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Toxicology |
Deparment of Zoology,4 North Carolina State University, Raleigh, North Carolina 27695
U.S. Environmental Protection Agency,5 National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, Endocrinology Branch, Research Triangle Park, North Carolina 27711
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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androgen receptor, male reproductive tract, male sexual function, testosterone, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One example of a PE that alters sex differentiation of the male rat is butyl benzyl phthalate (BBP). BBP is a plasticizer used in some vinyls and other types of plastics. The two principal metabolites of BBP are monobenzyl phthalate and monobutyl phthalate [9]. Although mechanistic work on BBP is limited, it is thought to act in a similar fashion to other PEs, such as dibutyl phthalate (DBP) and diethylhexyl phthalate (DEHP) [8–10]. As DBP and BBP share the same metabolites and as BBP, DEHP, and DBP share a similar profile of malformations due to developmental exposure, it is likely that BBP also decreases fetal T synthesis during sex differentiation.
An example of a weak AR antagonist is the urea-based herbicide, linuron. Linuron binds to the androgen receptor and competitively inhibits AR-DNA binding and gene activation [11, 12]. Linuron exposure in utero also reduces fetal testis T production, but unlike BBP, linuron does not affect insl3 mRNA levels. Male rat offspring exposed in utero display modest reductions in neonatal AGD and, when necropsied as adults, ventral prostate weight, seminal vesicle, and epididymal weights are reduced and there are malformations of the epididymides and testis. Interestingly, the profile of effects induced by linuron in utero more closely resembles that produced by a PE than an AR antagonist. When administered at 100 mg/kg/day from Gestational Day (GD) 14 to 16, the severity of effect on the epididymides is much greater than expected for an AR antagonist whereas the external genitalia are only occasionally malformed. This profile of malformations is not indicative with pure AR antagonists, like flutamide, and more closely resembles the profile produced by the PE DBP [7]. This observation led us to hypothesize that linuron may not only be an AR antagonist but it also may decrease fetal T synthesis like the PEs [11, 12].
Endocrine-disrupting chemicals (EDC) have been identified in a wide range of different sources, including pesticides, industrial chemicals, and pharmaceuticals, and commonly exist in the environment as mixtures [5, 13–15]. However, research on EDCs traditionally focused on singular drug exposures [16, 17]. A National Research Council panel advised that risk assessment of EDCs encompass multiple pesticides that cause a common toxic effect [18]. Currently, androgen receptor (AR) antagonists, such as vinclozolin and procymidone, may be assessed for cumulative toxicity, while it is unclear at the present if chemicals from different classes, such as the thiourea herbicide linuron and PE like BBP, would be considered as well.
This study examined four main questions. First, we wished to elucidate the alterations in fetal endocrine parameters with BBP and linuron exposure singularly and in combination. Second, developmental reproductive effects of linuron and BBP in the male rat were described. Third, the potential for developmental exposure to a combination of linuron and BBP to induce cumulative fetal endocrine and reproductive alterations in the adult male rat was examined. Could these two chemicals, with seemingly different mechanisms of action, act in an additive, antagonistic, or synergistic fashion? Finally, using a chemical dose expected to induce alterations in the adult animal, antiandrogen-induced changes in early androgen-organized endpoints (neonatal AGD and juvenile areola retention) were investigated to determine if they were predictive of future adult reproductive alterations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals Pregnant Sprague-Dawley rats (Charles River Breeding Laboratory, Raleigh, NC) were shipped on the day after mating and housed individually in clear plastic cages (20 x 25 x 47 cm) with laboratory-grade pine shavings as bedding (Northeastern Products, Warrensburg, NY). The day after mating was designated Day 1 of gestation. Animals were provided Purina Rat Chow (5001) and filtered (5 µm) water, ad libitum, in a room with a 14:10D (lights off at 2300 h EST) photoperiod and temperature of 20–22°C with a relative humidity of 45–55%. All experiments were conducted following the animal care guidelines described in the National Research Council publication "Guide for the Care and Use of Laboratory Animals" [19] and conducted under an IUCAC-approved laboratory-animal protocol.
Maternal dosing On Postnatal Day (PND) 13, animals were weight ranked and assigned randomly to treatment in a manner that provided similar means in body weights for the different treatment groups. Laboratory-grade corn oil (CAS 8001-30-7, lot 107h1649; Sigma, St. Louis, MO) was the vehicle used for the dosing solutions. Four different treatment groups were used in this study—namely, 1) corn oil control, 2) linuron (CAS Number: 330-55-2, lot 225, 100% tech grade; DuPont) at 75 mg/kg/day, 3) BBP (CAS 85-68-7, 30,8501, lot 08523JQ; Aldrich) at 500 mg/kg/day, and 4) both linuron (75 mg/kg/day) and BBP (500 mg/ kg/day) coadministered to the combination treatment group. Doses of linuron and BBP were selected to induce minimal rates of malformations of the external genitalia, including hypospadias based on the literature and prior experience in the laboratory [10–12]. In contrast, we anticipated that male offspring exposed to a combination of both chemicals would display high incidences of hypospadias and vaginal pouch. Treatments were administered by oral gavage from GD 14 to 18 (sperm detected on GD 1) in 2.5 µl/g body weight of corn oil adjusted daily for body-weight gain.
Fetal Hormone Study
This study consisted of three experimental blocks with two controls, two BBP, and two linuron-treated dams per block. Dams were killed on GD 18 with a total of 6 litters per treatment.
Fetal dissections On GD 18, dams were anesthetized using carbon dioxide and killed by decapitation. Fetuses were removed, anesthetized, and killed on ice and sex determined by gonad identification using a dissecting microscope. Female fetuses were identified and retained for extraction and subsequent whole-body T measurement for blocks 2 and 3; no female fetuses were taken from block 1. Testes were removed and either used to measure T production (n = 16–24 males per treatment), progesterone production (n = 19–22 per treatment), or testicular T concentrations (n = 15–20 per treatment). After removal of testes, the carcass was frozen and kept at –20°C until extracted for determination of T concentrations (n = 35–51 males per treatment). Dams were killed in a random fashion alternating between control and treated females. Dissections were conducted within a 4-h period between 800 and 1200 h EST.
Ex vivo testicular T production The tunica for each testis was carefully torn using a pair of forceps. The testes were then incubated in 400 µl (GD 17, 18, 20) of M-199 medium (Hazleton Biologics, Inc., Lenexa, KS) + 10% steroid-stripped serum (charcoal/dextran-treated fetal bovine serum; Hyclone Laboratories, Logan, UT) for 3 h in a 37°C incubator on a rocker. At the end of the incubation, the media was removed and placed in a microcentrifuge tube, frozen on dry ice, and stored at –20°C until T and P4 radioimmunoassay (RIA) analysis.
Testicular T extraction Each testis was placed in a 12- x 75-mm glass test tube with 100 µl of distilled water. One ml (0.5 ml, 2x) of ethyl ether (99% pure) was added and the testis was crushed using a plastic pestle. The homogenized sample was placed in an acetone/dry-ice bath until the aqueous portion was frozen. The ethyl ether fraction was poured off into a clean 12- x 75-mm glass test tube and the two extractions were pooled and evaporated to dryness in a fume hood overnight. Tubes were parafilm sealed and kept at room temperature until RIA analysis for T, which was no longer than 2 wk.
Whole-body T extraction The fetus was weighed (testes already removed), placed in a 15-ml Falcon tube (Becton Dickinson, Lincoln Park, NJ), to which was added 0.5 ml of double-distilled deionized water and homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). After homogenization, ethyl ether (2 ml, 2x) was added and samples were vortexed for at least 30 sec and centrifuged at 2200 rpm (1000 x g) for 10 min. After centrifugation, the sample was placed in an acetone/dry-ice bath until the aqueous portion was frozen. The ethyl ether fraction was poured off into a clean 12- x 75-mm glass test tube and allowed to evaporate in a fume hood overnight. The extraction efficiency of radiolabeled T from whole-body homogenate was between 65% and 68%. Tubes were parafilm sealed and kept at room temperature until RIA analysis for T, which was no longer than 2 wk.
RIA analysis for T
Testosterone concentrations in media and from fetal tissue extracts were measured by RIA using a Coat-a-Count total T kit according to specifications (Diagnostic Products Corporation, Los Angeles, CA). Tubes containing dried fetal extract were resuspended in 70 µl of zero standard, vortexed for 30 sec, and a 50-µl aliquot was assayed for T. Testosterone cross-reactivity with 5
-dihydrotestosterone was 7.9%. The interassay coefficient of variation was 7%. Extracted testicular tissues were assayed for T by RIA as previously described [20–22]. Briefly, dried testes extracts were resuspended in 300 µl of phosphate buffered saline with 1% gelatin (PBSG) and vortexed for 30 sec and incubated for 10 min in a 45°C waterbath. Six hundred microliters of PBSG containing T antibody (1:10 000) and tritiated T (10 000 disintegrations per min/100 µl, 1 mCi/ml, DuPont NEN) was added to this suspension. Data are presented as litter means of 1) ng T produced per testis per 3 h, 2) ng T per testis, and 3) ng T per fetus.
Developmental Study
Neonatal and pubertal data Treated and control females were allowed to deliver naturally and on PND 2 (morning after delivery was defined as PND 1) pups were sexed by the external genitalia and their body weight recorded. AGD measurements were taken to the nearest 0.15 mm by a single experienced observer, blinded to treatment, on male and female pups using a dissecting microscope with an ocular micrometer. The AGD was defined as the distance between the base of the genital papilla and the rostral end of the anal opening. Male and female pups were individually identified within each litter with tattoo ink and placed back in their cages.
At PND 13, infant male rat offspring were reweighed and examined by an observer blinded to treatment for presence or absence of areolae/ nipples (dark areas lacking hair found in the region of the developing nipple bud). Male rats normally do not display retained nipples due to the effects of androgens on the nipple anlagen in utero. On PND 28–30, pups were weaned and males were weighed and housed with 2–3 siblings.
Necropsy At about 3 mo of age, males were killed by decapitation and necropsied. The ventral surface of each animal was shaved and examined for presence of nipples. AGD was measured with calipers and external malformations such as cleft prepuce, cleft phallus, hypospadias, exposed os penis, vaginal pouch, and incomplete preputial separation were noted. Internally, animals were examined for epididymal agenesis, testicular atrophy, undescended and ectopic testes, fluid-filled testes, and prostatic and vesicular agenesis. During necropsy, weights were taken for the following tissues: glans penis, ventral prostate, seminal vesicles, whole epididymis, right cauda, right caput and corpus, testes, levator ani plus bulbocavernosus muscle (LABC), adrenals, liver, and kidneys.
Necropsy of dams All dams in this study were weighed and then killed by decapitation. Uteri were removed and the number of implantation scars counted.
Malformations and correlations between developmental markers, malformations, and reproductive tissue weights Correlations were generated using individual values for AGD and total areolar number in infant animals. Malformations were noted as either presence or absence of effect for each animal. Dihydrotestosterone (DHT)-dependent endpoints included hypospadias, nipples, presence of a vaginal pouch, and exposed os penis. Testosterone-dependent endpoints included seminal vesicles, testicular malformations including ectopic and fluid-filled testes, and epididymal malformations (agenesis). Correlations were established for AGD by including all endpoints in both T and DHT endpoints. Nipple retention was not included among the reproductive tract malformations when analyzing for areola number correlations with adult reproductive malformations.
For areola data, animals were separated into seven categories. The categories were as follows: 0, 2, 4, 6, 8, 10, and 12 areolae present on PND 13. Likewise, AGD was separated into six different categories, with category 1 being a neonatal AGD of 1.8 mm and smaller, category 2 = 1.8–2.2 mm, category 3 = 2.2–2.6, category 4 = 2.6–3.0 mm, category 5 = 3.0–3.4 mm, category 6 = 3.4 mm and larger. Adult nipple retention also was not included among the reproductive tract malformations when analyzing for AGD or areola correlations with the malformations in adulthood.
Statistical analysis of the data Data were analyzed using a two-way ANOVA on PROC GLM from Statistical Analysis Software (SAS) for main effects of linuron and BBP and the interaction of linuron and BBP. When the interaction term is not significant (P > 0.15), this indicates that the main effects of linuron and BBP were cumulative and statistically/ effect additive. This was the case for a few endpoints, whereas for most endpoints, the interaction term was statistically significant, indicating that the effects were cumulative but not effect-additive in a mathematical or statistical sense. For the endpoints in which the interaction term is significant in all cases, the effects appeared to be dose additive, being greater than, rather than less than, effect additive. In these cases, the litter means for each of the three treated groups were compared by least-square means using a two-tailed t-test (P < 0.05) to the control value. One should not equate the presence of a significant interaction and the absence of statistical/effect additivity with synergy because the data may be dose-additive but not effect-additive. A t-test is appropriate because we hypothesized that these chemicals would increase the incidence of malformations while reducing reproductive tract tissue weights. When analysis of the block effect was not significant, this factor was not included in the final model. For analysis of all treatment effects on organ weights, litter means were used as the sample size versus the number of animals. AGD and organ weights also were analyzed using body weight as a covariate. Tissues that displayed complete agenesis were not used to calculate litter means for the organ weight in the overall analysis, such that the reduction in mean organ weight indicates that those rats that had a tissue, on the average, had smaller organs. Correlation analyses were run using the PROC CORR option on SAS. For these correlation analyses, individual values were used as the sample size.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetotoxicity Neither of the treatments, alone or in combination, significantly affected fetal male or female viability at necropsy on GD 18. Weights of fetal males ranged from 0.84 ± 0.03 g for controls to 0.82 ± 0.01 (BBP), 0.75 ± 0.2 (linuron), and 0.78 ± 0.03 (BBP + linuron) in the treated groups. Hence, the dramatic endocrine changes below, especially in the BBP + linuron groups, do not result from general fetotoxicity.
Testicular T production Testicular T production (T [ng] produced/testis/3 h) was significantly reduced in all three treatment groups versus control with a 45% reduction for BBP exposure (P < 0.0004), 34% for linuron exposure (P < 0.004), and 67% for BBP + linuron exposure (P < 0.0001) (Fig. 1A). These results continue the trend observed with whole-body T concentrations and testis T concentrations in that the greatest reduction in T production was in the BBP + linuron exposure group. This reduction is approximately 20% less than the concentrations measured in the BBP and linuron exposure groups, which suggests an effect-additive response of the two chemicals in combination. The reduction in T production was greatest with BBP + linuron exposure and was also significantly different as compared with both BBP (P < 0.04) and linuron (P < 0.004). These results were assessed using litter means (six litters per treatment) with 16–24 observations per treatment.
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Testicular T concentrations The concentration of T in the extract of testicular tissue (T [ng]/testis) was also reduced by BBP (P < 0.016), linuron (P < 0.018), and BBP/ linuron (P < 0.0001) exposure as compared with control (Fig. 1B). As in the whole-body T concentrations, the reduction in testicular T concentration was greatest in the BBP + linuron treatment, being 69% lower than controls and approximately 50% less than the concentrations measured for BBP and linuron administered singularly, with testicular T concentrations reduced by 35% for both BBP and linuron as compared with control. The reduction in testicular T concentrations was also significantly different as compared with both BBP (P < 0.02) and linuron (P < 0.02). These results were assessed using litter means (six litters per treatment) with 15–20 observations per treatment.
Fetal whole-body T concentrations and concentrations
Testosterone (T) concentrations (T [ng]/fetus) and concentrations (T [ng]/g fetus) in extracts of male fetal tissues (whole body, minus testes) were significantly reduced by BBP (P < 0.01), linuron (P < 0.0005), and BBP and linuron in combination (BBP/linuron; P < 0.0001) (Fig. 1C). The reduction in T concentrations were greatest in the BBP/ linuron treatment, being 
59% lower than controls, and these levels were similar to the normal low concentrations of T reported for the control female treatment group. Further, T concentrations for the BBP + linuron treatment were significantly different as compared with BBP treatment (P < 0.006). BBP was 71% of control T concentrations. However, there was no significant difference in T concentrations with linuron treatment (52% of control) as compared with BBP or with BBP/linuron. Data are litter means (six litters per treatment for males) with 35–51 males observed per treatment (5–10 males per litter).
Testicular progesterone production Testicular progesterone (P4) production (P [ng] produced/testis/3 h) was significantly reduced in all three treatment groups, with a 33% reduction for BBP exposure (P < 0.002), 43% for linuron exposure (P < 0.009), and 46% for BBP + linuron exposure (P < 0.0001) as compared with control (Fig. 1D). The reduction in P4 production was greatest with BBP + linuron exposure and was significantly different as compared with both BBP (P < 0.003) and linuron (P < 0.001). These results were assessed using litter means (six litters per treatment) with 19–22 observations per treatment.
Developmental Study
Maternal and pregnancy data Dams treated with BBP did not display any significant signs of maternal toxicity in terms of reduced body weight gain over the dosing period, decreased litter size, increased fetal or pup loss (Table 1). Linuron, in comparison, induced decreases in maternal weight gain over dosing period, as well as smaller litter sizes (Table 1). The mixture of BBP and linuron also had a decrease in the maternal weight gain from GD 14 to 18 (Table 1).
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Neonatal and infant data Male pup body mass was reduced by 13% in the linuron group (6.86 ± 0.25 g; P < 0.01) and by 15% in the linuron + BBP group (6.63 ± 0.3 g; P < 0.001) group as compared with controls (7.85 ± 0.19 g). Female body weights were also affected with linuron treatment, causing a 9% reduction and the linuron + BBP group experiencing 12% reduction (data not shown). Statistical significance was not achieved for treatment effects on male body weight in the juvenile and adult animals. However, linuron- and BBP + linuron-treated males tended to be smaller than controls throughout the experiment (data not shown).
AGD, in males, analyzed both with and without adjustment for pup body weight, was significantly reduced from controls in linuron (25%), BBP (13%), and BBP + linuron (43%) treatment groups as compared with control male values (Fig. 2A). Treatment with BBP, linuron, and BBP + linuron significantly increased the retention of areolae in male offspring (Fig. 2B).
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Necropsy data Adult AGD was significantly reduced in linuron (10%) and BBP + linuron (25%) (Fig. 2C). This AGD effect was significant, considering both analyses with and without body weight as a covariate. Treated animals also displayed permanent nipples. BBP animals had, on average, 1.29 nipples, linuron animals had 2.16 nipples, while BBP + linuron animals had 8.24 nipples (Fig. 2D).
No malformations of the genitalia were noted for the BBP, linuron, or control groups. The only anomaly in the linuron group was a low incidence of incomplete preputial separation (24.2%) (Fig. 3). Although not a normal condition, incomplete preputial separation was not considered a malformation because it is occasionally seen in control males. BBP + linuron-treated animals had cleft prepuce (73.4%), cleft phallus (63.2%), hypospadias (56.0%), exposed os penis (61.4%), vaginal pouch (40.2%), and incomplete preputial separation (83.2%) (Fig. 3).
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Several different internal malformations were noted in all treatment groups. Of greatest prevalence were the malformations of the ventral prostate (adhesions, agenesis), seminal vesicle (agenesis, small lobes), epididymis (agenesis), and testis (undescended-ectopic or fluid filled) (Table 2). Each of these malformations had the greatest incidence in the mixture treatment group with linuron-treated animals displaying the next highest incidence (Table 2). In addition, a low incidence (1/34) of BBP animals displayed gubernacular agenesis while another (1/34) displayed an elongated filamentous gubernaculum (34 mm in length). Unlike the effect of BBP on androgen-dependent tissues, gubernacular agenesis (an insl3-dependent process) was not enhanced by coadministration of linuron.
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Reproductive organ weights were reduced in many of the treatment groups, with the mixture group experiencing the greatest reductions in weight. Specifically, in the linuron and mixture group, the glans penis, ventral prostate, all epididymal measures, seminal vesicle, levator ani plus bulbocavernosus muscle, and paired testis weight were all reduced (Table 3). BBP treatment alone reduced ventral prostate weight, epididymal tissues, seminal vesicle, and levator ani bulbocavernosus (Table 3).
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Correlation of early developmental parameters Overall analysis showed that neonatal AGD was predictive of adult AGD (r = 0.80, P < 0.0001), infant areolae (r = –0.72, P < 0.0001), and reproductive organ weights including ventral prostate (r = 0.48, P < 0.0001), seminal vesicle (r = 0.58, P < 0.0001), LABC (r = 0.70, P < 0.0001), cauda epididymis (r = 0.61, P < 0.0001), and paired testis mass (r = 0.67, P < 0.0001) (Fig. 4, A–E). In addition, neonatal AGD was predictive of external malformations, including vaginal pouch (r = –0.47, P < 0.0001) and hypospadias (r = –0.52, P < 0.0001) (Fig. 4F). Infant areola number was also a significant predictor of adult nipple retention (r = 0.77, P < 0.0001), neonatal AGD (r = –0.72, P < 0.0001), and reproductive organ weights, including ventral prostate (r = –0.50, P < 0.0001), seminal vesicle (r = –0.65, P < 0.0001), LABC (r = –0.74, P < 0.0001), cauda epididymis (r = –0.62, P < 0.0001), and paired testis mass (r = –0.65, P < 0.0001) (Fig. 5, A–E). Finally, infant areolae were also predictive of external malformations, including vaginal pouch (r = 0.47, P < 0.0001), and hypospadias (r = 0.56, P < 0.0001) (Fig. 5F). Although correlation coefficients for both endpoints proved to be highly significant at predicting adult alterations for all endpoints examined, areola number had higher correlation coefficients (Table 4). In addition, this endpoint is not confounded by decreases in body weight, as is AGD at Day 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The reduction in T with linuron exposure seen here replicates the observation of Wilson et al. [1]. In addition, linuron binds antagonistically to the AR and decreases androgen-dependent organ weights in the Hershberger assay [25]. While it is uncertain as to which mechanism of action for linuron is causally related to the alterations of sexual differentiation of the male tract, we suspect that the inhibition of T synthesis is the primary mode of action because the profile of reproductive malformations includes high incidences of malformed epididymides and testes but a low incidence of hypospadias [7], whereas the relatively low-dose levels of AR antagonists, like flutamide, procymidone, and vinclozolin, induce a high incidence of hypospadias with epididymal and testicular lesions only occurring at much higher dosage levels. Taken together, the results of this study suggest that linuron disrupts the development of androgen-dependent tissues primarily by inhibiting T synthesis by the fetal testis, and acting as an AR antagonist.
In utero exposure to BBP altered sexual differentiation in the male Sprague-Dawley rat. Treatment-affected reproductive endpoints included reduced neonatal AGD, retention of areolae in the juvenile, adult nipple retention, and reductions in reproductive tissue weights. These results are consistent with those reported previously [9, 10, 26]. In accordance with earlier reports [9, 26], 500 mg/kg/day of BBP was not sufficient to induce malformations of the genitalia. However, a treatment-related reduction in neonatal AGD, retention of infant areolae, and permanent nipples were observed. In comparison, BBP animals were more affected internally than externally, as has been reported by Gray et al. [10]. This difference between internal and external alterations appears to be symptomatic of the effects of many of the PEs. This difference in profile of malformations initially led Gray et al. [7] and Mylchreest et al. [8] to conclude that the PEs DEHP and DBP were antiandrogenic, but acting through a mechanism of action different from a classic AR antagonist.
Although more potent than the BBP at 500 mg/kg/day, linuron at 75 mg/kg/day also was effective in altering internal but not external reproductive differentiation in the male rat. Linuron appears to be about 10-fold more potent than is BBP in reducing fetal androgen synthesis and in inducing epididymal agenesis. Currently, dose-response studies are ongoing with these chemicals to more accurately determine the relative potency of linuron to the PEs and linuron. As seen in earlier studies [7, 11], linuron significantly affected many DHT-dependent tissues but did not induce hypospadias or vaginal pouch. The high incidence of malformations of the internal structures such as the epididymal agenesis (62.5%) and testicular malformations (62.5%), is PE-like and in concordance with the hypothesis that linuron acts both as an AR antagonist and reduces fetal T synthesis.
BBP + linuron animals had a high incidence of severe malformations and alterations in both the external genitalia and internal reproductive tissues. All alterations observed were to a greater degree and severity in the mixture-exposed animals than either of the individual treatments of BBP or linuron. In fact, in some cases, the mixture group was the only treatment group to display effects due to treatment. Initial examination of the data revealed treatment effects that were at least effect additive in nature. In the case of effect additivity, when two chemicals are added, the sum of their combined effects should be represented by an arithmetic sum of the individual components (effect addition or effect summation). If adopted for the data presented herein, most of the effects observed would appear to be more than additive, considered by some to be synergistic. However, when comparing outcomes due to mixtures of chemicals, it is critical to consider the dose-response curves for each of the endpoints and individual constituents of the combination in question [17]. This often reveals that the effect is dose additive and not necessarily effect additive. This difference arises from characteristics of the dose-response curves. Use of the effect summation analysis requires that the two or more chemicals can be adjusted for the same potency and both display individual linear dose-responses for the endpoint in question [17].
In this study, many endpoints in the linuron + BBP group displayed effects that appeared to be greater than effect summation of the individual chemicals. However, in the case of AGD and ventral prostate, suspected to show a linear dose-response at the lower doses [7], the summation of the individual effects does approximate the combined results. In other endpoints, such as incidence of genital malformations and internal tissue weights, previous literature has suggested a threshold—thereby producing effects seemingly greater than additive [7].
In the male rat, the AGD is organized by DHT in the prenatal period, and accordingly, males have AGD regions approximately twice the distance of females [7, 27, 28]. Prenatal exogenous antiandrogens decrease AGD [5, 7]. In this study, changes in neonatal AGD accurately predicted the incidence of alterations in the adult animal across treatments. Areolae, indicative of adult nipples, also are sensitive to prenatal androgens alterations. In the males, prenatal androgens cause the regression of these structures; male rat pups prenatally exposed to antiandrogens have increased numbers of retained areolae and adult nipples [7, 8]. Areola expression was generally more predictive of adult alterations than changes in neonatal AGD. However, to accurately predict reproductive alterations in the adult male rat, there is justification in measuring both endpoints. Gray et al. [7] discuss the importance of measuring both endpoints to determine endocrine activity when the fetal exposure to a compound results in a loss of body weight. Reduction in body weight can cause confounding factors in the analysis of AGD. However, because the areolae are free from the effects of body weight changes, they make a suitable addition to AGD information.
In the past, the utility and justification for considering either a reduction in neonatal AGD or induction of retained nipples/areolae in males as adverse indicators of development was unknown. Concerns were raised about the true meaning of a reduction in AGD or the presence of extra areolae. Were these alterations really adverse conditions and worth consideration for the screening of potential EDCs? Consideration of the significant correlation between AGD or areolae and adverse conditions in the adult suggests these endpoints should continue to be used to assist in making accurate risk assessments for chemicals with antiandrogenic activity. A previous study demonstrated that antiandrogen-induced reductions of AGD and induction of retained nipples were representative of permanent alterations and were suggestive, but not predictive, of altered development [29]. However, in this study, these alterations were predictive of adverse adult reproductive condition. Taken together, these results indicate that treated males with the shorter AGDs and higher numbers of retained nipples are more likely to also display serious reproductive tract malformations; in this regard, it is evident that AGD and areolae measurements are useful biomarkers of antiandrogen action, if not adverse effects in themselves as they often are permanent effects.
In summary, linuron and BBP, alone or in combination, reduced fetal T production and tissue concentrations. This demonstrates that linuron disrupts the development of androgen-dependent tissues by two mechanisms (androgen receptor antagonist and fetal T synthesis inhibitor) and explains why the linuron malformation profile resembles that of the PEs for the androgen-dependent, but not the insl3-dependent, tissues.
In this study, two antiandrogens, with mixed mechanisms of action, were capable of acting in a cumulative and apparently dose-additive fashion to alter sexual differentiation in the rat. Further, some results obtained from the mixture treatment could be misinterpreted as greater than additive if consideration of the dose-response curves is not included in the analysis.
Finally, the utility of measuring both neonatal AGD and juvenile areola retention as early biomarkers for alterations in the adult animal was demonstrated. Examination of the adult AGD and nipples show that these changes are not necessarily transient and in many cases are reflective of permanent changes in adult phenotype or physiology. Continued use of these endpoints is definitely warranted for future screening and testing of endocrine-active chemicals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: L. Earl Gray, Jr., U.S. EPA, NHEERL, RTD (MD-72), RTP, NC 27711. FAX: 919 541 4017; gray.earl{at}epamail.epa.gov
3 Current address: Department of Develop & Reprod Toxicology, Merck Research Lab, WP 45-119, West Point, PA 19486. FAX: 215 652 7758; louise_saldutti{at}merck.com
Received: 7 May 2004.
First decision: 4 June 2004.
Accepted: 21 July 2004.
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  H. M. Koch and A. M. Calafat Human body burdens of chemicals used in plastic manufacture Phil Trans R Soc B, July 27, 2009; 364(1526): 2063 - 2078. [Abstract] [Full Text] [PDF]
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 C. V. Rider, V. S. Wilson, K. L. Howdeshell, A. K. Hotchkiss, J. R. Furr, C. R. Lambright, and L. E. Gray Jr Cumulative Effects of In Utero Administration of Mixtures of "Antiandrogens" on Male Rat Reproductive Development Toxicol Pathol, January 1, 2009; 37(1): 100 - 113. [Abstract] [Full Text] [PDF]
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 A. K. Hotchkiss, C. V. Rider, C. R. Blystone, V. S. Wilson, P. C. Hartig, G. T. Ankley, P. M. Foster, C. L. Gray, and L. E. Gray Fifteen Years after "Wingspread"--Environmental Endocrine Disrupters and Human and Wildlife Health: Where We are Today and Where We Need to Go Toxicol. Sci., October 1, 2008; 105(2): 235 - 259. [Abstract] [Full Text] [PDF]
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K. L. Howdeshell, V. S. Wilson, J. Furr, C. R. Lambright, C. V. Rider, C. R. Blystone, A. K. Hotchkiss, and L. E. Gray Jr A Mixture of Five Phthalate Esters Inhibits Fetal Testicular Testosterone Production in the Sprague-Dawley Rat in a Cumulative, Dose-Additive Manner Toxicol. Sci., September 1, 2008; 105(1): 153 - 165. [Abstract] [Full Text] [PDF]
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K. L. Howdeshell, J. Furr, C. R. Lambright, V. S. Wilson, B. C. Ryan, and L. E. Gray Jr Gestational and Lactational Exposure to Ethinyl Estradiol, but not Bisphenol A, Decreases Androgen-Dependent Reproductive Organ Weights and Epididymal Sperm Abundance in the Male Long Evans Hooded Rat Toxicol. Sci., April 1, 2008; 102(2): 371 - 382. [Abstract] [Full Text] [PDF]
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K. L. Howdeshell, J. Furr, C. R. Lambright, C. V. Rider, V. S. Wilson, and L. E. Gray Jr Cumulative Effects of Dibutyl Phthalate and Diethylhexyl Phthalate on Male Rat Reproductive Tract Development: Altered Fetal Steroid Hormones and Genes Toxicol. Sci., September 1, 2007; 99(1): 190 - 202. [Abstract] [Full Text] [PDF]
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C. R. Blystone, C. S. Lambright, K. L. Howdeshell, J. Furr, R. M. Sternberg, B. C. Butterworth, E. J. Durhan, E. A. Makynen, G. T. Ankley, V. S. Wilson, et al. Sensitivity of Fetal Rat Testicular Steroidogenesis to Maternal Prochloraz Exposure and the Underlying Mechanism of Inhibition Toxicol. Sci., June 1, 2007; 97(2): 512 - 519. [Abstract] [Full Text] [PDF]
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A. K. Hotchkiss, C. S. Lambright, J. S. Ostby, L. Parks-Saldutti, J. G. Vandenbergh, and L. E. Gray Jr Prenatal Testosterone Exposure Permanently Masculinizes Anogenital Distance, Nipple Development, and Reproductive Tract Morphology in Female Sprague-Dawley Rats Toxicol. Sci., April 1, 2007; 96(2): 335 - 345. [Abstract] [Full Text] [PDF]
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 L. Aksglaede, A. Juul, H. Leffers, N. E. Skakkebaek, and A.-M. Andersson The sensitivity of the child to sex steroids: possible impact of exogenous estrogens Hum. Reprod. Update, July 1, 2006; 12(4): 341 - 349. [Abstract] [Full Text] [PDF]
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