**6. The "endocrine disruptor" hypothesis**

Organisms encounter many environmental compounds that approximate, diminish or enhance the activity of sex-steroids. As explained in the sections on fish and amphibians, exposure to sex-steroids can morphologically and functionally reverse the sex of many vertebrate species. Here, a brief history of subsets of key events characterizing the development and expansion of the endocrine disruptor hypothesis is outlined. Emphasis is placed on the types of compounds that have received attention due to their observed effectiveness, abundance or distribution in this regard. Arguments in this section are also described with reference to some of the hormone-treatment experiments leading to current perspectives regarding the endocrine disruption of normal reproductive behavior, development and function in wildlife and laboratory species.

Naturally-occurring compounds as well as anthropogenic compounds released into the environment due to human activity have been hypothesized to affect endocrine function in vertebrates by mimicking the action of endogenous hormones (Colborn & Clement 1992), thereby 'disrupting' normal endocrine settings. In cases of endocrine disruption, exposure levels are typically too low to have toxic or acute effects on adults (Colborn et al. 1993), but affect organisms during critical organizational periods of early life stages (Guillette et al. 1995). Tissue contaminant levels previously considered safe are sufficient to alter endogenous chemical mediation in fish (Sumpter & Jobling 1995; Jobling et al. 2006), amphibians (Hayes et al. 2002; Hayes et al. 2006; Hayes et al. 2010), reptiles (Crews et al. 1995; Guillette et al. 2000), birds (Ottinger et al. 2009), and mammals (Colborn et al. 1993).

With respect to ontogenetic development of sexual dimorphism in individuals, the effects of sex-steroids are commonly discussed using the "organizational" vs. "activational" nomenclature (Phoenix et al. 1959b; a; Arnold & Breedlove 1985). For a given structure, "organizational" typically refers to effects of sex-steroid exposure during a critical time window in early development which determines the type of response or morphology that the affected structure will have. "Activational" refers to acute effects of sex-steroids on the structure after the critical window during which the structure was organized (Whalen & Edwards 1967). The type of activation is dependent on the organizational effect that

Naturally occurring dramatic sexual dimorphism in vertebrate brains are exemplified in canaries (*Serinus canaria*) and zebra finches (*Taeniopygia guttata*) where three vocal control areas in the brain are strikingly larger in males (Nottebohm & Arnold 1976). Sex steroid effects on sexually dimorphic brain development was originally investigated in mammals (Raisman & Field 1973) and have since been described in the broader central nervous system (Breedlove & Arnold 1983b; a) for all vertebrate classes (Norris & Lopez 2011a). Correspondingly, sexual dimorphisms of brain neurotransmitter systems are also now evident (De Vries et al. 1984) (Simerly et al. 1985). Note that while this classical view of sex-steroid involvement in the dimorphic brain development is very robust, the paradigm has shifted to incorporate sex differences due to gene expression which occur before gonadal differentiation and subsequent

Organisms encounter many environmental compounds that approximate, diminish or enhance the activity of sex-steroids. As explained in the sections on fish and amphibians, exposure to sex-steroids can morphologically and functionally reverse the sex of many vertebrate species. Here, a brief history of subsets of key events characterizing the development and expansion of the endocrine disruptor hypothesis is outlined. Emphasis is placed on the types of compounds that have received attention due to their observed effectiveness, abundance or distribution in this regard. Arguments in this section are also described with reference to some of the hormone-treatment experiments leading to current perspectives regarding the endocrine disruption of normal reproductive behavior,

Naturally-occurring compounds as well as anthropogenic compounds released into the environment due to human activity have been hypothesized to affect endocrine function in vertebrates by mimicking the action of endogenous hormones (Colborn & Clement 1992), thereby 'disrupting' normal endocrine settings. In cases of endocrine disruption, exposure levels are typically too low to have toxic or acute effects on adults (Colborn et al. 1993), but affect organisms during critical organizational periods of early life stages (Guillette et al. 1995). Tissue contaminant levels previously considered safe are sufficient to alter endogenous chemical mediation in fish (Sumpter & Jobling 1995; Jobling et al. 2006), amphibians (Hayes et al. 2002; Hayes et al. 2006; Hayes et al. 2010), reptiles (Crews et al. 1995; Guillette et al. 2000), birds (Ottinger et al. 2009), and mammals (Colborn et al. 1993).

occurred during the critical window of development (Guillette et al. 1995).

organizational effects of sex-steroids (Mccarthy & Arnold 2007; Arnold 2009).

**6. The "endocrine disruptor" hypothesis** 

development and function in wildlife and laboratory species.

**5. Applied considerations** 

**5.1 Brain dimorphism** 

Pasture-specific variations in plant-derived estrogens (Walker & Janney 1930) affect livestock fertility according to grazing location. Hormones and hormone analogs are routinely used to manage production of agricultural animals (Brooks et al. 1986) and byproducts of steroid analogs used in livestock production have endocrine activity and are likely to affect wildlife (Orlando et al. 2004; Soto et al. 2004; Durhan et al. 2006). Sex-steroids or their analogs are routinely applied in aquaculture and agriculture to manipulate the sex ratios, behavior and physiology of commercially reared vertebrates.

Metabolites of steroids applied to livestock have biological activity that may affect animals at sites removed from the source of application (Shelton 1990; Lone 1997; Meyer 2001). Similarly, effluent from pulp mills, paper mills and sewage treatment plants affecting the endocrinology of aquatic vertebrates are transported far from sites of entry into the environment (Jobling & Tyler 2003a; b).

A key feature in the development of the endocrine disruptor hypothesis was the concept of "environmental estrogens". The environmental estrogen was a hallmark of the hypothesis because estrogenic properties of the compounds first identified as endocrine disruptors were well described. For example, widespread use of the synthetic estrogen diethylstilbestrol (DES) in humans (Morrell 1941; Smith & Smith 1949b; a) continues to be one of the most well studied examples of endocrine disruption where effects in the exposed mothers are minimal compared to effects in their offspring who were exposed during critical windows of development (Giusti et al. 1995). Organochlorine pesticides and environmentally persistent chlorinated hydrocarbons designed to be harmless to exposed vertebrates tended to bioaccumulate and affect offspring of exposed animals. Key players in the environmental estrogen story included the synthetic estrogen DES; the organochlorine pesticides, DDT and methoxychlor; a host of polychlorinated biphenyls (PCB's); and the plasticizers bisphenol-A, nonylphenol, and octylphenol. Compounds of concern were identified based partially on their ability to bind to nuclear estrogen receptors (Blair et al. 2000), and activity observed using *in vivo* biological assessments of estrogen activity such as the rat uterotropic assay (Gray et al. 2004), and production of the egg yolk protein vitellogenin (Sumpter & Jobling 1995). *In vitro* induction of estrogen-responsive breast cancer cell lines (Klotz et al. 1996) and tests of transcription in response to estrogen receptor binding (Ernst et al. 1991) were also primary means of screening compounds for estrogenic activity. With mechanistic knowledge came distinctions between feminizing and demasculinizing effects, and the concept of anti-androgens were more carefully considered (Wilson et al. 2008) in addition to estrogens or anti-estrogens. Key anti-androgens were originally characterized by their antagonism of androgen receptor (AR). Further characterization was based on extensive batteries of *in vivo* reproductive tract and secondary sex character examinations in the laboratory rat. In these studies, androgen action *in utero*, or on pubertal development were examined (Gray et al. 2004; Owens et al. 2007). Recognized antiandrogens fell into classes including dicarboximide (Gray et al. 1994; Kelce & Wilson 1997) and imidazole fungicides (Vinggaard et al. 2002; Noriega et al. 2005), organochlorine insecticides (Kelce et al. 1995), urea-based herbicides (Gray et al. 1999; Lambright et al. 2000), phthalate esters used as plasticizers (Parks et al. 2000; Howdeshell et al. 2007), and polybrominated diphenyl ethers (PBDEs) used as flame retardants (Stoker et al. 2005).

Natural hormones (Stumm-Zollinger & Fair 1965) and birth control agents (Tabak et al. 1981) occurring in wastewater prompted concern over sewage effluents. Some compounds, such as the phthalate esters, diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP) and benzylbutyl phthalate (BBP), originally considered estrogenic (Jobling et al. 1995), also exhibited anti-androgenic properties by decreasing testosterone production in fetal testes

Evolutionary Perspectives on Sex Steroids in the Vertebrates 17

Fig. 3. Phallus abnormalities in adult male rats receiving a 5-day *in utero* exposure to prochloraz. Panels are arranged from left to right according to dosage group indicated by numbers to the left of the diagram. **A** and **B**) Variation within controls. **C** and **D**) Animals with a maternal dose of 125 mg/kg per day. **E**–**G**) Animals with a maternal dose of 250 mg/kg per day. gl, glans penis; pr, prepuce; ur, urethral opening; os, os penis. Hypospadias is evident in the two highest dosage groups and (**C**–**E**) show examples of incomplete preputial separation. Severe phallus clefting and exposure of the os penis is evident in the highest dosage group (**F** and **G**). (Noriega et al. 2005) modified with permission from the

Society for the Study of Reproduction.

and reducing the expression of steroidogenic genes after *in utero* exposure (Parks et al. 2000; Wilson et al. 2004).

Although effects of endocrine disruptors are well documented, as explained earlier in the chapter, complex endocrine interactions with other steroids, peptides and lipids are integral to the function of sex-steroids in living vertebrates. Therefore it is critical to consider the enormous range of physiological variation that occurs in vertebrates under "normal" conditions that would be considered "uncontaminated" (Orlando & Guillette 2007). Using terms like "estrogenic" or "androgenic" may limit the scope of investigation because androgens and estrogens have well-characterized function in a very small percentage of species. In addition, phylogenetic assessments discussed earlier in this chapter may expand the scope of sex-steroid considerations outside of the vertebrates. For example, the recently characterized ER in molluscs is not responsive to steroid ligands (Thornton et al. 2003) (Keay et al. 2006; Matsumoto et al. 2007) and the cephalochordate ER acts as a constitutive repressor of estrogen response element (ERE) function (Bridgham et al. 2008; Paris et al. 2008). It is important to note that although compounds may be defined based on a given outcome or mechanism of action, almost all chemicals influence multiple physiological systems and influence the way physiological systems interact with each other. For example, compounds such as linuron (a urea-based herbicide) and prochloraz (an imidazole fungicide) act as anti-androgens via multiple mechanisms of action (Lambright et al. 2000; Wilson et al. 2004; Noriega et al. 2005).

#### **6.1 A case study using prochloraz**

Any number of compounds can be used to demonstrate endocrine disruption via an endocrine active chemical (EAC). However, prochloraz provides a highly illustrative example because it affects development of external mammalian genitalia with which readers will have prior familiarity. Much of the reason that an imidazole fungicides like prochloraz kill fungi is because they affect members of the diverse cytochrome P450 enzyme family (Mason et al. 1987; Riviere & Papich 2009), a group of enzymes catalyzing electron transfer in representatives of all classes of cellular life (Nebert et al. 1989; Lewis et al. 1998; De Mot & Parret 2002; Nelson 2011). These enzymes have been evolutionarily co-opted for vertebrate steroidogenesis, as well as the metabolism of steroids and xenobiotics (Gibson et al. 2002). In addition to affecting steroidogenic cytochrome P450 enzymes and reducing androgen production, prochloraz is an androgen receptor antagonist (Vinggaard et al. 2002; Noriega et al. 2005) and inhibits testicular expression for insulin-like hormone 3 (insl3), which affects gubernacular development (Wilson et al. 2004). An example of laboratory-administered *in utero* prochloraz exposure (Noriega et al. 2005) can be used for a discussion on effects of a non-steroidal compound on sex-steroid action as viewed throughout the historical development of the endocrine disruptor hypothesis. Namely:


and reducing the expression of steroidogenic genes after *in utero* exposure (Parks et al. 2000;

Although effects of endocrine disruptors are well documented, as explained earlier in the chapter, complex endocrine interactions with other steroids, peptides and lipids are integral to the function of sex-steroids in living vertebrates. Therefore it is critical to consider the enormous range of physiological variation that occurs in vertebrates under "normal" conditions that would be considered "uncontaminated" (Orlando & Guillette 2007). Using terms like "estrogenic" or "androgenic" may limit the scope of investigation because androgens and estrogens have well-characterized function in a very small percentage of species. In addition, phylogenetic assessments discussed earlier in this chapter may expand the scope of sex-steroid considerations outside of the vertebrates. For example, the recently characterized ER in molluscs is not responsive to steroid ligands (Thornton et al. 2003) (Keay et al. 2006; Matsumoto et al. 2007) and the cephalochordate ER acts as a constitutive repressor of estrogen response element (ERE) function (Bridgham et al. 2008; Paris et al. 2008). It is important to note that although compounds may be defined based on a given outcome or mechanism of action, almost all chemicals influence multiple physiological systems and influence the way physiological systems interact with each other. For example, compounds such as linuron (a urea-based herbicide) and prochloraz (an imidazole fungicide) act as anti-androgens via multiple mechanisms of action (Lambright et al. 2000;

Any number of compounds can be used to demonstrate endocrine disruption via an endocrine active chemical (EAC). However, prochloraz provides a highly illustrative example because it affects development of external mammalian genitalia with which readers will have prior familiarity. Much of the reason that an imidazole fungicides like prochloraz kill fungi is because they affect members of the diverse cytochrome P450 enzyme family (Mason et al. 1987; Riviere & Papich 2009), a group of enzymes catalyzing electron transfer in representatives of all classes of cellular life (Nebert et al. 1989; Lewis et al. 1998; De Mot & Parret 2002; Nelson 2011). These enzymes have been evolutionarily co-opted for vertebrate steroidogenesis, as well as the metabolism of steroids and xenobiotics (Gibson et al. 2002). In addition to affecting steroidogenic cytochrome P450 enzymes and reducing androgen production, prochloraz is an androgen receptor antagonist (Vinggaard et al. 2002; Noriega et al. 2005) and inhibits testicular expression for insulin-like hormone 3 (insl3), which affects gubernacular development (Wilson et al. 2004). An example of laboratory-administered *in utero* prochloraz exposure (Noriega et al. 2005) can be used for a discussion on effects of a non-steroidal compound on sex-steroid action as viewed throughout the historical

1. Exposure (ingestion) over a time course (5 days) and dosage that produced no observable effects on directly exposed mothers, was sufficient to produce severe abnormalities in offspring of those mothers (figure 3) through secondary *in utero*

2. Abnormalities such as vaginal morphology in males (figure 4), that might have initially been classified as "feminization" in the early history of the field are more accurately described as extreme cases of de-masculinization towards a default female morphology

exposure during critical windows for sex-steroid sensitive development;

Wilson et al. 2004).

Wilson et al. 2004; Noriega et al. 2005).

**6.1 A case study using prochloraz** 

for the species in question.

development of the endocrine disruptor hypothesis. Namely:

Fig. 3. Phallus abnormalities in adult male rats receiving a 5-day *in utero* exposure to prochloraz. Panels are arranged from left to right according to dosage group indicated by numbers to the left of the diagram. **A** and **B**) Variation within controls. **C** and **D**) Animals with a maternal dose of 125 mg/kg per day. **E**–**G**) Animals with a maternal dose of 250 mg/kg per day. gl, glans penis; pr, prepuce; ur, urethral opening; os, os penis. Hypospadias is evident in the two highest dosage groups and (**C**–**E**) show examples of incomplete preputial separation. Severe phallus clefting and exposure of the os penis is evident in the highest dosage group (**F** and **G**). (Noriega et al. 2005) modified with permission from the Society for the Study of Reproduction.

Evolutionary Perspectives on Sex Steroids in the Vertebrates 19

The terminology used in discussions of endocrinology, evolution and behavior is changing in light of the growing body of knowledge regarding the complexity of hormonal interactions. This chapter is intended to provide the reader with a panoramic snapshot of sex-steroid function compared to what is normally encountered in specialized fields of study. The summaries of concepts presented here will hopefully be catalysts for further investigation of topics in more detail than presented here. Existing paradigms regarding "disruption", "variation" and "adaptation" are increasingly seen as parts of a continuum. Thus the scope of "endocrine disruptor" assessment has already expanded beyond currently established definition parameters (Guillette 2006; Marty et al. 2011; Norris & Lopez 2011a). For example, the term "Endocrine Active Chemical" (EAC) is now used in favor of terms

The claim has long been made that distinctions such as endocrine system vs. nervous system are arbitrary (Roth et al. 1986). Evolutionary constraints lead to reduced variation in reproductive strategies used by birds and mammals compared to evolutionarily older clades represented by fishes, reptiles and amphibians. However, the evolution of complex neuroendocrine systems may provide time and context-specific behavioral avenues for adaptive radiation in the face of external influences on peripheral hormone levels (Wingfield et al. 1997; Adkins-Regan 2008). We have only a brief window of perspective on the cycle of extinction and adaptive radiation. The ability to include expansive, as well as reductionist perspectives may facilitate new thresholds in our evaluation of environmental, social,

ancSR = Ancestral Steroid Hormone Receptor; AR = Androgen Receptor; CG = Chorionic Gonadotropin; ER = Estrogen Receptor; FSH = Follicle Stimulating Hormone; GR = Glucocorticoid Receptor; hCG = Human Chorionic Gonadotropin; HPA = Hypothalamic-Pituitary-Adrenal; HPG = Hypothalamic-Pituitary-Gonad; HPT = Hypothalamic-Pituitary-Thyroid; insl3 = Insulin-like hormone 3; LH = Lutenizing Hormone; MR = Mineralocorticoid Receptor; SHBG = Steroid Hormone Binding Globulin; TSH = Thyroid Stimulating

Adkins-Regan, E. (2008). Review. Do hormonal control systems produce evolutionary

Adkins, E. K. (1975). Hormonal basis of sexual differentiation in the Japanese quail. *Journal of comparative and physiological psychology* Vol. 89, No. 1, pp (61-71), 0021-9940 Adkins, E. K. (1976). Embryonic exposure to an antiestrogen masculinizes behavior of female quail. *Physiology & Behavior* Vol. 17, No. 2, pp (357-359), 0031-9384 Arnold, A. P. (2009). The organizational-activational hypothesis as the foundation for a

*sciences* Vol. 363, No. 1497, pp (1599-1609), 0962-8436

*behavior* Vol. 55, No. 5, pp (570-578), 1095-6867

inertia? *Philosophical transactions of the Royal Society of London. Series B, Biological* 

unified theory of sexual differentiation of all mammalian tissues. *Hormones and* 

**7. Conclusion** 

**8. Abbreviations** 

Hormone;

**9. References** 

implying "disruption" (Norris & Lopez 2011a).

behavioral and clinical aspects of sex-steroid biology.

Fig. 4. Prochloraz-induced vaginal morphology in adult male rats receiving a 5-day *in utero* exposure. **A**) Panoramic view of a vaginal pouch (forceps inserted) in a male from the 250 mg/kg maternal dosage group. **B**) A close-up of a control female phallus and vaginal opening. **C** and **D**) Vaginal pouch and phallus deformity variations in males from the 250 mg/kg maternal dose group. **E**–**G**) The most severely affected animal from the 125 mg/kg maternal dose group. In this male, an ejaculatory plug (**F**) was found embedded (**E**) in the vaginal opening (**G**). (Noriega et al. 2005) modified with permission from the Society for the Study of Reproduction.
