number was then divided by the testis weight to give spermatids per gramme of testes. Developing spermatids spend 4.84 days in steps 1416 during spermatogenesis in the mouse. Thus, the values for the number of spermatids per testis and spermatids per gramme of testis were divided by 4.84 to obtain daily sperm production and efficiency of sperm production (per gramme of testis) respectively (Robb et al., 1978
; Joyce et al., 1993
).
Prostate androgen receptor assay
In addition to measuring androgen receptors (AR), protein was measured as a reference for expressing AR content, and the amount of DNA was also measured to discern between hypertrophy and hyperplasia. Prostates were homogenized on ice in running buffer [13% glycerol and 83 mmol/l TrisHCl (pH 6.8) with 0.67 mg/ml sodium dodecyl sulphate and 21 mg/ml dithiothreitol], and boiled at 100°C for 3 min. After samples were taken for DNA and protein assay (see below) the homogenates were centrifuged at 10 000 r.p.m. (9300 g) for 5 min at 4°C. Homogenization of prostates in any buffer other than the denaturing running buffer was accompanied by losses in the labile AR protein.
To determine total protein and DNA concentrations directly in this homogenate, the following procedure (Brown et al., 1989, modified) was used. A 50 µl aliquot of prostate homogenate was added to 440 µl of double-distilled water, after which 50 µl of 0.15% (w/v) sodium deoxycholate was added and mixed. After incubation for 10 min at 25°C, 60 µl of 80% (w/v) trichloroacetic acid (TCA; final concentration 8.0%) were then added, the tubes were vortexed, and centrifuged at 3000 g for 15 min at 25°C. The supernatants were discarded and the pellets washed with 500 µl 8% TCA, centrifuged, and supernatants were carefully removed again. The pellets were dissolved by brief vortexing or sonication in 462.5 µl of added 10 mmol/l EDTA (pH 12.4), and then neutralized to pH 7.2 with 37.5 µl of 0.77 mol/l KH2PO4 for a total volume of 500 µl. Protein and DNA concentrations in this solution were determined in 50 µl aliquots by using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) and by using the Hoechst dye 33258 fluorimetric DNA assay (Labarca and Paigen, 1980; Taylor et al., 1995
). The standard curve samples for the assays [50 µl protein, or 50 µl DNA with 50 µl 5% bovine serum albumin (BSA) carrier] were subjected to the same precipitation and extraction procedure as were homogenate samples.
After determination of protein, each sample was diluted to a constant protein concentration (µg protein/µl) with loading buffer containing bromophenol blue dye at 0.25 mg/ml. Samples were then loaded onto 8% SDSpolyacrylamide mini-gels (20 µg protein in 20 µl buffer per lane), electrophoresed for 2 h at 10 mA/gel, and electroblotted onto nitrocellulose membranes using a semi-dry transfer apparatus (BioRad, Hercules, CA, USA) for 45 min at 12 V. Each gel contained a sample from one prostate of each treatment group, and a pooled standard of prostates from five to seven month-old animals was loaded in two lanes on each gel as a reference standard for comparisons between gels.
The membrane was blocked with 5% BSA (fraction IV) in 10 mmol/l Tris (pH 7.5) at room temperature for 1 h, washed in Tris/Tween (0.02% Tween, 10 mmol/l Tris), then incubated with 1:10 000 monoclonal rat anti-androgen receptor (Affinity BioReagents, Neshanic Station, NJ, USA) in 10 mmol/l Tris (pH 7.5) overnight at room temperature. The membrane was washed in Tris/Tween for 30 min and incubated in 5% normal rabbit serum (Sigma) at room temperature for 90 min, washed, and incubated in rabbit anti-rat IgG horseradish peroxidase conjugate (Sigma), washed for 1 h and reacted with Lumi-glo chemiluminescence kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Membranes were exposed to film, and films were scanned and analysed for band density using Kodak 1D imaging software. Band densities for each lane of a gel were divided by the average density of the two pooled standard bands, and then expressed for each group as a percentage of the pooled standard control values run in duplicate on each gel (band density % of standard per µg protein). Since these data were based on an equal amount of loaded protein per lane, the band densities could be multiplied by total protein per prostate to express as total AR per prostate (band density % standard per prostate), as well as AR per µg protein. Total AR per prostate were divided by µg DNA per prostate to yield AR per µg DNA (band density % of standard per µg DNA; roughly proportional to receptors per cell). Periodically, an assay involving serial dilution of the control samples was conducted to verify that the assay was operating within its linear range.
Statistical analyses
Statistical analyses were conducted using the general linearized model (GLM) procedure in the Statistical Analysis System (SAS). Since only one animal from each litter was used in an experiment, correction for litter effects was not necessary. Homogeneity of variance between groups was assessed using Levene's test. For those variables showing heterogeneous variance, a logarithmic transformation eliminated the heterogeneity, and thus the statistical analysis was performed on the log-transformed data. If body weight was significantly (P < 0.05) correlated with organ weight, analysis of co-variance (ANCOVA) was conducted, with body weight as the co-variate, in order to assess group differences. If body weight was not significantly correlated with organ weight, then the data were analysed using analysis of variance (ANOVA).
Planned pairwise comparisons were made following a significant (P < 0.05) omnibus ANOVA (or ANCOVA) by the least significant difference (LSD) test. In presenting the results, the results of the overall ANOVA (or ANCOVA) are reported first, followed by results of planned comparisons. To control the experiment wise error rate, the statistical significance of the pairwise differences was limited to the significance level of the overall test. The primary comparisons of interest were between the control and experimental (EE-exposed) groups.
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Results |
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DSP and DSP/g testis
Daily sperm production was analysed both without correction for testis weight (DSP) and after correction for testis weight (DSP/g testis). At 50 days of age, neither value correlated significantly with body weight. All doses of EE at 50 days of age resulted in significantly (P < 0.01) reduced DSP and DSP/g testis relative to controls, although these effects were not more severe at higher doses (Figure 1; Table I
). At 5 months, both DSP and DSP/g testis were significantly (P < 0.05) correlated with body weight. However, no significant group differences in DSP or DSP/g testis at 5 months were detected.
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Although these three AR variables were highly correlated (r = 0.920.98, P < 0.001) and showed generally similar dose-related patterns of response, a significant (P = 0.04) quadratic term was found only for AR per prostate. A follow-up LSD comparison indicated that the 0.02 µg/kg group had a significantly (P < 0.05) increased number of AR per prostate (~50%) relative to controls (Figure 3). Slight elevations in AR per DNA and AR per protein were seen in the 0.02 µg/kg/day group relative to controls, although these increases were not statistically significant. Moreover, total protein and total DNA per prostate were also somewhat, but not significantly, elevated in the 0.02 µg/kg/day group (Table II
).
Body weight and weights of testis, epididymis, seminal vesicle, and coagulating gland
Analysis of variance indicated no difference in body weight between groups at 50 days of age. Similarly, there were no differences in other reproductive organ weights in 50-day-old animals, none of which was significantly correlated with body weight (Table I). At 5 months of age, all treatment groups showed some increase in body weight compared with controls, but the increases were only statistically significant (P < 0.05) in the 0.002 and 2 µg/kg/day groups (Table II
). Similar to findings in 50-day-old males, there were no overall differences at 5 months between groups in seminal vesicle, coagulating gland, epididymal or testis weights (Tables I and II
).
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Discussion |
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Because the total number of Sertoli cells accounts for most of the variability (<85%) in DSP in adult rats (Berndtson and Thompson, 1990), one hypothesis is that prenatal exposure to oestrogen may permanently reduce Sertoli cell numbers, resulting in subsequent decreases in adult sperm output (Sharpe and Skakkebaek, 1993
). Sertoli cell proliferation begins on embryonic day 14 (Vergouwen et al., 1991
) and ends early in postnatal life in the mouse (postnatal days 1217, depending on the strain) with no additional proliferation in adulthood (Kluin et al., 1984
; Vergouwen et al., 1991
). Therefore, a decrease in DSP resulting from a decrease in Sertoli cells is predicted to be a permanent effect. However, in the current study, DSP was decreased by EE at 50 days of age, but was not different from controls when males in most dose groups were 5 months old. This finding suggests that the effects of EE during fetal life on DSP are not due to a decreased Sertoli cell numbers.
Our finding that a specific very low maternal dose of EE produced an `imprinted' increase in prostatic AR in male offspring was not unexpected. It has been reported previously that circulating oestradiol in male mouse fetuses is positively correlated with adult prostate size and numbers of prostatic AR (Nonneman et al., 1992), which was confirmed when prostate size, number of prostatic glands and prostatic AR were increased in male mice in response to a very small experimental increase in circulating oestradiol during fetal life (while testosterone concentrations remained constant) (vom Saal et al., 1997
). This finding has recently been confirmed (Gupta, 2000
). Interestingly, in the present study an increase in AR levels was only observed in the 0.02 µg/kg/day EE dose group, despite increased prostate weight at all doses examined. These observations suggest qualitative differences in mechanisms contributing to the increased prostate weight across the dose range examined, although further research is needed to test this hypothesis. It is generally accepted that in moving from very low to much higher doses of a hormone, the hormone can potentially interact with response systems for other hormones, thus leading to qualitatively different, rather than just quantitatively different, outcomes (Kavlock and Ankley, 1996
).
The elevated body weight in the 5-month EE-dosed groups was a potentially complicating factor in the evaluation of prostate and other organ weight. Although alternative approaches are possible (e.g. organ/body weight ratios), it is felt that the most appropriate method for adjusting organ weight for the influence of body weight is ANCOVA, because the correction for body weight is based on the observed relationship calculated from the data rather than on the use of a specific, proportional (one-to-one) relationship between organ and body weight. Moreover, for those organ weights showing no association with body weight, body-weight adjustment is neither needed nor appropriate.
Although there have been virtually no prior experimental animal studies to determine whether exposure to clinically relevant doses of EE during prenatal life can disrupt the development of reproductive organs, several studies have shown that prenatal exposure to DES, at the same very low doses examined here, produce similar effects on prostatic growth described here for EE (vom Saal et al., 1997; Gupta, 2000
). This is not surprising, since EE and DES are very similar in their oestrogenic activity in rodents (Branham et al., 1988
). For example, oral doses of 0.02, 0.2 and 2.0 µg/kg/day of DES during days 1117 of pregnancy in mice resulted in a permanently enlarged prostate in male offspring that were examined in adulthood (vom Saal et al., 1997
). An important additional finding is that administration of a 200 µg/kg/day dose of DES to pregnant female mice resulted in an inhibition of normal prostate development (vom Saal et al., 1997
), revealing that low doses of oestrogenic chemicals exert a stimulating effect, while high doses have an opposite, inhibitory, effect on prostate development in mice. These findings concerning the effects of low versus high doses of DES on prostate development have recently been confirmed in CD-1 mice with administration to pregnant females and with direct application of DES to urogenital sinus explants, demonstrating direct effects of oestrogen on prostate development independent of effects on the mother or placenta (Gupta, 2000
).
Female offspring are also affected by maternal administration of these low doses of DES: administration of a 0.01 µg/kg/day dose of DES to pregnant mice was associated with subfertility and a decrease in the number of ovulated oocytes in response to a superovulating dose of gonadotrophins in female offspring (McLachlan et al., 1982). In contrast to the inhibitory effects that prenatally administered very low doses of DES or EE have on the gonads (ovaries or testes), we and others (Newbold et al., 1999
), have found that similar to the prostate, the response of the uterus to oestrogen stimulation in female offspring is enhanced by prenatal exposure to very low doses of DES (0.0010.1 µg/kg/day). In sharp contrast, normal development of both the uterus in female offspring and prostate in male offspring is inhibited by maternal administration of high doses of DES (100 µg/kg/day) (vom Saal et al., 1997
; Alworth et al., 1999
; Newbold et al.,1999). In the present study, only male offspring were examined, but based on these recent findings with DES, future studies focusing on the effects of maternal administration of EE on female offspring are warranted.
In summary, in the present study it was found that prenatal exposure to low doses of the oestrogen used in OC in the USA, ethinyl oestradiol, can alter the development of the prostate and testes in male mice. Characterizing the effects of low-dose exposure to oestrogen is not only of interest to clinicians, but has become of significant issue to those in the environmental community. Over the past several years there has been increasing concern that certain chemicals in the environment can mimic oestrogen action, and if exposure occurs during critical periods in organ development, these might potentially contribute to a variety of human health problems (Colborn and Clement, 1992). These chemicals are termed oestrogenic endocrine disrupting chemicals (EEDC), and have been discussed in the context of being a potential factor contributing to human epidemiological findings of decreases in sperm concentration, increases in hypospadias, cryptorchidism, and hormonally mediated cancers, such as prostate and testicular cancer (Colborn and Clement, 1992
; Toppari et al., 1996
; Swan et al., 1997
; Paulozzi, 1999
). One of the most controversial aspects of the EEDC debate is whether exposure to low doses of chemicals can disrupt development. The data presented here show that effects of EE occur at doses considerably below those previously assumed to be without effect.
Hence, it is proposed that the risk to offspring exposed in utero to EE (as well as progestins in OC) should be further explored. One of the most important lessons learned from DES, that the absence of observable malformations at birth did not serve as a predictor of reproductive system (and other) abnormalities later in life, should be applied to the assessment of risk associated with in-utero exposure to EE in OC. Until there are appropriate human studies with outcomes other than just malformations to assess the actual risk to human fetuses of exposure to the low doses of EE in OC, an emphasis on education and public awareness is suggested. That is, increased efforts should be devoted to educating the millions of women using OC with regard to the potential risks (as suggested by animal studies) that are associated with exposing their fetuses to even very low, subclinical doses of steroids used in OC. This should also raise awareness of the need for appropriate human studies to provide more definitive data to determine the actual risks.
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Acknowledgements |
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Notes |
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References |
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Akpoviroro, J. and Fotherby, K. (1980) Assay of ethinyl oestradiol in human serum and its binding to plasma proteins. J. Steroid Biochem., 13, 773779.[ISI][Medline]
Alworth, L., Howdeshell, K., Ruhlen, R. et al. (1999) Uterine response to estradiol: low-dose facilitation and high-dose inhibition due to fetal exposure to diethylstilbestrol and methoxychlor in CD-1 mice. Paper presented at the Environmental Hormones meeting. Tulane University, New Orleans, LA.
Bern, H.A., Edery, M., Mills, K.T. et al. (1987) Long-term alterations in histology and steroid receptor levels of the genital tract and mammary gland following neonatal exposure of female balb/ccrgl mice to various doses of diethylstilbestrol. Cancer Res., 47, 41654172.[Abstract]
Berndtson, W.E. and Thompson, T.L. (1990) Changing relationships between testis size, Sertoli cell number and spermatogenesis in Sprague-Dawley rats. J Androl., 11, 429435.
Bibbo, M., Gill, W.B., Azizi, F. et al. (1977) Follow-up study of male and female offspring of DES-exposed mothers. Obstet. Gynecol., 49, 18.[Abstract]
Blair, P.B., Noller, K.L., Turiel, J. et al. (1992) Disease patterns and antibody responses to viral antigens in women exposed in utero to diethylstilbestrol. In Colborn, T. and Clement, C. (eds), Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection. Princeton Scientific Publishing Company, Inc., Princeton, pp. 283288.
Branham, W.S., Zehr, D.R., Chen, J.J. et al. (1988) Uterine abnormalities in rats exposed neonatally to diethylstilbestrol, ethynylestradiol, or clomiphene citrate. Toxicology, 51, 201212.[ISI][Medline]
Brown, R.E., Jarvis, K.L. and Hyland, K.J. (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem., 180, 136139.[ISI][Medline]
Colborn, T. and Clement, C. (eds) (1992) Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection. Princeton Scientific Publishing Company, Inc., Princeton.
Cooke, P.S., Hess, R.A., Porcelli, J. et al. (1991) Increased sperm production in adult rats after transient neonatal hypothyroidism. Endocrinology, 129, 244248.[Abstract]
DeSesso, J.M. (1997) Comparative embryology. In Hood, R.D. (ed.), Handbook of Developmental Toxicology. CRC Press, New York, pp. 111174.
Dunn, T. and Green, A. (1963) Cysts of the epididymis, cancer of the cervix, granular cell myoblastoma, and other lesions after estrogen injection in newborn mice. J. Natl Cancer Inst., 31, 425438.[ISI][Medline]
Gill, W.B., Schumacher, G.F. and Bibbo, M. (1976) Structural and functional abnormalities in the sex organs of male offspring of mothers treated with diethylstilbestrol (DES). J. Reprod. Med., 16, 147153.[ISI][Medline]
Giusti, R.M., Iwamoto, K. and Hatch, E.E. (1995) Diethylstilbestrol revisited: a review of the long-term health effects. Ann. Intern. Med., 122, 778788.
Gray, L.E. (1992) Chemical-induced alterations of sexual differentiation: A review of effects in humans and rodents. In Colborn, T. and Clement, C. (eds), Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection. Princeton Scientific Publishing Company, Inc., Princeton, pp. 203230.
Gupta, C. (2000) Reproductive malformation of the male offspring following maternal exposure to estrogenic chemicals. Proc. Soc. Exp. Biol. Med., 224, 6168.
Harada, S., Takayama, S., Shibano, T. et al. (1991a) Fertility study of oral contraceptives dt-5061 and dt-5062 (1/35) in rats. Yakuri To Chiryo, 19, 157196.
Harada, S., Takayama, S., Shibano, T. et al. (1991b) Teratogenicity study of oral contraceptives dt-5061 and dt-5062 (1/35) in rats. Yakuri To Chiryo, 19, 197231.
Herbst, A.L. (1981) Diethylstilbestrol and other sex hormones during pregnancy. Obstet. Gynecol., 58, 35S40S.[Medline]
Herbst, A.L., Scully, R.E. and Robboy, S.J. (1979) Prenatal diethylstilbestrol exposure and human genital tract abnormalities. Natl Cancer Inst. Monogr., 2535.
Hines, M. (1992) Surrounded by estrogens? Considerations for neurobehavioral development in human beings. In Colborn, T. and Clement, C. (eds), Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection. Princeton Scientific Publishing Company, Inc., Princeton, pp. 261282.
Hite, R.C., Bannemerschult, R., Fox-Kuchenbecker, P. et al. (1999) Large observational trial of a new low-dose oral contraceptive containing 20 micrograms ethinylestradiol and 100 micrograms levonorgestrel (Miranova) in Germany. Eur. J. Contracept. Reprod. Health Care, 4, 713.[Medline]
Joyce, K.L., Porcelli, J. and Cooke, P.S. (1993) Neonatal goitrogen treatment increases adult testis size and sperm production in the mouse. J. Androl., 14, 448455.
Kallen, B., Mastroiacovo, P., Lancaster, P.A. et al. (1991) Oral contraceptives in the etiology of isolated hypospadias. Contraception, 44, 173182.[ISI][Medline]
Kavlock, R.J. and Ankley, G.T. (1996) A perspective on the risk assessment process for endocrine-disruptive effects on wildlife and human health. Risk Anal., 16, 731739.[ISI][Medline]
Kluin, P.M., Kramer, M.F. and de Rooij, D.G. (1984) Proliferation of spermatogonia and Sertoli cells in maturing mice. Anat. Embryol., 169, 7378.[ISI][Medline]
Labarca, C. and Paigen, K. (1980) A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem., 102, 344352.[ISI][Medline]
Li, D.K. (1998) Reply to `Prenatal exposure to sex hormones: a case-control study' (letter). Teratology, 58, 1.[ISI][Medline]
Li, D.K., Daling, J.R., Mueller, B.A. et al. (1995) Oral contraceptive use after conception in relation to the risk of congenital urinary tract anomalies. Teratology, 51, 3036.[ISI][Medline]
Martinez-Frias, M.L., Rodriguez-Pinilla, E., Bermejo, E. et al. (1998) Prenatal exposure to sex hormones: a case-control study. Teratology, 57, 812.[ISI][Medline]
McLachlan, J.A., Newbold, R.R., Shah, H.C. et al. (1982) Reduced fertility in female mice exposed transplacentally to diethylstilbestrol (DES). Fertil. Steril., 38, 364371.[ISI][Medline]
Mittendorf, R. (1995) Teratogen update: carcinogenesis and teratogenesis associated with exposure to diethylstilbestrol (DES) in utero. Teratology, 51, 435445.[ISI][Medline]
Nagel, S.C., vom Saal, F.S. and Welshons, W.V. (1998) The effective free fraction of estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity. Proc. Soc. Exp. Biol. Med., 217, 300309.[Abstract]
National Research Council (1999) Hormonally active agents in the environment. National Academy Press, Washington, DC.
Newbold, R. (1995) Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens. Environ. Health Perspect., 103, 8387.[ISI][Medline]
Newbold, R.R., Jefferson, W.N. and Banks, E.P. (1999) Developmental exposure to low doses of diethylstilbestrol (DES) results in permanent alterations in the reproductive tract. Poster presented at the Endocrine Society. San Diego, CA.
Nonneman, D.J., Ganjam, V.K., Welshons, W.V. et al. (1992) Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice. Biol. Reprod., 47, 723729.[Abstract]
Paulozzi, L.J. (1999) International trends in rates of hypospadias and cryptorchidism. Environ. Health Perspect., 107, 297302.[ISI][Medline]
Raman-Wilms, L., Tseng, A.L., Wighardt, S. et al. (1995) Fetal genital effects of first-trimester sex hormone exposure: a meta-analysis. Obstet. Gynecol., 85, 141149.
Reel, J.R., Lamb, I.J. and Neal, B.H. (1996) Survey and assessment of mammalian estrogen biological assays for hazard characterization. Fundam. Appl. Toxicol., 34, 288305.[ISI][Medline]
Robb, G.W., Amann, R.P. and Killian, G.J. (1978) Daily sperm production and epididymal sperm reserves of pubertal and adult rats. J. Reprod. Fertil., 54, 103107.[Abstract]
Shah, H.C. and McLachlan, J.A. (1976) The fate of diethylstilbestrol in the pregnant mouse. J Pharmacol. Exp. Ther., 197, 687696.[Abstract]
Sharpe, R.M. and Skakkebaek, N.E. (1993) Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet, 341, 13921395.[ISI][Medline]
Slikker, W., Jr., Bailey, J.R., Newport, D. et al. (1982) Placental transfer and metabolism of 17 alpha-ethynylestradiol-17 beta and estradiol-17 beta in the rhesus monkey. J. Pharmacol. Exp. Ther., 223, 483489.[Abstract]
Smithells, R.W. (1981) Oral contraceptives and birth defects. Dev. Med. Child Neurol., 23, 369372.[ISI][Medline]
Stillman, R.J. (1982) In utero exposure to diethylstilbestrol: adverse effects on the reproductive tract and reproductive performance and male and female offspring. Am. J. Obstet. Gynecol., 142, 905921.[ISI][Medline]
Swan, S.H., Elkin, E.P. and Fenster, L. (1997) Have sperm densities declined? A reanalysis of global trend data. Environ. Health Perspect., 105, 12281232.[ISI][Medline]
Swan, S.H. and vom Saal, F.S. (2001) Alterations in male reproductive development: the role of endocrine disrupting chemicals. In Metzler, M. (ed.), Endocrine Disruptors in the Environment. Springer-Verlag, Heidelberg (in press).
Takasugi, N. and Bern, H. (1964) Tissue changes in mice with persistent vaginal cornification induced by early postnatal treatment with estrogen. J. Natl Cancer Inst., 33, 855865.[ISI]
Taylor, J.A., Grady, L.H., Engler, K.S. and Welshons, W.V. (1995) Relationship of growth stimulated by lithium, estradiol and EGF to phospholipase C activity in MCF-7 human breast cancer cells. Breast Cancer Res. Treat., 34, 265277.[ISI][Medline]
Toppari, J., Larsen, J.C., Christiansen, P. et al. (1996) Male reproductive health and environmental xenoestrogens. Environ. Health Perspect., 104 (Suppl. 4), 741803.[ISI][Medline]
Vergouwen, R.P., Jacobs, S.G., Huiskamp, R. et al. (1991) Proliferative activity of gonocytes, Sertoli cells and interstitial cells during testicular development in mice. J. Reprod. Fertil., 93, 233243.[Abstract]
vom Saal, F.S., Timms, B.G., Montano, M.M. et al. (1997) Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proc. Natl Acad. Sci. USA, 94, 20562061.
Walker, A.H., Bernstein, L., Warren, D.W. et al. (1990) The effect of in utero ethinyl oestradiol exposure on the risk of cryptorchid testis and testicular teratoma in mice. Br. J. Cancer, 62, 599602.[ISI][Medline]
Wilcox, A.J., Baird, D.D., Weinberg, C.R. et al. (1995) Fertility in men exposed prenatally to diethylstilbestrol. N. Engl. J. Med., 332, 14111416.
Yasuda, Y., Kihara, T. and Tanimura, T. (1985a) Effect of ethinyl estradiol on the differentiation of mouse fetal testis. Teratology, 32, 113118.[ISI][Medline]
Yasuda, Y., Kihara, T., Tanimura, T. et al. (1985b) Gonadal dysgenesis induced by prenatal exposure to ethinyl estradiol in mice. Teratology, 32, 219227.[ISI][Medline]
Yasuda, Y., Konishi, H., Matuso, T. et al. (1986a) Accelerated differentiation in seminiferous tubules of fetal mice prenatally exposed to ethinyl estradiol. Anat. Embryol., 174, 289299.[ISI][Medline]
Yasuda, Y., Konishi, H. and Tanimura, T. (1986b) Leydig cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol. Teratology, 33, 281288.[ISI][Medline]
Yasuda, Y., Konish, H. and Tanimura, T. (1987) Ovarian follicular cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol. Teratology, 36, 3543.[ISI][Medline]
Yasuda, Y., Ohara, I., Konishi, H. et al. (1988) Long-term effects on male reproductive organs of prenatal exposure to ethinyl estradiol. Am. J. Obstet. Gynecol., 159, 12461250.[ISI][Medline]
Submitted on August 15, 2000; accepted on February 14, 2001.