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Cumulative Effects of In Utero Administration of Mixtures of "Antiandrogens" on Male Rat Reproductive Development
1 MD-72, Endocrinology Branch, Reproductive Toxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, RTP, North Carolina, USA Correspondence: L. Earl Gray Jr., MD-72, Endocrinology Branch, Reproductive Toxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27713, USA; e-mail:gray.earl{at}epa.gov.
Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act (FQPA) required the Environmental Protection Agency (EPA) to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures to provide a framework for assessing the cumulative effects of "antiandrogenic" chemicals. Rats were dosed during pregnancy with antiandrogens singly or in pairs at dosage levels equivalent to about one half of the ED50 for hypospadias or epididymal agenesis. The pairs include: AR antagonists (vinclozolin plus procymidone), phthalate esters (DBP plus BBP and DEHP plus DBP), a phthalate ester plus an AR antagonist (DBP plus procymidone), and linuron plus BBP. We predicted that each chemical by itself would induce few malformations; however, by mixing any two chemicals together, about 50% of the males would be malformed. All binary combinations produced cumulative, dose-additive effects on the androgen-dependent tissues. We also conducted a mixture study combining seven "antiandrogens" together. These chemicals elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibition of androgen synthesis). In this study, the complex mixture behaved in a dose-additive manner. Our results indicate that compounds that act by disparate mechanisms of toxicity display cumulative, dose-additive effects when present in combination.
Key Words: reproductive system • male reproduction • endocrine disrupters
There is now widespread awareness that humans (Calafat et al. 2008; Eskenazi et al. 1999; Landrigan et al. 1999; Silva, Barr et al. 2004; Silva, Reidy et al. 2004; Wolff, Britton et al. 2008; Wolff, Engel et al. 2007; Wolff, Engel et al. 2008), fish (Ankley et al. 2007; Jobling et al. 1998; Jobling and Tyler 2006; Jobling et al. 2006), and wildlife (Hall and Thomas 2007) are exposed to multiple contaminants on a continuous basis. The chemicals found in some aquatic systems include not only pesticides (Hela et al. 2005; Jaspers et al. 2006) and industrial chemicals (Hall and Thomas 2007), but also pharmaceuticals and hormones (Durhan et al. 2006; Kolpin et al. 2002). As a result, the field of "mixtures toxicology" is emerging as an area of increasing scientific and regulatory focus in the United States and abroad. For example, in 1996 the U.S. Environmental Protection Agency (EPA) began considering the cumulative risk of chemicals that act via a common mechanism of toxicity as mandated in the Food Quality Protection Act (FQPA). The U.S. EPA’s Offices of Water (OW) and Research and Development (ORD) and the U.S. EPA Superfund, Solid Waste, and Air Programs also have ongoing programs in this area. In 2003, the U.S. EPA National Center for Environmental Assessment (NCEA), ORD, published a "Framework" report that initiated a long-term effort to develop cumulative risk assessment guidance (http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=54944). The report identifies the basic elements of the cumulative risk assessment process and provides a flexible structure for conducting and evaluating cumulative risk assessment, and for addressing scientific issues related to cumulative risk. It is intended that the NCEA Framework report will serve as a foundation for developing future guidance. In this regard, the research from our laboratory, described herein, is intended to contribute to the development of a guidance framework for assessing cumulative risks to reproduction and development from exposure during pregnancy. Although many studies have examined the effects of mixtures in vitro or in short-term in vivo assays with mature animals, few studies have examined the effects of mixtures of chemicals on mammalian reproductive development. Since the early 1980s, our laboratory has been studying the effects of pesticides and toxic substances administered in utero on fetal and postnatal rodent reproductive development. We have conducted dose response studies on the postnatal reproductive effects of environmental estrogens, androgens, antiandrogens, dioxins and PCBs, and germ cell toxicants. Along with these long-term in vivo studies, we also have conducted mechanistic studies in vitro and in vivo to identify the mechanisms and modes of action of these different toxicants. Currently, we are using this information to design mixture studies to examine how members of one of these classes of toxicants, the "antiandrogens," interact when they are administered during sexual differentiation of the laboratory rat. Toxicants studied include pesticides and phthalates that disrupt sexual differentiation by acting as androgen receptor (AR) antagonists and/or inhibitors of fetal testosterone synthesis. The review that follows will: (1) describe the chronology of events that led us to initiate a "mixtures" research program with vinclozolin and procymidone and then phthalates and the pesticides prochloraz and linuron; (2) describe the modes of action in vitro and in vivo of the individual chemicals that we selected to study as the program evolved; (3) describe the mathematical modeling procedures that we now use; (4) present the results of our completed mixture studies; (5) describe our future research plans; and (6) present an alternative framework for selecting chemicals for inclusion in cumulative assessments.
Our First Mixture Study In the late 1990s the Agency began an examination of whether some or all members of the dicarboximide class of fungicides, which includes vinclozolin, iprodione, and procymidone, shared a common mechanism of toxicity. At this time, the scientific information on the mechanisms of toxicity of this class of fungicides was incomplete. For this reason, the EPA concluded in 2000 that "The Agency does not currently have a fully developed understanding of whether vinclozolin shares a common mechanism of toxicity with iprodione and procymidone because the androgen system is highly complex. As a result, the Agency has not determined if it would be appropriate to include them in a cumulative risk assessment. Therefore, for the purposes of this assessment, the Agency has assumed that vinclozolin does not share a common mechanism of toxicity with iprodione and procymidone" (http://www.epa.gov/opp00001/reregistration/vinclozolin/). In addition, in a risk assessment on procymidone in 2005, the Agency concluded that "EPA has not made a common mechanism of toxicity finding and therefore, has not assumed that procymidone has a common mechanism of toxicity with other substances for the purposes of this tolerance action" (http://www.epa.gov/oppsrrd1/REDs/procymidone_tred.pdf). At the encouragement of the program office, we initiated studies at EPA’s National Health and Environmental Effects Laboratory to better elucidate the mechanism of toxicity for these antiandrogenic fungicides as well as mixture studies on how they interact. Since then, several studies from our laboratory and other laboratories have been completed that address these uncertainties. These studies demonstrate that vinclozolin and procymidone share a common mechanism of toxicity and interact in a cumulative manner.
Our Mixture Studies with Phthalates
Concerns about some phthalates in toys led to passage of the 2008 US Consumer Protection Agency Modernization Act (Public Law No: 110-314, section 108), which prohibits the sale of certain products containing phthalates (DEHP, DBP, benzyl butyl-[BBP], di-iso-nonly phthalate [DINP], di-iso-octyl phthalate DIOP and di-n-octyl phthalate [DNOP]). Additionally, the law established the Chronic Hazard Advisory Panel charged with examining "the potential health effects of each of these phthalates both in isolation and in combination with other phthalates" and "to consider the cumulative effect of total exposure to phthalates, both from children’s products and from other sources, such as personal care products."
Our Mixture Studies with Pesticides and Phthalates with Diverse Modes of Toxicity
Mechanisms and Modes of Action of the Individual Chemicals Used in the Mixture Studies
Modes of Action of the Individual Chemicals Used in Our Mixture Studies Peripubertal administration of antiandrogens can alter the onset of pubertal landmarks and reduce androgen-dependent organ weights in the young male rat (Monosson et al. 1999). In a Hershberger assay using castrated immature testosterone-treated male rats, vinclozolin and procymidone (0, 25, 50, and 100 mg/kg/d) alone or in combination inhibited testosterone-induced growth of androgen-dependent tissues (ventral prostate, seminal vesicles, and levator ani-bulbocavernosus muscles) in a dose-additive fashion (Gray et al. 2001; Nellemann et al. 2003). Administration of vinclozolin during sexual differentiation demasculinizes and feminizes the male rat offspring such that treated males display female-like AGD at birth, retained nipples, hypospadias, suprainguinal ectopic testes, a blind vaginal pouch, and small to absent sex accessory glands (Gray et al. 1994). In contrast to the phthalates and linuron, even at high dosage levels (200 mg/kg/d), epididymal hypoplasia was rare and gubernacular agenesis was not displayed in vinclozolin-treated male offspring. At low doses, vinclozolin administration (0, 3.125, 6.25, 12.5, 25, 50, or 100 mg/kg/d from gestational day 14 to postnatal day 3) reduces neonatal AGD and increases the incidence of retained nipples/areolae in infant male rats (Gray, Ostby et al. 1999). In adult life, ventral prostate weight is permanently reduced (at 6.25, 25, 50, and 100 mg/kg/d) and male offspring display permanent female-like nipples. Treatment at 50 and 100 mg/kg/d induces hypospadias and other reproductive tract malformations (Gray, Ostby et al. 1999; Hellwig et al. 2000). The most sensitive period of development to the disruptive effects of vinclozolin is GD 16–17, with less severe effects seen in males exposed to vinclozolin on GD 14–15 and GD 18–19 (Wolf et al. 2000). When procymidone is administered from day 14 of pregnancy to day 3 after birth at 25, 50, 100, or 200 mg/kg/d, AGD is shortened in male pups, and the males display retained nipples, hypospadias, cleft phallus, a vaginal pouch, and reduced sex accessory gland size (Ostby et al. 1999). Hypospadias was displayed by males in the 50 mg/kg/d dose group and above and ectopic, undescended testes displayed at 200 mg/kg/d. These two dicarboximide pesticides not only induce reproductive tract malformations and permanent reductions in androgen-dependent organ weights, but they also program the differentiating prostatic and vesicular tissues abnormally such that F1 male offspring develop high rates of inflammation in these tissues later in life. In 1999, we observed that in utero procymidone treatment induced fibrosis, cellular infiltration, and epithelial hyperplasia in the dorsolateral and ventral prostatic and seminal vesicular tissues in the offspring at 50 mg/kg/d and above when examined as adults (Ostby et al. 1999). More recently, similar effects were seen in males exposed to vinclozolin in utero (Cowin et al. 2008). One hundred percent of male rats exposed to 100 mg/kg/d during sexual differentiation displayed prostatitis after puberty. The authors also reported that prostatic inflammation "was not associated with the emergence of premalignant lesions, such as prostatic intra-epithelial neoplasia or proliferative inflammatory atrophy, and hence mimics nonbacterial early-onset prostatitis that commonly occurs in young men" (Cowin et al. 2008). Furthermore, when global gene expression was interrogated in the ventral prostate, Rosen et al. (2005) found identical alterations after either short-term procymidone or vinclozolin treatments. In summary, the effects of procymidone and vinclozolin are identical in vivo and in vitro.
Inhibitors of Fetal Reproductive Development and Testis Hormone Production: Phthalates In utero, some phthalate esters alter the development of the male rat in an antiandrogenic manner. Prenatal exposure to DBP, BBP, DINP, and DEHP treatment cause a syndrome of effects, including underdevelopment and agenesis of the epididymis and other androgen-dependent tissues and testicular abnormalities (Foster et al. 2001; Gray et al. 2000) characterized as the "Phthalate Syndrome." Prenatal exposure to DBP from day 10 to day 22 of gestation produces effects nearly identical to those seen with DEHP, with effects occurring at dosage levels of 50–100 mg/kg/d (Mylchreest and Foster 2000; Mylchreest et al. 1999). Among the antiandrogenic EDCs, the phthalates are unique in their ability to induce agenesis of the gubernacular cords, a tissue whose development is dependent upon the peptide hormone insulin-like peptide-3 and critical for testis descent.
A Pesticide with Dual Modes of Toxicity: Linuron (herbicide) Linuron administration to the dam or in vitro also inhibits fetal male rat testosterone synthesis during sexual differentiation, demonstrating that linuron is antiandrogenic via dual mechanisms of action (Table 1), inhibiting androgen synthesis and as an AR antagonist (Wilson et al. 2004) (Wilson et al. unpublished). When administered in utero, linuron exposure causes malformations in male rat offspring. More than half of the males exposed to 100 mg linuron/kg/d (GD14–18) displayed epididymal and testicular abnormalities (Gray, Ostby et al. 1999), with effects seen at dosage levels as low as 12.5 mg/kg/d (exposed from GD 10–22) (McIntyre et al. 2000). The testicular effect seen in adult F1 males results from epididymal lesions rather than a direct effect of linuron on testis morphology. When male rat fetuses or offspring were necropsied on GD 17, 19, and 21, and postnatal days (PND) 7 and 14, epididymal malformations were not observed in fetuses from linuron-treated dams but were seen in linuron-exposed male offspring on PND 7 and 14 (McIntyre, Barlow, Sar et al. 2002). The testicular lesions are seen only in adults and not in younger animals. These lesions develop as a consequence of pressure atrophy induced by fluid accumulation in the postpubertal testis in animals with epididymal lesions. Testicular lesions were not observed at any time point during fetal or infant life. In contrast to the effects of vinclozolin and procymidone, malformed external genitalia and undescended testes were rarely displayed by linuron-exposed males. The syndrome of effects induced by linuron is atypical of an AR antagonist and more closely resembles the Phthalate Syndrome.
A Pesticide with Dual Modes of Toxicity: Prochloraz (fungicide) In a transgenerational study, prochloraz treatment from GD 14 to 18 at doses of 62.5, 125, 250, and 500 mg/kg/d delayed parturition and altered reproductive development in the male offspring in a dose-related manner (Noriega et al. 2005). Treated males displayed reduced AGD and female-like areolas (33%, 71%, and 100% in 62.5, 125, and 250 mg/kg groups, respectively), and males in the 250 mg/kg treatment group displayed hypospadias. However, the epididymides and gubernacular ligaments were relatively unaffected. In male rat offspring, the profile of effects induced by prenatal prochloraz appears to more closely resemble that of an AR antagonist, like vinclozolin, rather than an inhibitor of fetal testosterone synthesis, like a phthalate or linuron.
Dose-response Analysis of Individual Chemicals Over the past several decades, research in our laboratory has focused on defining the effects of individual chemicals on the reproductive development of male rats. However, recent advancements in analytical techniques have drawn attention to the prevalence of environmental chemical mixtures, which has shifted focus from individual chemical effects to mixtures effects (CDC 2008; Kolpin et al. 2002; Squillace et al. 2002). Now, individual chemical data are input into mathematical models of mixture toxicity to make predictions about the potential effects of mixtures on male reproductive tract development. Predicted mixture responses are compared to observed data generated from mixture exposures to determine the type of joint action (dose addition, response addition, synergy, antagonism) exhibited by the mixture. The mixture toxicity models we use require dose-response data from individual chemical exposures. These data were compiled from studies conducted in our laboratory over the past twenty years. Assumptions were introduced when dose-response data were incomplete. For example, we previously evaluated the effects of only one dose of BBP on reproductive development; the BBP dose coincided with the dose-response data from DBP, thus we assumed that BBP had a similar dose-response curve to DBP. Historical data included studies conducted by different researchers with several rat strains and slightly different dosing schedules. However, all studies included exposure to the chemical during the critical window of reproductive tract differentiation in utero. Once the raw data were compiled, we transformed the data to fit a 0-to-100 scale. For continuous end points (AGD and organ weights), we converted the data to percentage change from the control value. For malformation data, we presented the data as percentage incidence. We then graphed the data on a log-linear scale and fit the data with a logistic equation (see example in Figure 1):
where R is the response, D is the chemical dose,
Modeled versus Observed Responses
where R is the response to the mixture, Di is the concentration of chemical i in the mixture, ED50i is the concentration of chemical i that causes a 50% response, and We also calculated mixture responses with the toxic equivalency approach. The toxic equivalency approach is often associated with dioxin-like compounds (Safe 1990). For this approach, a reference chemical was selected for each end point based on the strength of the individual chemical dose response data for that end point. For example, we used vinclozolin as a reference chemical for hypospadias in one mixture study because it had the most complete dose-response data of the chemicals in the mixture (Rider et al. 2008). Relative potency factors were calculated by dividing the ED50 of the reference compound by the ED50 of each of the other mixture components. The dose of each chemical present in the mixture was then multiplied by the corresponding potency factor. The converted doses were added to get the total mixture dose in terms of the reference chemical, which could then be inserted into the logistic equation for the reference chemical to calculate the predicted mixture response. The response-addition model, also referred to as independent-action model, was first introduced by Bliss (1939) and has been used to describe mixtures of chemicals with different mechanisms of action. However, there has been discussion of whether it is possible for chemicals to have completely independent action at a common target tissue given the complexity of biological systems (Hermens and Leeuwangh 1982). The equation for response addition is based on probability theory and is expressed as:
The integrated addition model, introduced relatively recently by several different groups (Altenburger et al. 2005; Rider and Leblanc 2005; Teuschler et al. 2004), combines the dose and response addition models. In this approach, chemicals with the same mechanism of action are grouped, and the total dose associated with each group is calculated using dose addition. The groups are then combined using response addition. The integrated addition model is expressed mathematically as:
Results of Our Mixture Studies
Mixtures with Chemicals That Have the Same Mechanism of Toxicity
Vinclozolin plus Procymidone Mixtures
Phthalate Mixture Studies In both binary phthalate mixture studies, exposure to the individual chemicals resulted in no malformations or low incidences of malformations, and the combination exposures typically resulted in 50% or greater incidences of malformations (Figures 3 and 4) (Howdeshell et al. 2007).
In a more complex mixture study, we assessed the cumulative effects on fetal testosterone production following in utero exposure to a mixture of five phthalates: DBP, di-iso-butyl phthalate (DiBP), BBP, DEHP, and DPP (Howdeshell et al. 2008). First, we characterized the individual chemical dose-response relationships as described above (Figure 5). We then dosed animals on GD 8–18 with a mixture of the five phthalates. The mixture was designed such that every chemical would contribute equally to the reduction in fetal testosterone production. Finally, observed mixture responses were compared to responses predicted based on a model of dose addition (Figure 6).
From these mixture studies, we conclude that chemicals that target the androgen signaling pathway via the same mechanism of action are dose additive when present in a mixture. Currently, cumulative risk assessments have not been performed on the antiandrogenic chemicals. This work indicates that these chemicals would be good candidates for cumulative risk assessment and supports the use of the dose-addition model for determining the effects of these mixtures.
Mixtures with Chemicals That Have Different Mechanisms of Toxicity The first binary mixture consisted of a fetal testosterone inhibitor (BBP) and an antiandrogen with multiple mechanisms of action (linuron) (Hotchkiss et al. 2004). The second binary mixture consisted of DBP and the AR antagonist procymidone. In these studies, pregnant rats were dosed on GD 14–18 with either the individual compounds or the binary mixture at a dose level equivalent to approximately one half of the ED50 value for malformations (Table 2).
BBP plus Linuron
Di-n-butyl Phthalate plus Procymidone In the procymidone plus DBP study, procymidone, or DBP alone induced low incidences of hypospadias (1.5% and 0%, respectively) and vaginal pouch (0% and 0%, respectively), whereas the males treated with the combination of procymidone and DBP displayed 49% and 27% incidences of hypospadias and vaginal pouch, respectively, indicating that the interaction was at least dose additive (Figure 8). We are currently conducting an expanded binary study including multiple doses of a fixed-ratio mixture of DBP and procymidone. Initial results demonstrate that responses to the binary mixture conform to a model of dose addition, not response addition (Hotchkiss et al. unpublished data).
Seven-chemical-mixture Study Vinclozolin, Procymidone, Prochloraz, Linuron, and Three Phthalates (DBP, BBP, DEHP) To further test our hypothesis, we designed a study with seven antiandrogenic chemicals with diverse mechanisms of action including AR antagonists (vinclozolin and procymidone), mixed-mechanism chemicals that bind to the AR and decrease testosterone production (linuron and prochloraz), and testosterone synthesis–inhibiting phthalates (BBP, DBP, and DEHP) (Rider et al. 2008). According to the current mixtures paradigm, this seven-antiandrogenic-chemical mixture should conform to a model of integrated addition. However, we found that models of integrated addition or response addition consistently underestimated the effects of our seven-antiandrogenic-chemical mixture. The dose-addition model and the related toxic equivalency approach provided estimates of mixture responses that approximated the observed responses (Figure 9). For example, hypospadias was seen in 100% of the high-dose animals, and whereas dose addition and toxic equivalency models predicted 70% affected, integrated and response-addition models predicted 0% affected.
Our future studies will be designed to answer questions concerning the characteristics of chemicals that contribute to making them dose additive. Currently, the focus of cumulative-risk assessments is on chemicals with the same mechanism of action. However, we have demonstrated that chemicals with different mechanisms of action that target a common signaling pathway can also display dose additivity. Our future studies will be aimed at refining our experimental designs for mixture assessments and testing the boundaries of dose additivity with chemicals that target male reproductive tract development. In future studies, we will continue testing mixtures of chemicals that target androgen signaling in studies designed to provide clear distinctions among predictions based on each of the mixture models. For example, we are currently building on our previous study with antiandrogens by increasing the number of chemicals included in the mixture to ten. The goal of increasing the number of chemicals is to have all mixture components present in the mixture at doses clearly below their individual no observable adverse effect (NOAEL) levels. This scenario allows for a greater distinction between models of response addition (where chemicals below their NOAELs do not contribute to the mixture toxicity) and dose addition (where chemicals below their NOAELs do contribute to the overall mixture toxicity). To date, the chemicals tested in our mixtures studies target male reproductive tissues through interference with androgen signaling. Other chemicals disrupt male reproductive tissue development through mechanisms that are not fully understood. For example, in utero exposure to TCDD results in epididymal malformations in male rats that do not involve AR antagonism or testosterone synthesis inhibition. Currently, we are assessing a binary mixture of TCDD and DBP to ascertain whether these chemicals act in a dose-additive manner to elicit epididymal malformations.
Our binary mixture studies were designed to combine pairs of chemicals at doses where each chemical would produce few if any malformations, but doubling the dose of one would induce malformations in about 50% of the males. If the chemicals behaved in a dose-additive, cumulative fashion, then the mixture would produce malformations in 50% of the males, but if they interacted independently, then few males would be malformed. Our results clearly show that all binary combinations produced cumulative, dose-additive effects on the androgen-dependent tissues. We also conducted a complex mixture study combining seven "antiandrogens" together. These chemicals elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibition of androgen synthesis). In this study, the complex mixture also behaved in a dose-additive manner. Our results indicate that compounds that act by disparate mechanisms of toxicity display cumulative, dose-additive effects when present in combination. The results also suggest that a modification of the approach for cumulative risk assessments from one based upon "common mechanism of toxicity" to one that includes the cumulative assessment of chemicals that disrupt development of the same reproductive tissues during sexual differentiation would result in target organ- and timing-based approach rather than on a narrow mechanism of toxicity. We propose that the primary focus should be on the biological system (e.g., androgen signaling pathway) rather than the mechanism of toxicity and that a cumulative risk assessment could potentially include all chemicals that target that system during the same critical developmental period.
We would like to recognize the excellent scientific collaboration and support that we have received with these research projects from Gerald LeBlanc and Paul Foster.
3 Current address: NCEA, ORD, US Environmental Protection Agency, RTP, North Carolina, USA  The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, ORD, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.
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Abstract
Introduction
is the power or Hill slope of the curve, and ED50 is the exposure dose eliciting a 50% response. The parameters (Hill slope and ED50) generated from the logistic fit to the individual chemical data were used in models to make predictions of the mixture responses. 
