Developmental stage sensitivity and mode of action information for androgen agonists and antagonists

The Science of the Total Environment 274 Ž2001. 103᎐113

Developmental stage sensitivity and mode of action information for androgen agonists and antagonists 夽 Susan Y. EulingU , Carole A. Kimmel National Center for En¨ ironmental Assessment (8623D), Ariel Rios Bldg., 1200 Pennsyl¨ ania A¨ e., NW, US En¨ ironmental Protection Agency, Washington, DC 20460, USA Received 27 July 2000; accepted 27 October 2000

Abstract The response from exposure to a toxic agent during development may vary depending on the dose, time of exposure and the mode of action. The mode of action and developmental stage sensitivity are established only for a limited number of chemical classes. Some aspects of developmental stage sensitivity that appear to affect the response to androgen agonists and antagonists are the levels and distribution of endogenous androgens and the androgen receptor at particular times during development. This information is summarized and discussed as it relates to two critical windows of development: the period of male reproductive tract differentiation, and the peripubertal period when male sexual maturation occurs. Developmental stage sensitivity and mode of action data for the androgen antagonist vinclozolin are reviewed. Vinclozolin acts by binding to and activating the androgen receptor and affects a number of endpoints of reproductive tract differentiation as well as pubertal maturation. Approaches to incorporating mode of action, developmental stage sensitivity, and doserpotency information into risk assessment, as well as the additional data needed for using mode of action information, both qualitatively and quantitatively, in risk assessment are discussed. These issues are also considered in the context of combining the risks of exposure to two or more chemicals with similar modes of action. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Critical windows; Mode of toxicity; Mechanism of action; Developmental toxicity; Vinclozolin; Androgens; Antiandrogenic; Mode of action; Male sexual development; Risk assessment; Androgen receptor

夽 This paper is a modified summary of a talk given by S. Euling at the 2000 Conference on Topics in Toxicology and Risk Assessment, Approaches for the 21st Century in Kings Island, Ohio, April 2000. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names of commercial products does not constitute endorsement or recommendation for use. U Corresponding author. Fax: q1-202-565-0078. E-mail address: [email protected] ŽS.Y. Euling..

0048-9697r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 0 1 . 0 0 7 3 6 - 7

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1. Introduction Information on developmental stage sensitivity and the mode of action for agents that cause developmental toxicity will improve risk assessment for children. Defining the developmental times when exposures lead to a response, so-called critical windows of exposure, and the times of exposure that lead to the greatest response, the most sensitive window of exposure, for a developmental toxic agent provides information for determining chemical potency for different developmental stages. Determining a chemical’s mode of action also informs the prediction of responses to a toxic agent at untested doses and the response from exposure to multiple chemicals that act via the same mode of action. Additionally, mode of action information may aid in predicting critical windows of exposure. For example, if a chemical’s mode of action is through androgen receptor ŽAR. binding, developmental periods that depend on androgen action are likely candidates for critical windows. The Food Quality Protection Act ŽFQPA, 1996. mandates cumulative risk assessment of pesticides that act via a common mechanism of toxicity; chemicals shown to share the same mechanism or mode of action may be considered effectively the same chemical, although varying in potency. In considering chemical co-exposure, we asked the following questions: Is the combined response from exposure to two chemicals that affect the same mode of action always predicted by calculating dose-addition using relative potencies at all dose combinations? Does developmental stage have an impact on the response to single or combined chemical exposure? To this end, we were interested in defining various components of developmental stage. Dihydrotestosterone ŽDHT., testosterone, and AR concentrations are components of developmental stage that may affect the level of response to androgen agonists and antagonists.

2. Mode of action information Although FQPA uses the term ‘mechanism of

action,’ it is more likely that ‘mode of action’ can be determined for a given agent. Mode of action and mechanism of action are defined differently. For the purposes of this review, mechanism of action or mechanism of toxicity encompasses all of the mechanistic steps from the initial interaction with the target site to the ultimate cellular and molecular consequence of chemical exposure ŽFig. 1.. In contrast, the mode of action or mode

Fig. 1. An illustration of the mechanism of action and target cell response after vinclozolin treatment. The horizontal open arrowhead indicates the dependence of target cell interaction and subsequent response on the developmental stage of vinclozolin exposure. Closed vertical arrowheads indicate a progression of events. Neither all molecular events nor affected endpoints are shown. The thicker bold box indicates the mode of action for vinclozolin. The mode of action data are from Kelce et al. Ž1994. and Wong et al. Ž1995.. The data on affected endpoints in male rats are from Gray et al. Ž1994.. Abbreviations: ARs androgen receptor; AGDs anogenital distance.

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of toxicity is the key event or events that occur at the target site after chemical interaction ŽIPCS, 1999.. The key event is the one change upon which all other changes depend. Fig. 1 illustrates the distinction between mode and mechanism of action for an anti-androgenic chemical, vinclozolin. The mode of action is binding to the AR; all other steps, including the endpoints of hypospadias, nipple retention, and altered anogenital distance ŽAGD., require that the chemical bind to the AR, inhibiting androgen action. The distinction between mechanism of action and mode of action is made because the complete mechanism of action is rarely known, but for some developmental toxic agents there are data supporting a particular mode of action. As a starting point for incorporating mode of action and developmental stage information into the risk assessment paradigm, the literature was reviewed for chemicals that cause developmental toxicity with information linking these two areas. Two limitations to the approach were the lack of mechanistic data in the literature for many developmental toxic agents and the assumption that each chemical affects a single mode of action. Five mode of action classes of developmental toxic chemicals were identified and evaluated for the available data supporting the mode of action, particularly the link between the mode of action and specific developmental endpoints, data available on human health effects, and quantifiable dose-response data in a reliable animal model. The five mode of action classes of developmental toxic agents reviewed were: Ž1. the dioxins and dioxin-like chemicals; Ž2. the weak organic acids; Ž3. chemicals that act by binding to and activating the estrogen receptor ŽER.; Ž4. chemicals that act by binding to and activating the AR; and Ž5. the distal cholesterol biosynthesis inhibitors. The details of the review and analysis are presented elsewhere ŽEuling and Kimmel, in preparation.. After judging the relative availability of data for these mode of action chemical classes, chemicals that bind to and activate the AR were selected for further consideration. 2.1. Androgens and antiandrogens Androgens are critical to male sexual differen-

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tiation and their mode of action is linked to many androgen-dependent events and endpoints. Androgen agonists act by increasing the total androgen activity. Androgen antagonists or anti-androgenic chemicals inhibit or completely block the response to androgens by binding to the AR without activating it. Examples of androgen agonists are the endogenous steroid hormones, testosterone and DHT, and the pharmaceuticals, danazol, methandriol, and methyltestosterone ŽSchardein, 2000.. Antiandrogenic chemicals include several pesticides, e.g., the vinclozolin metabolites M1 and M2 ŽKelce et al., 1994; Wong et al., 1995; Kelce et al., 1997., the dichloro-diphenyl-trichloroethane ŽDDT. metabolite p, p⬘1,1-dichloro-2,2-bis Ž p-chlorophenyl. ethylene Ž p, p⬘-DDE. ŽKelce et al., 1995, 1997; Maness et al., 1998; Gray et al., 1999a., the methoxychlor metabolite 2,2-bisŽ p-hydroxyphenyl.-1,1,1-trichloroethane ŽHPTE. ŽManess et al., 1998., and procymidone ŽHosokawa et al., 1993; Ostby et al., 1999; Gray et al., 1999a., and the pharmaceutical, flutamide ŽSchardein, 2000.. The AR binding mode of action class is interesting for two additional reasons. First, since some chemicals of the class are androgen antagonists and others, androgen agonists, an opportunity exists to assess two chemicals with the same mode of action but opposing effects. Second, exposure to an androgen agonist effectively adds, and exposure to an androgen antagonist subtracts from endogenous androgen levels. Since androgen levels vary throughout development, what impact does this have on the effect of androgen or antiandrogen chemical exposure at different times of development?

3. Developmental stage information 3.1. The role of androgen action in normal de¨ elopment Two events regulated at least partially by androgens are urogenital tract sexual differentiation beginning at approximately 8 weeks of gestation in humans, and male puberty and sexual maturation at approximately 12᎐17 years of age. In the

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rat, androgens also regulate urogenital tract differentiation, which begins approximately on gestation day ŽGD. 14, and the time of male puberty measured by balanopreputial separation, which occurs from approximately postnatal day ŽPND. 42᎐46 ŽClark, 1999.. During the first 8 weeks of gestation in humans, the male and female gonads are indistinguishable and are called indifferent gonads; the urogenital tract includes two duct systems, the Mullerian and Wolffian ducts ŽGeorge and Wilson, 1994.. After this time in males, the Wolffian duct differentiates into the male reproductive tract and the Mullerian duct degenerates. In females, the Mullerian duct differentiates into the uterus and Fallopian tubes while the Wolffian duct remains but does not undergo further differentiation. The requirement for androgens in male urogenital tract differentiation was deduced from a classic gonad ablation experiment in rabbits ŽJost, 1953, 1972.. In this experiment, the gonads were removed from male and female rabbits at the indifferent gonad stage. Development of the ducts progressed such that all animals, regardless of their chromosomal sex, developed a female urogenital tract. This experiment showed that the male gonad is required to induce male urogenital tract differentiation and that the testes produce the inductive agent, that was later determined to be testosterone and its metabolites. Androgens are at least partially responsible for triggering a number of other key prenatal developmental events, such as external genitalia differentiation and testes descent, as well as male pubertal developmental events. Table 1 lists some of the male prenatal and pubertal developmental events, the stage of development in humans, and the endogenous levels of serum and tissue testosterone reported at each stage. Testosterone production by the testes is first detectable at 8 weeks of gestation, reaching a maximum at 12᎐13 weeks, in humans ŽPasqualini and Kincl, 1985.. During gestation, total testosterone in cord blood decreases by approximately 5᎐6-fold from the first trimester to birth ŽTable 1.. DHT and testosterone concentrations decrease in human prostate tissue from ; 16 to 41 weeks of gestation ŽZondek et al., 1986.. Male total serum testosterone continues to decrease

after birth and plateaus until ; 10 years of age, when testosterone levels begin to increase peripubertally ŽByrd et al., 1998; Table 1.; testicular tissue testosterone levels are well-correlated with serum testosterone levels ŽBidlingmaier et al., 1983.. Total serum testosterone increases from approximately 50 to 100-fold from prepuberty to adulthood ŽTable 1.. Relatively high testosterone levels correspond to the two periods of androgen-regulated development discussed here: the period of male sexual differentiation of the urogenital tract, and the time around male sexual maturation and puberty ŽPasqualini and Kincl, 1985.. In mammals, virilization of the external genitalia is DHT-mediated whereas Wolffian duct differentiation is testosterone-mediated. Thus, the levels of DHT as well as testosterone may be important developmental stage components when considering the effects of androgen agonist or antagonist exposure. However, since total testosterone levels do not reflect biologically active testosterone, further defining developmental stages requires free testosterone measurements. In the fetus and prepubertal child, the majority of androgen is bound to albumin and sex hormonebinding globulin ŽSHBG.. Free or unbound testosterone has been estimated as 1᎐2% of total testosterone in adults ŽFisher, 1998.. However, there are developmental changes in the percentage of free testosterone and the percentage of SHBG-bound testosterone. In cord blood, free testosterone was measured to be approximately 3% of total testosterone ŽForest et al., 1974.. In boys from 0.5᎐14 years, an increase of approximately threefold in the mean % free testosterone and a decrease in the mean serum SHBG was reported ŽBelgorosky and Rivarola, 1987.. Androgens act on development by binding to the AR in target cells during sensitive developmental stages. The testes produce and secrete testosterone, which circulates in the blood. In the androgen-responsive cells, testosterone can enter the nucleus or first, be converted in the cytoplasm to DHT by the enzyme 5␣-reductase. In the nucleus, testosterone and DHT bind to the AR and this androgen᎐AR complex undergoes an activation step involving a conformational change. The

Table 1 Serum and tissue testosterone concentrations during different stages of human male development Time of development

Mean or range of total Ta concentration Žvariance.b

ReferenceŽs.c

Wolffian duct differentiation, external genitalia and fetal prostate development, scrotum formation, phallus elongation to become penis

8᎐18 weeks of pregnancy in a male fetus

3.70 ŽS.D. 0.92. ngrml cord blood

Diez d’Aux and Murphy, 1974

External genitalia and fetal prostate development, scrotum formation, testicular descent, growth of penis

12᎐13 weeks of pregnancy in a male fetus

2.55 ŽS.D. 0.99. ngrml cord blood

Abramovich and Rowe, 1973

Testicular descent, growth of penis

13᎐15 weeks of pregnancy in a male fetus

2.35 ŽS.D. 0.99.ngrml cord blood

Abramovich and Rowe, 1973

Testicular descent, growth of penis

15᎐18 weeks of pregnancy in a male fetus

2.65 ŽS.D. 0.90. ngrml cord blood

Abramovich and Rowe, 1973

Testicular descent, growth of penis

18᎐22 weeks of pregnancy in a male fetus

1.17 ngrml cord blood

Abramovich and Rowe, 1973

Birth

Term

0.39 ŽS.D. 0.11. ngrml cord blood; 0.68 ŽS.D. 0.59. ngrml cord blood; - 0.8 ngrml cord blood; 0.84 ŽS.D. 0.17. ngrml cord blood;

Forest et al., 1974; Forest et al., 1973;

0.22 ŽS.E.M. 0.04. ngrml umbilical vein; 0.14 ŽS.E.M. 0.02. ngrml umbilical artery

Diez d’Aux and Murphy, 1974; Abramovich and Rowe, 1973; Dawood and Saxena, 1977; Dawood and Saxena, 1977

Fetal and neonatal prostate

16᎐41 weeks gestation

13 ŽS.D. 24. pgrmg tissue wet weight Žrange: 0.7᎐135.

Zondek et al., 1986

Newborn

1᎐15 day

0.68 ŽS.D. 0.60. ngrml

Forest et al., 1974

Child

10᎐11 yr

0.05᎐0.50 ngrml

Fisher, 1998

S.Y. Euling, C.A. Kimmel r The Science of the Total En¨ ironment 274 (2001) 103᎐113

Developmental stageŽs.reventŽs.

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Table 1 Ž Continued. Time of development

Mean or range of total Ta concentration Žvariance.b

ReferenceŽs.c

Prepubertal child

Mixed ages

0.066 ŽS.D. 0.025. ngrml

Forest et al., 1974

Prepuberty: no pubic hair and no growth of genitalia

Tanner stage Id

5᎐95 percentiles: - 0.058᎐0.260 ngrmle ; 0.02᎐0.23 ngrml

Andersson et al., 1997; Fisher, 1998

Puberty: sparse pubic hair development, enlarged scrotum and testes

Tanner stage IId

5᎐95 percentiles: - 0.058᎐3.87 ngrmle ; 0.05-0.70 ngrml

Andersson et al., 1997; Fisher, 1998

Puberty: further pubic hair development, growth of penis and further growth of testes and scrotum

Tanner stage IIId

5᎐95 percentiles: 0.260᎐6.114 ngrmle ; 0.15᎐2.80 ngrml

Andersson et al., 1997; Fisher, 1998

Puberty: adult-type pubic hair with incomplete coverage, further growth of penis, testes, scrotum, and development of glans

Tanner stage IVd

5᎐95 percentiles: 2.221᎐7.643 ngrmle ; 1.05᎐5.45 ngrml

Andersson et al., 1997; Fisher, 1998

Adult genitalia and pubic hair

Tanner stage Vd

5᎐95 percentiles: 3.259᎐9.315 ngrmle ; 2.65᎐8.00 ngrml

Andersson et al., 1997; Fisher, 1998

Late Puberty

15-17 yr

2.2᎐8.0 ngrml

Fisher, 1998

᎐᎐᎐᎐᎐᎐

Adult

2.6᎐10.0 ngrml; 7.57 ŽSD 0.074. ngrml; 5.72 ŽSD 1.35. ngrml; 4.1 ŽSD 1.38. ngrmle

Fisher, 1998; Belgorosky and Rivarola, 1987; Forest et al., 1974; Luppa et al., 1997

a

T s testosterone. Values refer to serum concentrations unless otherwise noted. c In rows with data from multiple references, each value is on the same line as it’s reference. d Tanner staging for male puberty from Marshall and Tanner, 1970. e Values converted from original measurement units to these units. b

S.Y. Euling, C.A. Kimmel r The Science of the Total En¨ ironment 274 (2001) 103᎐113

Developmental stageŽs.reventŽs.

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activated androgen᎐AR complex is capable of binding to androgen-response elements ŽAREs., regulatory sequences upstream of genes whose expression is androgen-regulated. As a result, particular genes are turned on or off in response to androgen activity in the cell. Androgen antagonist chemicals bind to the AR, thereby reducing or blocking androgen action, depending upon the potency, dose, and timing of antiandrogen exposure. For determining AR activity levels, the concentration or density of unbound AR in the target tissue is measured but such measurements are difficult. The timing of appearance of AR expression in some male organs has been determined in the rat. AR expression was first detected at GD 14 in the rat testes and the reproductive tract mesenchyme near the testes, and in the urogenital sinus on GD 16 ŽBentvelsen et al., 1995.. Studies of the timing of AR expression in male reproductive organs in the mouse found that AR expression begins in the Wolffian ducts and urogenital sinus on approximately GD 16 and AR expression begins in epithelium of the epididymis and ductus deferens by GD 19 ŽCooke et al., 1991.. Thus, the time period when androgens and AR are detected, and the events of male sexual differentiation are correlated with one another. 3.2. Critical windows for exposure to androgen agonists and antagonists Response data from exposure to a particular chemical at different developmental stages, various exposure intervals, multiple doses, and for all affected endpoints are often incomplete. Critical windows of exposure are defined as intervals of developmental time when an organ or system is particularly vulnerable to chemical exposure. Critical windows of exposure are presumed to correspond to times of cellular differentiation, the transition from the presumptive to the determined cell fate, and occur just before or during the time of appearance of a particular developing structure ŽWilson, 1965; reviewed in Selevan et al., 2000.. For example, the critical window for

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possible effects on development of the external genitalia is from approximately the 6th to the 12th week of gestation in humans, a timeframe that overlaps the time of external genitalia development and differentiation ŽMoore and Persaud, 1998.. The external genitalia are at an indifferent stage from the 4th to 7th weeks of gestation and the appearance and development of male external genitalia occurs between the 9th and 12th weeks and is dependent on androgen secretion from the testes ŽMoore and Persaud, 1998.. Female genitalia differentiate during this same period but in the absence of androgens. In utero exposure of the developing female to exogenous androgens during this time can result in masculinization of external genitalia or the more severe pseudohermaphroditism ŽSchardein, 2000.. Synthetic progestogens, which can be converted into androgens in the fetus and by the mother, effectively have the same mode of action as androgens. Examples of chemicals that can cause these effects in human females are the androgens methandriol, danazol, and methyltestosterone, and the synthetic progestogens, ethisterone and norethindrone. From greater than 250 cases of in utero exposure to progestogens or androgens, the critical period has been approximated to be the first 10 weeks of gestation, with the most sensitive period being the 8th week of gestation ŽSchardein, 2000.. This critical window is similar to the time of endogenous androgen action on male sexual differentiation.

4. Vinclozolin Vinclozolin is a carboximide fungicide used on many crops. According to a 1990s pesticide usage study, the four US crops to which the greatest number of pounds of vinclozolin per year were applied were lettuce, green beans, strawberries, and peaches ŽUSGS, National Water Quality Assessment, 1998.. Human exposure to vinclozolin may occur via ingestion of pesticide residues on foods as well as via dermal and inhalation routes from crops andror during application of the pesticides.

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4.1. Mode of Action The vinclozolin metabolites, M1 and M2, bind to the human and rodent ARs ŽWong et al., 1995. and compete with DHT for AR binding in vitro ŽKelce et al., 1994.. Vinclozolin treatment in vivo reduces AR-dependent gene expression ŽKelce et al., 1997.. Vinclozolin treatment affects both DHT- and testosterone-mediated developmental events in rats, predicting that endogenous DHT and T levels will affect the response to vinclozolin. 4.2. Critical windows for ¨ inclozolin exposure A great deal of information on developmental stage sensitivity and affected endpoints is available for vinclozolin exposure in the rat. When rats are exposed to vinclozolin perinatally, from GD 14-PND 3 at 100 or 200 mgrkg per day, effects in male offspring can include feminized AGD, retained nipples, cleft phallus, hypospadias, suprainguinal ectopic scrotartestes, vaginal pouch, epididymal granulomas, and small to absent sex accessory glands ŽGray et al., 1994.. Additionally, the organ weights of seminal vesicle, ventral prostate, and epididymis, as well as sexual behavior, are altered in male rats ŽGray et al., 1994.. This time period of exposure, GD 14 to PND 3, corresponds to the time of sexual differentiation in the rat. To determine the most sensitive vinclozolin exposure interval within this period, animals were treated for shorter intervals. Results from perinatal vinclozolin treatment, at 400 mgrkg per day, suggest that GD 16᎐17 is the most sensitive two-day period for exposure to vinclozolin to produce male sexual differentiation effects in the rat ŽWolf et al., 2000.. Additionally, Wolf et al. Ž2000. found that 200 mgrkg per day vinclozolin treatment from GD 14᎐19 leads to effects on male reproductive development in all males, suggesting that this is the critical period. This corresponds to the developmental stage when the male urogenital tract tissues express AR ŽBentvelsen et al., 1995. and secrete androgens from the testes ŽPasqualini and Kincl, 1985.. Balanopreputial separation, which occurs at approximately PND 42-46, is a good marker for

male puberty in the rat ŽClark, 1999.. Peripubertal vinclozolin treatment of rats for 35 days beginning at weaning ŽPND 21. with 100 mgrkg per day produced effects in males including delayed puberty, reduced epididymis, seminal vesicle and ventral prostate weights, and increases in serum testosterone, 5␣-androstanediol, and luteinizing hormone ŽLH. levels ŽMonosson et al., 1999; Ashby and Lefevre, 2000.. These data demonstrate that there are at least two critical windows of exposure to vinclozolin, one prenatally and one peripubertally, for a number of different male sexual developmental effects. Additionally, data suggest that male sexual differentiation is very sensitive to low doses of vinclozolin in rodents since a sensitive marker for sexual differentiation, AGD, was reduced in males treated with doses as low as 12.5 mgrkg per day ŽGray et al., 1999b..

5. Incorporating mode of action and developmental stage information into risk assessment Mode of action information has been used qualitatively for classifying chemicals and evaluating their hazards in several cases, e.g. dioxins and organophosphate pesticides. Using vinclozolin as an example of an agent that alters development through its androgen antagonist activity, we considered how mode of action and developmental stage information could be incorporated into risk assessment. The mode of action for vinclozolin and some of the downstream molecular changes of the mechanism of action have been established. For the risk assessment of developmental toxic agents, in vivo dose response data are needed at several different developmental stages and with different intervals of exposure to define critical windows; this type of information exists for vinclozolin for a number of endpoints ŽGray et al., 1994; Monosson et al., 1999.. Vinclozolin’s critical windows of exposure for effects on males at sexual differentiation and puberty, and the times of androgen-regulated differentiation events are well correlated, indicating that developmental stage of exposure is very important in determin-

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ing hazard ŽFig. 1.. The critical window during sexual differentiation for vinclozolin exposure for most sexual differentiation effects was identified as GD 14-19 and this interval corresponds to a time of increased or detectable androgen and AR levels ŽGray et al., 1994; Monosson et al., 1999; Wolf et al., 2000; George and Wilson, 1994.. Using developmental stage information, such as levels and distribution of testosterone, DHT, and AR during rodent and human development, one can predict the potential for the effect of an androgen agonist or antagonist at different developmental stages. This information, along with relative potency, can then be used for developing a dose-response model for an androgen antagonist; such a model could be used to predict the probability of responses at untested dose levels, and at different developmental stages. Since vinclozolin’s mode of action is to compete with androgens for binding sites on the AR and its binding affinity for the AR is known, this information can be used to quantify its potency and predict its in vivo effects. Some information about aspects of male developmental stages, such as DHT and AR levels, can be quantified and differences can be used to predict effects of vinclozolin at various developmental stages. These three pieces of information, doserpotency, mode of action, and developmental stage, are necessary to inform and refine the risk assessment model. Human exposure information will then allow the risk assessment to focus in on populations that may be at increased risk. The US Environmental Protection Agency’s Office of Pesticide Programs currently divides the population into 6 subpopulations for estimating dietary exposure to pesticides: children ages 0᎐1; children ages 1᎐6; children ages 7᎐12; women of childbearing age, ages 13᎐50; men ages 13᎐50; and senior men and women ages 55 q ŽJ. Rowland, personal communication.. Obtaining exposure information for the different subpopulations would allow for determination of the most sensitive subpopulations. Given the critical window and mode of action of vinclozolin, women of childbearing age and adolescent males are likely to be populations at risk. Thus, information about mode of action and developmental stage sensitivity can, at the very least,

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suggest subpopulations for further evaluation and characterization. 5.1. What additional data are needed for risk assessment for ¨ inclozolin? Top priorities for vinclozolin data needs are target dose and exposure measurements. Vinclozolin dose information in various target organs as well as data on vinclozolin pharmacokinetic differences between humans and rodents are needed to more precisely determine the effective dose for extrapolation. It is possible that vinclozolin, as is the case for other agents that cause developmental toxicity, may affect additional modes of action at different dose levels andror at different developmental stages. Thus, it is important to generate dose-response data at a number of dose levels and developmental stages in order to sort out whether additional modes of action may be affected and also, whether target dose is always proportional to exposure level. Human vinclozolin exposure estimates during various stages of fetal and childhood development are needed. Further information is needed on species differences between rodent and human sensitivity, exposure during fetal and postnatal development, and relative timing of developmental events. While testosterone levels during development are presented herein, measures of free DHT, the ratio of testosterone to DHT and particularly, AR levels at different developmental stages would enhance the understanding of developmental stage components that are likely to impact the response to an androgen agonist or antagonist. Then, a comparison between the male developmental stages and their corresponding AR, DHT, and testosterone levels in rodents and humans can be compiled. Since in vitro and in vivo response data exist for vinclozolin and its in vitro and in vivo potencies are well correlated ŽWong et al., 1995; Gray et al., 1994., it is possible to begin defining developmental components in an in vitro system. While the in vitro system is no substitute for the whole animal, one advantage is the ability to manipulate different developmental stage components, allowing for testing of the effect of vinclozolin

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exposure at different levels of testosterone, for example. Since vinclozolin and DHT have the same mode of action but opposing effects, this scenario also presents an interesting model in which to investigate cumulative risk. Results from DHT and vinclozolin co-treatment, using levels of DHT that correspond to endogenous DHT levels in vivo for each developmental stage, may lead to an understanding of the critical concentrations for DHT and vinclozolin that differentially impact various stages of development.

Acknowledgements This work was done when SYE was an American Association for the Advancement of Science ŽAAAS. Fellow at NCEA, US-EPA. We are grateful to AAAS for their support. We thank Elizabeth Wilson and Bill Kelce for numerous discussions. We thank anonymous reviewers for their comments and suggestions. References Abramovich DR, Rowe P. Foetal plasma testosterone levels at mid-pregnancy and at term: relationship to foetal sex. J Endocrinol 1973;56Ž3.:621᎐622. Andersson AM, Juul A, Petersen JH, Muller J, Groome NP, Skakkebaek NE. Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol levels. J Clin Endocrinol Metab 1997;82Ž12.:3976᎐3981. Ashby J, Lefevre PA. The peripubertal male rat assay as an alternative to the Hershberger castrated male rat assay for the detection of anti-androgens, oestrogens and metabolic modulators. J Appl Toxicol 2000;20Ž1.:35᎐47. Belgorosky A, Rivarola MA. Changes in serum sex hormonebinding globulin and in serum non-sex hormone-binding globulin-bound testosterone during prepuberty in boys. J Steroid Biochem 1987;27Ž1-3.:291᎐295. Bentvelsen F, Brinkmann A, van der Schoot P, van der Linden JE, van der Kwast TH, Boersma WJ, Shroder F, Nijman JM. Developmental pattern and regulation by androgens of androgen receptor expression in the urogenital tract of the rat. Mol Cell Endocrinol 1995;113Ž2.:245᎐253. Bidlingmaier F, Dorr HG, Eisenmenger W, Kuhnle U, Knorr D. Testosterone and androstenedione concentrations in human testis and epididymis during the first two years of life. J Clin Endocrinol Metab 1983;57Ž2.:311᎐315.

Byrd W, Bennett MJ, Carr BR, Dong Y, Wians F, Rainey W. Regulations of biologically active dimeric inhibin A and B from infancy to adulthood in the male. J Clin Endocrinol Metab 1998;83Ž8.:2849᎐2854. Clark RL. Endpoints of reproductive system development. In: Daston G, Kimmel C, editors. An Evaluation and Interpretation of Reproductive Endpoints for Human Health Assessment, ILSI Press: Washington, 1999:10᎐27. Cooke PS, Young P, Cunha GR. Androgen receptor expression in developing male reproductive organs. Endocrinology 1991;128Ž6.:2867᎐2873. Dawood MY, Saxena BB. Testosterone and dihydrotestosterone in maternal and cord blood and in amniotic fluid. Am J Obstet Gynecol 1977;129Ž1.:37᎐42. Diez d’Aux RC, Pearson Murphy BE. Androgens in the human fetus. J Steroid Biochem 1974;5Ž3.:207᎐210. Food Quality Protection Act of 1996. Public Law 104-170, 1996. Fisher DA, editor. Quest Diagnostics Manual: endocrinology test selection and interpretation, 2nd ed. San Juan Capistrano, CA: Quest Diagnostics Inc, 1998. Forest MG, Cathiard AM, Bertrand JA. Total and unbound testosterone levels in the newborn and in normal and hypogonadal children: use of a sensitive radioimmunoassay for testosterone. J Clin Endocrinol Metab 1973;36 Ž6.:1132᎐1142. Forest MG, Sizonenko PC, Cathiard AM, Bertrand J. Hypophyso᎐gonadal function in humans during the first year of life. 1. Evidence for testicular activity in early infancy. J Clin Invest 1974;53Ž3.:819᎐828. George FW, Wilson JD. Sex determination and differentiation. In: Knobil E, Neill JD, editors. The Physiology of Reproduction, 2nd ed. New York: Raven Press, 1994:3᎐28. Gray Jr. LE, Ostby JS, Kelce WR. Developmental effects of an environmental antiandrogen: the fungicide vinclozolin alters sex differentiation of the male rat. Toxicol Appl Pharmacol 1994;129Ž1.:46᎐52. Gray Jr. LE, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby J. Administration of potentially antiandrogenic pesticides Žprocymidone, linuron, iprodione, chlozolinate, p, p⬘-DDE, and ketoconazole. and toxic substances Ždibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate. during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health 1999a;15Ž1-2.:94᎐118. Gray Jr. LE, Ostby J, Monosson E, Kelce WR. Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation in the male rat. Toxicol Ind Health 1999b;15Ž1-2.:48᎐64. Hosokawa S, Murakami M, Ineyama M, Yamada T, Yoshitake A, Yamada H, Miyamoto J. The affinity of procymidone to androgen receptor in rats and mice. J Toxicol Sci 1993;18Ž2.:83᎐93. IPCS. IPCS workshop on Developing a Conceptual Framework for Cancer Risk Assessment, 16-18 February , Lyon, France, IPCSr99.6, Final Draft, 1999.

S.Y. Euling, C.A. Kimmel r The Science of the Total En¨ ironment 274 (2001) 103᎐113 Jost A. Problems in fetal endocrinology: the gonadal and hypophyseal hormones. Recent Prog Horm Res 1953;8:379᎐418. Jost A. A new look at the mechanism controlling sexual differentiation in mammals. Johns Hopkins Med J 1972;130:38᎐53. Kelce WR, Monosson E, Gamcsik MP, Laws SC, Gray Jr. LE. Environmental hormone disruptors: evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. Toxicol Appl Pharmacol 1994;126 Ž2 .: 276᎐285. Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Wilson EM. Persistent DDT metabolite p, p⬘-DDE is a potent androgen receptor antagonist. Nature 1995; 375Ž6532.:581᎐585. Kelce WR, Lambright CR, Gray Jr. LE, Roberts KP. Vinclozolin and p,p⬘-DDE alter androgen-dependent gene expression: in vivo confirmation of an androgen receptor-mediated mechanism. Toxicol Appl Pharmacol 1997;142 Ž1.:192᎐200. Luppa P, Bruckner C, Schwab I, Hauck S, Schmidmayr S, Birkmayer C, Paulus B, Hauptmann H. 7␣-biotinylated testosterone derivatives as tracers for a competitive chemiluminescence immunoassay of testosterone in serum. Clin Chem 1997;43Ž12.:2345᎐2352. Maness SC, McDonnell DP, Gaido KW. Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells. Toxicol Appl Pharmacol 1998;151Ž1.:135᎐142. Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in boys. Arch Dis Child 1970;45Ž239.:13᎐23. Monosson E, Kelce WR, Lambright C, Ostby J, Gray Jr. LE. Peripubertal exposure to the antiandrogenic fungicide, vin-

113

clozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol Ind Health 1999;15Ž1-2.:65᎐79. Moore KL, Persaud TVN. The Developing Human. Philadelphia: W.B. Saunders, 1998:563. Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray Jr. LE. 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;15Ž1-2.:80᎐93. Pasqualini JR, Kincl FA. Hormones and the fetus. New York: Pergamon Press, 1985. Schardein JL. Chemically induced birth defects, 3rd ed.. New York: Marcel Dekker, 2000:281᎐357. Selevan SG, Kimmel CA, Mendola P. Identifying critical windows of exposure for children’s health. Environ Health Perspect 2000;108Ž3.:449᎐597. US Geological Survey. National Water Quality Assessment, 1998. http:rrca.water.usgs.govrpnspruse92r Wilson JG. In: Wilson JG, Warkany J, editors. Embryological considerations in teratology. In Teratology: Principles and techniques, Chap. 10. Chicago: The University of Chicago Press, 1965:256. Wolf CJ, LeBlanc GA, Ostby JS, Gray Jr. LE. Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci 2000;55Ž1.:152᎐161. Wong C, Kelce WR, Sar M, Wilson EM. Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J Biol Chem 1995;270Ž34.:19998᎐20003. Zondek T, Mansfield MD, Attree SL, Zondek LH. Hormone levels in the foetal and neonatal prostate. Acta Endocrinol ŽCopenh. 1986;112Ž3.:447᎐456.