ToxSci Advance Access originally published online on April 9, 2007
Toxicological Sciences 2007 98(1):87-98; doi:10.1093/toxsci/kfm079
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Dysgenesis and Histological Changes of Genitals and Perturbations of Gene Expression in Male Rats after In Utero Exposure to Antiandrogen Mixtures
* Department of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark; and
The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom
1 To whom correspondence should be addressed. Fax: +45-72347001. E-mail: ulh{at}food.dtu.dk.
Received January 26, 2007; accepted March 12, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We investigated the ability of a mixture of three androgen receptor antagonists to induce disruption of male sexual differentiation after perinatal exposure. The aim was to assess whether the joint effects of vinclozolin, flutamide, and procymidone can be predicted based on dose-response data of the individual chemicals. Chemicals were administered orally to pregnant Wistar rats from gestational day 7 to postnatal day 16. Changes in reproductive organ weights and of androgen-regulated gene expression in prostates from male rat pups were chosen as end points for extensive dose-response studies. With all end points, the joint effects of the three antiandrogens were dose additive. Histological evaluations showed that dysgenesis and hypoplasia of prostates, seminal vesicles, and epididymis were seen with the highest mixture doses. No changes were observed in any single-compound low-dose group for these lesions, nor were there histopathological changes in the testes. Pronounced dysgenesis of external genitals was observed with all doses of the mixture, and severe dysgenesis was seen with a mixture for which the individual compounds caused no effects. A combination of doses of each chemical that on its own did not produce significant reductions in the weights of seminal vesicles and PBP C3 expression induced a marked mixture effect. Thus, antiandrogens cause additive effects on end points of various molecular complexities such as alterations at the morphological and the molecular level. Exposure to antiandrogens, which appears to exert only small effects when judged on a chemical-by-chemical basis, may induce marked responses in concert with, possibly unrecognized, similarly acting chemicals.
Key Words: mixtures; androgen receptor antagonist; vinclozolin; flutamide; procymidone; developmental toxicity; gene expression; rat; endocrine disrupters.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since the early 1990's, an increasing incidence of disorders such as cryptorchidism and hypospadia in newborn boys, decreased sperm counts in young men, and a rising incidence of hormone-related testicular cancer have been observed in the human population. Each of these endocrine disorders can be associated with subnormal androgen action in fetal life which may lead to these reproductive abnormalities, also commonly characterized as the testicular dysgenesis syndrome (TDS) (Skakkebaek et al., 2001). Environmental antiandrogens, which can arise from many different sources, including pesticides, industrial chemicals, pharmaceuticals, and phytochemicals, are potential endocrine disrupters. Such antiandrogens have the potential to perturb male reproductive development and act via a variety of mechanisms, including decreased androgen synthesis, disturbance of the pituitary-gonadal axis, and by blocking the androgen receptor (AR) (reviewed by Sharpe, 2006
). Although evidence of a causal relationship between prenatal exposure and TDS is inherently difficult to establish in human studies, a few studies have recently found correlations between human levels of environmental antiandrogens (i.e., phthalates) and anogenital distance (AGD) (Swan et al., 2005) and hormone levels in newborn boys (Main et al., 2006), indicating that exposure to endocrine disrupters may cause developmental effects in human infants.
Human exposure to single antiandrogens is generally considered low. However, as several antiandrogenic chemicals have been found to occur as mixtures in humans (Blount et al., 2000; Swan et al., 2005), including children (Brock et al., 2002; Main et al., 2006) and in wildlife (Guillette, 2000), the consequences of combined exposures to antiandrogens warrant attention in order to assess the human health risk. Only a few studies have addressed mixture effects of endocrine disrupting chemicals, focusing on antiandrogenic effects in vivo (Birkhoj et al., 2004; Gray et al., 2001; Hass et al., 2007; Nellemann et al., 2003). Until recently, little was known about the developmental effects of in utero and early postnatal exposure to multiple antiandrogenic chemicals. Consequently, we designed a mixture study of three AR antagonists: vinclozolin and procymidone, two fungicides that share a common antiandrogenic mechanism, and flutamide, a pharmaceutical used to treat prostate cancer, to study disruption of male sexual differentiation in male rat pups after gestational and lactational exposure (Hass et al., 2007). Common developmental effects of all three single chemicals after perinatal exposure of male rats include altered AGD, nipple retention (NR), hypospadias, reduced reproductive organ weights, and altered behavior in male offspring (Foster and McIntyre, 2002; Gray et al., 1994; Hellwig et al., 2000; Hib and Ponzio, 1995; Hotchkiss et al., 2002; McIntyre et al., 2001; Miyata et al., 2002; Ostby et al., 1999; Shimamura et al., 2002).
In a previous study (Hass et al., 2007), we investigated whether the joint developmental effects of these three antiandrogens could be predicted based on dose-response data of the individual chemicals by employing the concept of dose addition (Loewe and Muischnek, 1926). In the paper by Hass et al. (2007), the focus was on AGD and NR as the end points for assessment, and the results revealed that the combined effects of the three antiandrogens were dose additive for AGD and that the observed responses for NR were slightly higher than those expected on the basis of dose addition. Doses of each chemical that individually did not induce statistically significant changes in AGD led to marked effects when combined as a mixture. Furthermore, as individual doses correlated with only modest effects on NR, the mixture induced NR that approached complete "feminization" of the males (Hass et al., 2007). These results revealed that exposure to a mixture of antiandrogens with similar mechanism of action can induce marked effects even when each chemical is present at doses associated with only weak, if any, effects. This is consistent with theoretical expectations, and with earlier observations made with mixtures of estrogenic chemicals (Brian et al., 2005; Rajapakse et al., 2002; Silva et al., 2002; Tinwell and Ashby, 2004), and it highlights the importance of these findings for human and environmental risk assessment.
In the present paper, we broaden the range of end points relevant to antiandrogen action and present additional results from our original three-component mixture study with vinclozolin, flutamide, and procymidone. Our interest was to explore whether combinations of these antiandrogens followed dose additivity when end points representative of antiandrogen action at different levels of biological complexity, ranging from the molecular to the macroscopic, were chosen as the basis for evaluation. The end points selected for quantitative dose-response analysis included reproductive organ weights and perturbations of gene expression in the prostate. An additional aim was to determine whether small effects induced by low doses of single-mixture components would lead to exacerbations when the chemicals acted in concert, as previously seen for AGD and NR. To fulfill this goal, malformations of male external genitals were scored in addition to histological lesions and weights of reproductive organs as well as prostate gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Test compounds.
Vinclozolin, 99% pure (CAS no. 50471-44-8) (Bie & Berntsen, ChemService cat. no. PS-1049), flutamide, 99% pure (CAS no. 13311-84-7) (Sigma Aldrich, Brøndby, Denmark cat. no. F9397), and procymidone, 99% pure (CAS no. 32809-16-8) (Bie & Berntsen, ChemService cat. no. PS-2126) were used. Test compounds were dissolved in corn oil (Bie & Berntsen, Herlev, Denmark), which was employed as vehicle.
Studies and dose levels.
Dose-response studies for each test compound were performed prior to the mixture study in order to cover the entire range of effects, from no effect up to clear effects, without causing marked general toxicity to dams and offspring.
Dose-response study 1 involved a vehicle-dosed control group (16 dams), six doses of vinclozolin: 5, 10, 20, 40, 80, or 160 mg/kg/day and six doses of flutamide: 0.5, 1.0, 2.0, 4.0, 8.0, or 15 mg/kg/day (eight dams per dose group).
Dose-response study 2 included a vehicle-dosed control group (16 dams) and six doses of procymidone: 5, 10, 25, 50, 100, or 150 mg/kg/day (eight dams per dose group).
In the Mix study, a master mixture was prepared by combining doses of vinclozolin, flutamide, and procymidone that all induced a half-maximal degree of NR (six nipples) in male offspring. This approach was chosen in order to avoid that one single chemical contributed disproportionately to the overall mixture effect. The resulting mixture ratio of vinclozolin, flutamide, and procymidone was 0.62:0.02:0.36, and the master mixture contained 22.026 mg vinclozolin, 696.6 mg flutamide, and 12.675 mg procymidon in 600 ml corn oil. The master mixture was diluted into five dilutions termed ‘Mix1’ to ‘Mix5’. These five solutions gave rise to the following total doses of all three chemicals combined: 7.9, 19.7, 39.3, 70.8, or 106.2 mg/kg/day (16 dams per dose group). The Mix study also included a vehicle-dosed control group (16 dams) and a low and high dose of the three single compounds: vinclozolin (24.5 and 95.9 mg/kg/day), flutamide (0.77 and 3.9 mg/kg/day), and procymidone (14.1 and 68.1 mg/kg/day). For vinclozolin, 16 dams per dose group and 8 dams per dose group for the latter two compounds were included. It should be noted that the low doses of all three chemicals were combined to give one of the doses in the Mix study (‘Mix3’, 39.3 mg/kg/day).
Animals and dosing.
Time-mated young adult nulliparous Wistar rats (HanTac:WH, Taconic Europe, Denmark, body weight approximately 200g) were supplied at day 3 of pregnancy (gestational day [GD] 3). The day after arrival, at GD 4, the dams were randomly distributed into groups of 8 or 16 animals with similar body weight distribution. The animals were housed and handled as previously described (Hass et al., 2007). Test compounds and mixtures were administered by gavage from GD 7 to the day prior to expected birth (GD 21) and from postnatal day (PND) 1 to PND 16. Dams were weighed daily, and the health status of dams and offspring was monitored twice daily. All studies were divided into four blocks (one week between blocks), and each dose group was represented equally in all four blocks.
Autopsy of male pups PND 16.
The body weights of male pups were recorded, and an autopsy was performed at PND 16. Male external genitals were investigated, various organs were excised and weighed, and used for either histopathology or gene expression studies. Trunk blood was taken and pooled within litters.
Investigation of external genitals.
At PND 16, the external genitals were inspected blinded to the observer in all males from all litters. The changes were scored on a scale from 0 to 3 in order to investigate if male external genitals were demasculinized. The scores were as follows:
- Score 0 (no effect): normal genital tubercle, with the urethral opening found at the tip of the genital tubercle and the preputial skin intact. In the perineal area, thick fur extends caudally from the base of the genital tubercle and half the distance to the anus. A furless area circumscribes the anus.
- Score 1 (mild dysgenesis of the external genitals): a small cavity on the inferior side of the genital tubercle or a minor cleft in the preputial opening is observed, estimated 0.5–1.4 on an arbitrary scale. The size of the genital tubercle may be decreased. The furless area around the anus expands toward the base of the genital tubercle, but thick fur is still present at the base of the genital tubercle.
- Score 2 (moderate dysgenesis of the external genitals): the preputial cleft is larger, estimated 1.5–2.4 on an arbitrary scale. The urethral opening is situated halfway down toward the base of the genital tubercle (hypospadia). Partly furless e.g., thin fur is noted in the perineal area ranging from the base of the genital tubercle and caudally to the furless area circumscribing the anus.
- Score 3 (severe dysgenesis of the external genitals): the preputial cleft is large, estimated 2.5–3.5 on an arbitrary scale. The urethral opening is situated further than halfway down the inferior side of the genital tubercle to the base of the genital tubercle. At the base of the genital tubercle, a groove extending laterally is observed (similar to control females at PND 16). The rat is totally furless in the entire perineal area.
- Score 1 (mild dysgenesis of the external genitals): a small cavity on the inferior side of the genital tubercle or a minor cleft in the preputial opening is observed, estimated 0.5–1.4 on an arbitrary scale. The size of the genital tubercle may be decreased. The furless area around the anus expands toward the base of the genital tubercle, but thick fur is still present at the base of the genital tubercle.
Dissection and histopathology of organs.
From one male per litter at PND 16, the following organs were excised and weighed: testis, epididymides, ventral prostate, seminal vesicles, levator ani/bulbocavernosus muscle (LABC), bulbourethral glands, adrenals, kidney, and liver. From one or two males per litter, right or left testis was alternately fixed in Bouin's fixative. From males in the Mix study, the thyroid glands were excised and weighed as well. The remaining aforementioned organs, and one testis from litters with three or more males, were fixed in formalin. All fixed organs were embedded in paraffin, stained with hematoxylin and eosin, and used for the histopathological evaluation.
Gene expression levels.
When at least two males were in a litter, one pub was randomly selected and its ventral prostate was weighed and stored in RNAlater (Qiagen, Ballerup, Denmark) for gene expression analyses. The organs were homogenized, and total RNA was isolated using RNeasy-mini kit and RNase-Free DNase set (Qiagen). cDNA was synthesized from 0.5 µg total RNA using the Omniscript Reverse Transcription kit (Qiagen) with T16 oligonucleotides and a 18S rRNA primer. Samples were quantified on the 7900HT Fast Real-Time PCR System (Applied Biosystems, Naerum, Denmark) by standard TaqMan technology.
Expression levels of prostate-binding protein subunit C3 (PBP C3), ornithine decarboxylase (ODC), insulin-like growth factor I (IGF-I), complement component 3 (Compl.C3), testosterone-repressed prostate message 2 (TRPM-2), and AR were quantified in the prostate. Expression levels of each target gene were normalized to the expression level of the housekeeping gene 18S rRNA. For each sample, 2-µl cDNA (1.75 ng/µl) was amplified under universal thermal cycling parameters (Applied Biosystems) using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) in a total reaction volume of 10 µl. Three separate amplifications were performed for each gene, and when intraassay variation was above 15%, additional amplifications were performed. All genes were quantified from standard curves. Primers and probes for PBP C3, ODC, IGF-1, Compl.C3, TRPM-2, AR, and 18S rRNA have previously been published by Laier et al. (2006). The number of prostates analyzed from the control and the Mix1 to Mix5 group was 12, 11, 12, 11, 10, and 4, respectively. The number of prostrates from the low and high dose groups of single chemicals (Vin, Flu, and Pro) were 12, 10, 6, 4, 4, and 6, respectively. All prostates were from males belonging to different litters.
Statistical data analyses.
The weights of male and female pups were analyzed for all offspring. Statistical analyses of organ weights and macroscopic lesions were performed for one to four males per litter, and testes were analyzed from all offspring. The numbers of litters varied for the control and mixture groups from 11 to 13 and for the single-compound experiments from 4 to 10, respectively.
Continuous data were confirmed for normal distribution and homogeneity (Shapiro-Wilk's and Bartlett's tests). Dose-response effects different from controls were estimated by multiple testing methods (global error rate 
= 5%, two sided). When organ weights were analyzed, body weight was included as covariate, and statistical analyses were always adjusted using litter as an independent, random, and nested factor. Statistical significance was then assessed on the basis of contrast tests. All analyses were done using the SAS procedure PROC GENMOD, PROC MIXED, and PROC MULTTEST (SAS version 8, SAS Institute Inc., Cary, NC). Macroscopic and histological lesions were analyzed using Fisher's exact test with p values adjusted by permutation.
Statistical dose-response regression analyses for body weight data (seminal vesicle, ventral prostate, and LABC) and gene expression data (PBP C3/18S) were carried out by applying a best-fit approach (Scholze et al. 2001). Various nonlinear regression models (logit, probit Weibull, generalized logit) were fitted independently to the same data set, and the best fitting model was selected on the basis of a statistical goodness-of-fit criterion (information criterion of Schwarz). To control for litter effects, dose-response data were analyzed by using a generalized nonlinear mixed modeling approach (Vonesh and Chinchilli, 1996), with litter as a random effect modifier for individual organ weights and carried out using the SAS procedure PROC NLMIXED.
Prior to regression analysis, we analyzed whether there was a linear relationship between organ weights and body weight based on all control pups. This was indeed the case, and the corresponding regression lines went through the origin within their 95% confidence belts. Therefore, individual organ weights were normalized as the ratio between organ and body weight. Regression analyses were done for the single compounds on pooled effect data from the initial dose-response studies (Table 1) and the repeated doses that were run concurrently with the mixture study (Table 2).
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Under assumption of additive combination effects, a dose-response relationship for the mixture was predicted using the best-fit dose-response regression curves of the individual compounds and compared to the observed effects. Equation 1 allows calculation of any effect dose of a mixture under the hypothesis of dose additivity, provided the dose-response functions of all mixture components and the mixture ratio are known
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EDx1, EDx2, and EDx3 are the effect doses of vinclozolin, flutamide, and procymidone that on their own produce the same quantitative effect x as the mixture, and p1, p2, and p3 are the relative proportions of the corresponding individual doses present in the total mixture dose. The statistical uncertainty for the predicted mixture effects was determined by using the bootstrap method (Efron and Tibshirani, 1993) and expressed as 95% confidence limits for the predicted mean estimate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnancy Data, Postnatal Growth, and General Toxicity
No clinical signs of general toxicity were observed. The maternal body weight gain from GD 7 to PND 1, pregnancy length, litter size, birth weight of male and female offspring, sex ratio in the litters, and pup weight gain and survival were unaltered in all groups when compared to controls.
Malformations of the External Male Genitals
In the Mix study, external male genitals were investigated for malformations. For all low mixture doses (Mix1–Mix3), between 30 and 70% of the pups were diagnosed with a mild dysgenesis (score 1). At the higher doses, moderate (score 2) and severe dysgenesis (score 3) dominated, with up to 100% of the animals affected in the experiments with the two highest doses (Fig. 1A). Overall, the incidence as well as the severity of malformations increased with increasing mixture doses. For all three single compounds, pups with a mild dysgenesis were observed only at the low doses, while animals with a moderate (score 2) or severe (score 3) dysgenesis were diagnosed at the high doses.
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Concerning the number of affected males with macroscopic epididymal dysgenesis (uni- or bilateral), a clear dose-dependent effect was identifiable for the mixture (Fig. 1B). Mix3, a combination of 24.5 mg/kg vinclozolin, 0.77 mg/kg flutamide, and 14.1 mg/kg procymidone, yielded a clear increase in malformations. This response was considerably higher than any effect observed with the individual chemicals at the doses present in the combination.
Organ Weights
In the dose-response studies with the individual compounds, all reproductive organ weights showed a downward trend with increasing doses (Table 1). For vinclozolin, prostate, seminal vesicle, and epididymides weights were significantly reduced at 10 mg/kg and the remaining investigated organs, except kidneys, only at the higher doses (80 and 160 mg/kg). For prostate and seminal vesicle weights, these differences can be explained in terms of lower data variations with consequent lower statistical detection limits. A similar pattern became apparent for flutamide, although for all administered doses, the weights of the bulbourethral glands, adrenals, kidneys, and livers could not be detected as statistically different from the controls. For procymidone, seminal vesicle weights were affected at the lowest dose (5 mg/kg), weights of prostate, LABC, and bulbourethral glands were affected at 10 mg/kg, and epididymides weights were reduced at 25 mg/kg. Changes in the weight of the kidney and liver could not be identified.
In the Mix study, increasing doses both for the mixtures as well as for the single compounds decreased the reproductive organ weights (Table 2). A mixture dose of 19.7 mg/kg (Mix2) reduced weights of epididymides, prostates, and bulbourethral glands. The weight of the epididymides was the most sensitive parameter among all analyzed reproductive organs. At the Mix3, dose weights of all reproductive organs except for testes were reduced. In general, all high doses of single compounds affected reproductive organ weights, whereas low doses only affected prostate and LABC weights in a few cases (Table 2).
Histopathological Effects
Histopathological effects in the ventral prostate, seminal vesicles, epididymides, and in the testes were investigated in the Mix study. The ventral prostates from 16-day-old male rats are shown in the upper three Figures (2A for vehicle controls, 2B for mixture dose Mix4, 2C for mixture dose Mix5). Compared to controls (Fig. 2A), the three highest mixture doses and all high doses of the single compounds induced a dose-dependent hypoplasia of the ventral prostate, with a severe dysgenesis at the two highest mixture doses (Mix4 and Mix5). The acini were small, and the tall cuboidal epithelial cells lining the acini were flattened in some areas (Fig. 2B). The interstititial tissue was enlarged, and the acini were devoid of secretion (Fig. 2C).
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A general hypoplasia was also seen in the seminal vesicle at the two highest mixture doses (Figs. 2E and 2F). In contrast to controls (Fig. 2D), exposed males with hypoplastic seminal vesicles lacked large ducts with papillary and villous projections (Fig. 2E). The epithelial cells lining the duct were flattened, sometimes more cuboidal than columnar, sometimes even almost squamous. In Mix5, the duct was primitively developed and was surrounded by extensive interstitial connective tissue (Fig. 2F). In the epididymides, the epithelial cells lining the tubule were occasionally small with pyknotic nuclei. Furthermore, the cells were disarranged and the tubule had no lumen (data not shown).
The incidence of male pups with histological alterations in the prostates, seminal vesicles, and epididymides are summarized in Figure 3, demonstrating that these three organs were all affected at higher mixture doses (Mix4, Mix5). For epididymides, data indicate a low incidence even at the lowest administered mixture doses but no effect of the single compounds. No changes were observed in any of the single-compound low-dose groups for any of the three organs, and there were no histopathological alterations in the testes in any of the groups in the Mix study (data not shown).
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Gene Expression
The mRNA levels of the five androgen-regulated genes PBP C3, ODC, IGF-1, Compl.C3, and TRPM-2 as well as those for the AR were investigated in ventral prostates by real-time RT-PCR in the two dose-response studies. With the aim of screening for altered gene expression to select biomarkers for the Mix study, only controls and the highest doses were examined. The experiments revealed that PBP C3 and ODC mRNA were significantly down-regulated for vinclozolin and procymidone, and a tendency toward down-regulation was seen for flutamide as well. Furthermore, Compl.C3 mRNA was significantly elevated in the prostates of rats that received flutamide and procymidone (data not shown). In the Mix study, all five androgen-regulated genes were investigated in the ventral prostate for all dose levels (Fig. 4). As expected, PBP C3, ODC, and Compl.C3 mRNA were markedly affected in the mixture experiment. A significant down-regulation of PBP C3 mRNA was found for Mix2 doses and higher, a down-regulation of ODC mRNA was evident at the Mix4 dose and higher, and finally an up-regulation of Compl.C3 from the Mix2 dose. For the single compounds, PBP C3 and Compl.C3 were affected with the highest vinclozolin dose, PBP C3 was down-regulated by procymidone, whereas flutamide showed no effects.
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Observed versus Predicted Effect of Organ Weights and PBP C3 Expression
Dose-response relationships for organ weights (LABC, seminal vesicles, and prostate) and PBP C3 expression in prostates were constructed for each of the three compounds, vinclozolin, flutamide, and procymidone (Fig. 5, left column). These data enabled us to calculate response curves for the mixture of the three compounds, under the assumption of dose addition. The anticipated (additive) responses were then compared with the experimentally observed mixture effects (Fig. 5, right column). For all end points, the means of the observed mixture effects fell within the 95% confidence interval of the prediction curves, thus demonstrating good agreement with the expected additive effects of the compounds. It is safe to conclude that the overall combination effects were dose additive in all cases.
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For each of the three AR antagonists and their mixture, the investigated end points showed comparable sensitivity. Thus, flutamide was most effective in the dose range between 1 and 10 mg/kg and procymidone and vinclozolin between 10 and 100 mg/kg, no matter whether reproductive organ weights or PBP C3 expression was considered. Similarly, the investigated mixture was active between 10 and 100 mg/kg for all end points. Our previously published data show that AGD and NR exhibited comparable sensitivity to these three chemicals (Hass et al. 2007
). Thus, all five end points of antiandrogen action, representative of differing levels of biological complexity (from the molecular to the organ level), appear to be equally well suited for assessments of mixture effects. Finally, we tested whether doses of vinclozolin, flutamide, and procymidone that on their own were not effective or showed only small effects could induce significant responses in combination (Fig. 6). Administered on their own, vinclozolin (24.5 mg/kg), flutamide (0.77 mg/kg), and procymidone (14.1 mg/kg) did generally not induce effects statistically significantly different from vehicle-treated controls, the only exceptions being vinclozolin with respect to LABC weights (Fig. 6a) and ventral prostate weights and procymidone relative to ventral prostate weights (Fig. 6b). However, in all cases, the observed mixture effects were severe and statistically significant. Moreover, they were always higher than observed for the low doses of the individual compounds and were reasonably well predictable by dose addition.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early work with antiandrogens focused mainly on events surrounding AR binding and activation and has shown that combinations of these chemicals are able to act together in an additive fashion (Birkhoj et al., 2004
; Nellemann et al., 2003). These early studies were expanded upon by a recent paper addressing the question as to whether there are also joint effects with responses further removed from AR binding and activation, such as those related to male sexual differentiation (Hass et al., 2007). In this study, a fixed mixture ratio approach was applied for assessing combination effects on the disruption of male sexual development as determined by alterations of AGD and NR in newborn rat males. Using these morphological end points as a sign of demasculinization of the males, we have shown that vinclozolin, flutamide, and procymidone exhibited dose additivity (Hass et al., 2007). By making use of material from this study, we have investigated a wider range of end points indicative of antiandrogen action in 16-day-old male pups. The selected end points were morphological changes as determined by reproductive organ weights, dysgenesis of male external genitals, histological changes in the reproductive system, and changes at the molecular level as determined by gene expression levels in the prostate. It was our hypothesis that the joint action of vinclozolin, flutamide, and procymidone should also be dose additive in relation to these end points.
The results in our study show clearly that antiandrogens with a similar mode of action (AR antagonism) work together in an additive way for a broad spectrum of end points, ranging from reproductive organ weights to PBP C3 gene expression in the prostate. Our findings are consistent with the previously reported additivity of antiandrogens on other end points such as AR receptor activation in vitro and in vivo (Birkhoj et al., 2004; Nellemann et al., 2003) and demasculinization of newborn males exposed during gestation and lactation (Hass et al., 2007). Thus, the accumulated evidence points to the fact that receptor-mediated antiandrogenic effects follow the dose-addition principle for various end points of differing biological complexity, ranging from changes at the morphological level, tissue architecture, receptor level, to changes at the gene expression level. This is surprising, considering that the features of the dose-addition concept lend themselves particularly to the modeling of events close to receptor binding and molecular activation processes. At this level of biological complexity, the basic premise of dose addition, i.e., that one compound can be replaced by a fraction of an equieffective dose of another chemical, is readily interpreted in terms of molecular interactions. Our results indicate that antiandrogen action involves effector chains that feed through to higher levels of biological complexity without violating the principles of dose addition. This insight may be of relevance for human and clinical studies and has the potential to be exploited in future biomonitoring studies.
Although the primary aim of our work was to assess the predictability of mixture effects of antiandrogens, the results of our study also allow assessments of the question as to whether there are joint effects when all mixture components are present at doses that individually do not induce detectable effects. This phenomenon, termed "something from ‘nothing’" (Silva et al., 2002), has been observed with multicomponent mixtures of estrogenic agents in reporter-based assays (Rajapakse et al., 2002; Silva et al., 2002), the uterotrophic assay (Tinwell and Ashby, 2004), and vitellogenin induction in fish (Brian et al., 2005). The basis of this phenomenon derives from the theoretical assumptions that underlie the concept of dose addition: every agent at any dose contributes, in proportion to its toxic unit, to the overall effect of a mixture. Because every mixture component can be replaced totally or in part by an equal fraction of an equieffective dose of another, it does not matter whether the individual doses are also effective on their own. "Something from ‘nothing’" effects should occur even when individual toxicants are present at doses below effect thresholds, provided sufficiently large numbers of components sum up to a suitably high total effect dose. The results shown in Figure 6 support the idea that the "something from ‘nothing’" phenomenon also applies to the end points investigated in this study. A combination of the low doses of vinclozolin, flutamide, and procymidone induced an approximately 30 and 35% reduction of seminal vesicles weights and PBP C3 expression, respectively. The effects induced by each chemical on its own did not reach statistical significance when compared with untreated controls, and thus these data support the "something from ‘nothing’" phenomenon. For the other end points, the Mix3 dose caused a more pronounced effect than that observed with the single chemicals. Generally, these results show that lack of statistical significance cannot be equated with an absence of biological effects.
In conclusion, our results show that combinations of similarly acting antiandrogens are able to affect the male offspring of rats. These effects can be predicted fairly accurately on the basis of information about the potency of the individual mixture components by using the dose-addition concept. These data lend further support to the idea that antiandrogens act together to produce marked joint effects when combined at doses that individually produce small, statistically insignificant responses. The significance of these findings for human and environmental risk assessment must be emphasized; doses of endocrine active chemicals, which appear to exert only small effects when judged on their own, may induce marked responses when they act in concert with numerous, possibly unrecognized, similarly acting agents.
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ACKNOWLEDGMENTS |
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Heidi Letting, Birgitte Møller Plesning, Dorte Hansen, Ulla El-Baroudy, Lillian Sztuk, Trine Gejsing, and Bo Herbst are thanked for their excellent technical assistance. This work is funded by the European Commission and financially supported by the European Union as part of the EDEN-project "Endocrine Disrupters: Exploring Novel Endpoints, Exposure, Low Dose- and Mixture-Effects in Humans, Aquatic Wildlife and Laboratory Animal" (QLK4-CT-2002-00603) and by the Danish Research Council grant no. 2107-04-0006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Birkhoj M, Nellemann C, Jarfelt K, Jacobsen H, Andersen HR, Dalgaard M, Vinggaard AM. The combined antiandrogenic effects of five commonly used pesticides. Toxicol. Appl. Pharmacol. (2004) 201:10–20.[CrossRef][Web of Science][Medline]
Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson EJ, Lucier GW, Jackson RJ, Brock JW. Levels of seven urinary phthalate metabolites in a human reference population. Environ. Health Perspect. (2000) 108:979–982.[CrossRef][Web of Science][Medline]
Brian JV, Harris CA, Scholze M, Backhaus T, Booy P, Lamoree M, Pojana G, Jonkers N, Runnalls T, Bonfa A, et al. Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals. Environ. Health Perspect. (2005) 113:721–728.[Web of Science][Medline]
Brock JW, Caudill SP, Silva MJ, Needham LL, Hilborn ED. Phthalate monoesters levels in the urine of young children. Bull. Environ. Contam. Toxicol. (2002) 68:309–314.[CrossRef][Web of Science][Medline]
Foster PM, McIntyre BS. Endocrine active agents: Implications of adverse and non-adverse changes. Toxicol. Pathol. (2002) 30:59–65.[CrossRef][Web of Science][Medline]
Gray LE, Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DN, Wilson V, Price M, Hotchkiss A, et al. Effects of environmental antiandrogens on reproductive development in experimental animals. Hum. Reprod. Update (2001) 7:248–264.
Gray LE Jr, Ostby JS, Kelce WR. Developmental effects of an environmental antiandrogen: The fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. (1994) 129:46–52.[CrossRef][Web of Science][Medline]
Guillette LJ Jr. Contaminant-induced endocrine disruption in wildlife. Growth. Horm. IGF Res. (2000) 10(Suppl. B):S45–S50.[Web of Science]
Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard A, Axelstad M, Metzdorff S, Kortenkamp A. Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ. Health Perspect. (2007) (in press).
Hellwig J, van Ravenzwaay B, Mayer M, Gembardt C. Pre- and postnatal oral toxicity of vinclozolin in Wistar and Long-Evans rats. Regul. Toxicol. Pharmacol. (2000) 32:42–50.[CrossRef][Web of Science][Medline]
Hib J, Ponzio R. The abnormal development of male sex organs in the rat using a pure antiandrogen and a 5 alpha-reductase inhibitor during gestation. Acta Physiol. Pharmacol. Ther. Latinoam. (1995) 45:27–33.[Medline]
Hotchkiss AK, Ostby JS, Vandenburgh JG, Gray LE Jr. Androgens and environmental antiandrogens affect reproductive development and play behavior in the Sprague-Dawley rat. Environ. Health Perspect. (2002) 110(Suppl. 3):435–439.[Web of Science][Medline]
Laier P, Metzdorff SB, Borch J, Hagen ML, Hass U, Christiansen S, Axelstad M, Kledal T, Dalgaard M, McKinnell C, et al. Mechanisms of action underlying the antiandrogenic effects of the fungicide prochloraz. Toxicol. Appl. Pharmacol. (2006) 213:160–171.[CrossRef][Web of Science][Medline]
Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, Schmidt IM, Suomi AM, Virtanen HE, Petersen DV, et al. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ. Health Perspect. (2006) 114:270–276.[Web of Science][Medline]
McIntyre BS, Barlow NJ, Foster PM. Androgen-mediated development in male rat offspring exposed to flutamide in utero: Permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues. Toxicol. Sci. (2001) 62:236–249.
Miyata K, Yabushita S, Sukata T, Sano M, Yoshino H, Nakanishi T, Okuno Y, Matsuo M. Effects of perinatal exposure to flutamide on sex hormones and androgen-dependent organs in F1 male rats. J. Toxicol. Sci. (2002) 27:19–33.[CrossRef][Medline]
Nellemann C, Dalgaard M, Lam HR, Vinggaard AM. The combined effects of vinclozolin and procymidone do not deviate from expected additivity in vitro and in vivo. Toxicol. Sci. (2003) 71:251–262.
Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray LE Jr. The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro. Toxicol. Ind. Health (1999) 15:80–93.
Rajapakse N, Silva E, Kortenkamp A. Combining xenoestrogens at levels below individual no-observed-effect concentrations dramatically enhances steroid hormone action. Environ. Health Perspect. (2002) 110:917–921.[Web of Science][Medline]
Scholze M, Bödeker W, Faust M, Backhaus T, Altenburger R, Grimme L. A general best-fit method for concentration-response curves and the estimation of low-effect concentrations. Environ. Toxicol. Chem. (2001) 20:448–457.[CrossRef][Web of Science][Medline]
Sharpe R. Pathways of endocrine disruption during male sexual differentiation and masculinization. Best Pract. Res. Clin. Endocrinol. Metab. (2006) 20:91–110.[CrossRef][Medline]
Shimamura M, Kodaira K, Kenichi H, Ishimoto Y, Tamura H, Iguchi T. Comparison of antiandrogenic activities of vinclozolin and D,L-camphorquinone in androgen receptor gene transcription assay in vitro and mouse in utero exposure assay in vivo. Toxicology (2002) 174:97–107.[CrossRef][Web of Science][Medline]
Silva E, Rajapakse N, Kortenkamp A. Something from "nothing"—eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. (2002) 36:1751–1756.[Medline]
Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS, Redmon JB, Ternand CL, Sullivan S, et al. Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environ. Health Perspect. 113:1056–1061.
Tinwell H, Ashby J. Sensitivity of the immature rat uterotrophic assay to mixtures of estrogens. Environ. Health Perspect. (2004) 112:575–582.[Web of Science][Medline]
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