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The Society for Integrative and Comparative Biology
Welcome to the Revolution: Integrative Biology and Assessing the Impact of Endocrine Disruptors on Environmental and Public Health1
1 Laboratory for Integrative Studies in Amphibian Biology, Group in Endocrinology, Museum of Vertebrate Zoology, and Department of Integrative Biology, University of California, Berkeley, California 94720-3140
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Concern continues to grow over the negative impact of endocrine disrupting chemicals on environmental and public health. The number of identified endocrine disrupting chemicals is increasing, but biological endpoints, experimental design, and approaches for examining and assessing the impact of these chemicals are still debated. Although some workers consider endocrine disruption an "emerging science," I argue here that it is equally, a "merging science" developing in the tradition of integrative biology. Understanding the impact of endocrine disruptors on humans and wildlife is an examination of "context dependent development" and one that Scott Gilbert predicted would require a "new synthesis" or a "revolution" in the biological sciences. Here, I use atrazine as an example to demonstrate the importance of an integrative approach in understanding endocrine disruptors.
Atrazine is a potent endocrine disruptor that chemically castrates and feminizes amphibians and other wildlife. These effects are the result of the induction of aromatase, the enzyme that converts androgens to estrogens, and this mechanism has been confirmed in all vertebrate classes examined (fish, amphibians, reptiles, birds, and mammals, including humans). To truly assess the impact of atrazine on amphibians in the wild, diverse fields of study including endocrinology, developmental biology, molecular biology, cellular biology, ecology, and evolutionary biology need to be invoked. To understand fully the long-term impacts on the environment, meteorology, geology, hydrology, chemistry, statistics, mathematics and other disciplines well outside of the biological sciences are required.
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SCIENCE AND ITS HISTORY |
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It has been said that Aristotle, whose, "... studies on animals laid the foundations of the biological sciences; and ...were not superceded until more than two thousand years after his death" is the father of modern biology (Barnes, 1982, p. 9
). Aristotle was not just a biologist, however, but rather a mathematician who taught and wrote on subjects ranging from physics, chemistry, geography, astronomy, psychology, and meteorology to politics and economics. In his time, it was not unusual for scientists to study questions across broad disciplines. Even in 1831, Charles Darwin joined the Beagle on its expeditions as a geologist, botanist, zoologist, and general scientist, a role often filled by physicians in that time. In more recent years, however, science has moved away from the generalist approach and more defined disciplines within fields have developed. Not only are chemists, geologists, physicists, etc typically distinct from biologists, but botany, zoology, etc. are separate fields of study within biology. Even finer divisions emerged as developmental biologists became distinct from anatomists, biochemists, endocrinologists, ecologists, evolutionary biologists and so on. The discovery of the structure of DNA and technologies to study molecules introduced the field of molecular biology and many academic biology departments in the last couple of decades have divided into organismal biology/ecology and molecular/cellular biology. In addition, modern scientists began to identify themselves as "basic" or "applied" scientists. Within biology, some fields such as medicine clearly developed as more applied sciences, whereas others (evolutionary biology, for example) have been viewed as "basic" sciences. Even funding agencies focus on disciplines and commit to funding "basic" (e.g., National Science Foundation) or "applied" science (e.g., National Institutes of Health). More recently, defined disciplines have begun to merge, however, each realizing the benefit of the other. The use of DNA analysis, molecular markers, and the molecular clock has proven invaluable for evolutionary biologists. Likewise, the need to examine "whole animals" and their development and to conduct comparative studies is even more apparent now that entire genomes have been sequenced. The functions of the many newly discovered genes need to be assessed by cell, developmental, and organismal biologists. Regarding this "new modern synthesis," Scott Gilbert referred to the growing field of evolution and development (evo-devo) as a "revolution" citing many new discoveries, approaches, and new fields that will grow (or have grown) out of this merger.
In the current treatment, I briefly review how the syntheses of multiple fields ("reintegration") have provided insight into several problems in biology. In particular, I argue that examinations of pesticides as endocrine disruptors (e.g., atrazine) and the contribution of endocrine disrupting pesticides to amphibian declines requires a "re-integration" of biology and integration with many other fields extrinsic to biology.
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EVO-DEVO |
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The importance of evolution and development has been recognized since at least the 1970s. Although the popular name (Evo-Devo) suggests a synthesis of only two formerly separate fields of study (evolution and development), some consider evo-devo a true integration across multiple disciplines (medical sciences, ecology, development, and evolution) and a necessity ("revolution" even) in the biological sciences (Gilbert, 2003
). As pointed out by Brian Hall, "evolutionary developmental mechanisms may be genetic, cellular, developmental, physiological, hormonal or any combination of these levels" (Hall, 2003, p. 493), a realization that begs for just the new synthesis that Gilbert described. As examples, many important questions have been addressed using this approach including, but certainly not limited to: evolution of mammalian placentas (Endersa and Carterb, 2004), the evolution of feathers (Prum and Brush, 2002), evolution of primate skulls (Mitteroecker et al., 2004), the evolution of floral development (Jaramillo et al., 2004), the evolution and development of turtle shells (Gilbert et al., 2001), and the evolution and development of mammalian and fish brains (Wulliam and Mueller, 2004). |
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Evolutionary (comparative) endocrinology
Evo-Devo (in the strictest sense) is not the only synthetic approach that has proven fruitful. Evolutionary (comparative) endocrinology, has provided important insight into the role of hormones in evolution. The sex steroids are an important example. Sex steroids (androgens and estrogens) are ubiquitous in their control of sexual differentiation and reproduction in vertebrates (Couse and Korach, 1998
; Hayes, 1998b; Yamashita, 1998; Piferrer, 2001; Raman, 2002; Britt and Findlay, 2003; Elf, 2003; Lange et al., 2003; Uguz et al., 2003). Also, the steroid receptors that mediate activity of these hormones are highly conserved as are the response elements in the genes that these hormones regulate (Yamashita, 1998; Whitfield et al., 1999; Owen and Zelent, 2000; Baker, 2001a; Oberdorster et al., 2001; Thornton, 2001; Hahn, 2002; Thornton and Crews, 2003; Wu et al., 2003; Baker, 2004). Across vertebrates, the functional androgens, estrogens, progestins, and corticoids have similar structures (identical in most cases) and homologous receptors. Even in invertebrates (although the steroid hormones themselves are novel) synthetic enzymes and receptors are homologous with vertebrates (Oppermann et al., 1992; Katagiri and Kagawa, 1998; Lanisnik et al., 1999; O'Brien and Herschlag, 1999; Baker, 2001b; Breitling et al., 2001a; Breitling et al., 2001b, Lathe, 2002; Baker, 2004). In particular, several of the papers cited here examined the evolution and co-evolution of steroid enzymes and receptors that provided the modern diversity and specificity of these ancient signaling molecules. This task required compared endocrinology conducted in a phylogenetic context.
Developmental endocrinology
Likewise, examinations of endocrinology in the context of developmental biology have been very important. One of the most significant contributions in this regard is the understanding of "organizational" versus "activational" effects of hormones. Hormones that have reversible effects in adult organisms can have dramatic long-lasting permanent developmental effects. Excellent examples include the permanent role that sex steroids play in sex differentiation. In adult organisms, sex steroids have reversible physiological effects such as the increase in muscle mass in response to androgens (Michel and Baulieu, 1980; Danhaive and Rousseau, 1988), the induction of vitellogenin in non-mammalian vertebrates by estrogens (Tata, 1979; Chakravorty et al., 1992; Jones et al., 2000), and the cyclical effects of estrogen on the uterine lining in female mammals (Naciff et al., 2003). All of these effects are examples of reversible (non-permanent) effects. The developmental effects of these same hormones are quite different, however. In mammals, androgens induce growth of the phallus and growth and maintenance of the reproductive tract (Couse and Korach, 1998; Yamashita, 1998; Rivas et al., 2002) and laryngeal masculinization (Fuerst-Recktenwald et al., 2000) in males. These developmental effects are permanent: The genitalia, reproductive tract, and larynx do not regress if androgens are taken away after these features develop. Similarly, estrogens are important in female sex differentiation. In many ectotherms, estrogens can induce the development of ovaries (even in genetic males) (Chang and Witschi, 1955; Richards and Nace 1978; Hayes, 1998b; Piferrer, 2001; Raman, 2002). This developmental effect is irreversible, unlike the induction of vitellogenin in adults which ceases once estrogen exposure ends (Tata, 1979).
Evo-devo-endo
When we combine these fields (Evo-Devo-Endo), even more insight can be gained. Understanding the role of hormones in development in an evolutionary context can be quite revealing. Already, we have proposed that hormonal mechanisms may act as developmental constraints that prevent the evolution of neoteny in anurans and constrain sexual dimorphism in reedfrogs (Hyperolidae) (Hayes, 1997). Furthermore, in ongoing studies, we have shown that the evolution of accelerated metamorphosis in Pelobatid frogs is a derived feature that involves both early synthesis of thyroid hormones and earlier sensitivity to thyroid hormones during development of the rapidly metamorphosing species in the family (Hayes and Buccholz, unpublished).
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ENDOCRINE DISRUPTION |
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Thus, as suggested, new knowledge, understanding, and approaches to problems will emerge from this new "synthesis" or reintegration of biology. Furthermore, when a greater number of fields are included in the reintegration, even more insight will be gained. One valuable use of this information has arisen in the newly recognized problem of endocrine disruption. In fact, the interest in the "new synthesis" was in part stimulated by a recognition and interest in "context-dependent development," of which endocrine disruption is an example. As Gilbert pointed out, "Ecological developmental biology is interacting with evolutionary developmental biology in interesting ways. It is positioning itself to look at the proximate causes of life history strategies and to determine the epigenetic relationships between organisms. It is also forging links with medically oriented areas of developmental biology such as teratology and endocrine disruption." (Gilbert, 2003, p. 474
). Predictably, the re-integration of multiple disciplines (beyond evo-devo) has proven vital in understanding the action and impact of endocrine disrupting compounds in many ways.
My laboratory and others recently showed that atrazine, the most popular herbicide in the world and the most common contaminant of ground and surface water, is a potent endocrine disruptor that both chemically castrates and feminizes exposed male amphibians (Reeder et al., 1998; Hayes et al., 2002a, b, c; Mckoy et al., 2002, Tavera-Mendoza et al., 2002a, b; Carr et al., 2003; Miyahara et al., 2003; Hayes, 2004). Here, I focus on this example and how the merger of several fields has aided in understanding the effects and assessing the impact of atrazine.
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CONTRIBUTIONS OF MERGING FIELDS TO UNDERSTANDING ENDOCRINE DISRUPTION |
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Evolutionary (comparative) endocrinology
Because of our understanding gained through evolutionary (comparative) endocrinology, amphibian models can be used in the laboratory or as sentinel species in the wild to predict effects of endocrine disruptors on other animals. Because the structure of estrogen is identical across vertebrates and receptors homologous, compounds that bind the estrogen receptor and produce effects in amphibians, are likely to induce estrogen-dependent reproductive cancers (such as mammary cancer) in mammals, including humans. For example, estrogens that are known to induce division in mammary cancer cells, also induce estrogen-dependent color changes in the amphibian (Hyperolius argus) and anti-estrogens (such as tamoxifen) which block estrogen-dependent mammary cancer cells also prevent estrogen-dependent color change in this amphibian model (Hayes, 1998a
, 2000; Noriega and Hayes, 2000) (Fig. 1).
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Likewise, steroidogenic enzymes are homologous across vertebrates (Oppermann et al., 1992
; Katagiri and Kagawa, 1998; Lanisnik et al., 1999; O'Brien and Herschlag, 1999; Baker, 2001b; Breitling et al., 2001a, b; Lathe, 2002; Baker, 2004). Atrazine affects several steroidogenic enzymes (
imic et al., 1994; Wetzel et al., 1994; McDougal et al., 1997; McDougal and Safe, 1998; Eldridge and Wetzel, 1999; Cummings et al., 2000; Kniewald et al., 2000; Sanderson et al., 2000, 2001; Trentacoste et al., 2001; Bisson and Hontela, 2002; Friedmann, 2002; Hayes et al., 2002a, b; Matsushita, 2003; Miyahara et al., 2003; Spano et al., 2004) including the enzyme aromatase. As expected, this mechanism has been observed in every vertebrate class examined including fish (Spano et al., 2004), reptiles (Crain et al., 1997), birds (Matsushita, 2003), and mammals (Sanderson et al., 2000, 2001) in addition to amphibians (Miyahara et al., 2003). Although the physiological and developmental consequences differ: vitellogenin production in fish (Spano et al., 2004) and amphibians (Miyahara et al., 2003), hermaphroditism in amphibians (Reeder et al., 1998; Hayes et al., 2002a, b, c; Mckoy et al., 2002; Tavera-Mendoza et al., 2002a, b; Carr et al., 2003; Hayes, 2004), feminization of the reproductive tract in birds (Matsushita, 2003), and mammary cancer in mammals (Pintér et al., 1980; Eldridge et al., 1994; Stevens et al., 1994; Wetzel et al., 1994), these effects are all estrogen-mediated endpoints. Thus, amphibian models allow accurate predictions of effects in other classes once the underlying mechanisms of actions are understood. The new synthesis (revolution) places our findings of atrazine's effects on amphibians in a much broader light. The demasculinization and feminization of exposed males is not a species, or even a class-specific problem, but one that poses a threat across vertebrates and represents simultaneously an environmental and a public health concern.
Developmental endocrinology
Concepts from developmental endocrinology help put the effects of atrazine into new perspective as well. As mentioned, the role of hormones during critical developmental stages, can be much more dramatic than their physiological roles. Estrogen-induction of vitellogenin in exposed adults is a reversible effect, whereas exposure during sexual differentiation can bring about permanent (irreversible) alterations of secondary sex characters and even, in some vertebrate classes, complete, permanent sex reversal. Likewise, the more serious effects of endocrine disruptors (including atrazine) occur when animals are exposed developmentally. Vitellogenin production and chemical castration of adults (depletion of testosterone) caused by atrazine exposure are both reversible: animals return to normal when atrazine exposure is discontinued. Demasculinization of the larynges in male amphibians castrated by atrazine during larval development (Hayes et al., 2002a) (Fig. 2) and the production of hermaphrodites (Hayes et al., 2002a, b, c) and induction of testicular oogenesis as a result of larval atrazine exposure, however, appear to be permanent effects.
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Eco-devo
The example of atrazine disruption of sexual development in male amphibians also provides an example of why the separation of "basic" and "applied" biology is a hindrance. Understanding basic biology is vital to applications. For example, Morreale pointed out that conservation efforts for captive reproduction of endangered sea turtles was thwarted because, at the time, the effect of temperature on sex determination in turtles was not known. As a result, potentially all of the captive-reared turtles released into the wild were of the same sex (Morreale et al., 1982
). Similarly, in the case of atrazine's effects on amphibians, the true impact on wild amphibians would not be recognized without a basic understanding of normal sex differentiation in amphibians.
Carr and Solomon, proposed that hermaphroditism was a matter of normal variation in amphibians and occurred in the absence of atrazine exposure (Carr and Solomon, 2003). The authors cited a study by Emil Witschi which described a unique gonadal morphology identified in a limited number of animals. The described anomaly involved development of rostral ovaries in male European Ranid frogs followed by female secondary sex development of the reproductive ducts (Witschi, 1929). This morphology was identified in the field and propagated in the laboratory, but was not widespread, nor did it resemble the morphology induced by atrazine. Furthermore, Carr and Solomon cited several studies in which laboratory "controls" were contaminated with atrazine (Coady et al., 2004; Hecker et al., 2004) and field-collected animals were exposed to triazine levels 100 times higher than effective atrazine doses (Hayes, 2004) as evidence for a background incidence of hermaphroditism in frogs. Thus, they erroneously concluded that hermaphroditism was normal in amphibians.
Atrazine contamination is extensive and widespread as demonstrated in our studies (Hayes et al., 2002b) and many others (Müller et al., 1997; Battaglin and Goolsby, 1999; Boyd, 2000; Clark and Goolsby, 2000). As a result, atrazine-free areas (reference sites) are difficult to find. As the top selling pesticide in the U.S., between 60 and 120 million pounds of atrazine are applied per year (primarily in the Midwest) (USDA, 1994, Thurman and Cromwell, 2000). Atrazine can be found in rain water and snow (even in areas where it is not used) and even in clouds at levels that exceed its effective dose as an endocrine disruptor (Nations and Hallberg, 1992). As a result, atrazine contamination occurs in ground and surface waters in local areas, states, and even countries where it is not used. Furthermore, despite common belief, atrazine is quite persistent. A recent report revealed that atrazine can be measured in ground water in France even though it has been banned (and not used) since 1990 (Hennion et al., 2004). Thus, most amphibian populations are exposed. Surveys of amphibians in the wild would erroneously conclude that hermaphroditism occurred naturally, without knowledge of the extent and persistence of atrazine- contamination. Indeed reported hermaphroditism in Xenopus laevis in non-corn growing areas in South Africa (Du Preez et al., 2002; Smith et al., 2003a, b) and hermaphroditism in Rana pipiens in local areas and states in the U.S. where atrazine is not used (Hayes et al., 2002b, c) is associated with atrazine exposure as a result of widespread contamination. It is important to have a base or background data with regards to what is a normal developmental pattern for a species (or population). Carefully paired laboratory and field studies (Eco-Devo) are required to show that hermaphroditism does not occur in the absence of atrazine (Fig. 3). Without such studies, erroneous conclusion would be drawn and the true impact of atrazine on wild amphibians would not be realized.
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Endocrine mechanisms
The relationship between atrazine concentrations and the magnitude of the measured developmental responses is also an important question. Carr and Solomon challenged the observed dose response (Carr and Solomon, 2003
). The responses of amphibians to atrazine did not display a monotonic linear dose-response. Although monotonic linear dose responses may be expected for classical toxicological effects, such observations are not expected in cases where compounds act as endocrine disruptors.
In the case of demasculinization of the larynx, a threshold effect was observed regardless of whether the effect of atrazine dose was examined against laryngeal size or against the proportion of males affected in the exposed population (Fig. 4). This dose response is consistent with the observed effect, however. Atrazine does not reduce (shrink) the larynx, but rather reduces testosterone levels (Hayes et al., 2002a), thereby preventing laryngeal growth. In Xenopus laevis, males were castrated at an atrazine concentration of 1.0 ppb. Increasing the concentration beyond this threshold dose, could not increase an effect that has already occurred: There are not degrees of castration. Once castrated, the larynx is maintained at the "default" size, and increasing atrazine concentrations are not expected to reduce laryngeal size below the observed, because the larynx is not being reduced, but rather androgen-stimulation is prevented.
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In other measured effects of atrazine, lower doses may appear more potent than higher doses or the concentration response may be parabolic ("inverted U" concentration response), where low concentrations are effective, higher concentrations may be equally effective, but intermediate concentrations may produce greater effects. This type of response was observed for the production of hermaphrodites (Hayes et al., 2002a
, b), the induction of aromatase expression (Miyahara et al., 2003), and the induction of vitellogenin (Miyahara et al., 2003) by atrazine. Again, considering the underlying endocrine mechanisms (Fig. 5), this response is expected. At low doses, atrazine induces aromatase and estrogen expression. The estrogens subsequently induce ovarian development, testicular oogenesis, and vitellogenesis at low doses (but above some threshold). At higher doses, the effect may be dampened because estrogen production may be reduced as a result of negative feedback on the pituitary, so the effect (as measured by the chosen endpoint) decreases (Fig. 5). In fact, this concentration response pattern is observed for the induction of oogenesis in rodents exposed to phyto-estrogens (Almstrup et al., 2002) and in many other cases involving estrogenic compounds (Rozovsky et al., 2002; Skarda, 2002a, b; Duft et al., 2003; Köhlerová and karda, 2004), glucocorticoids (Love et al., 2003; Duclos et al., 2004), and others compounds that bind nuclear receptors (Cavieres et al., 2002; Kohn and Melnick, 2002).
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In summary, the important role of basic science is apparent in this applied example. Furthermore, the importance of Gilbert's "revolution," the re-integration of biological sciences is made clear. Without a true understanding of normal amphibian gonadal development in the laboratory and field the true impact of atrazine could not be addressed. Understanding the biochemical and molecular mechanisms of atrazine action and understanding the development and physiological response to estrogens across vertebrate classes (comparative, evolutionary endocrinology) allows us to predict impacts across vertebrates (environmental health) including humans (public health). Future examinations of behavioral and functional effects on reproduction will allow us to conduct better ecological risk assessments. Interactions beyond biology have proven and will unlikely continue to prove vital in completely understanding the impact of atrazine in the environment. Chemists (both laboratory and environmental), geologists, meteorologists, hydrologists, and statisticians are needed to integrate data regarding atrazine's transport and fate in the environment to completely assess its impact (Fig. 6).
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It has been said that endocrine disruption is an "emerging science" (Hosmer, 2003
). Given the evidence, I argue that this area is equally a "merging science," however. Regardless of the chosen terminology, the new knowledge gained through the reintegration of old knowledge and approaches is clear and necessary. The ability to use basic science and new discoveries to properly understand the observed effects and advance our understanding of the consequences remain vital. Regarding this necessity of merging and emerging sciences, recent political developments, threaten this critical approach. The recently passed Data Quality Act effectively blocks the use of new discoveries in regulatory decisions and policies (Weiss, 2004). In fact, the submission of our data on the effects of atrazine on amphibians was one of the first challenges filed under the Data Quality Act, leading to the EPA's conclusion that endocrine disruption could not be used as an endpoint for regulation. Thus, the very policies and agencies that depend on emerging/merging sciences are moving counter to the revolution that will produce the newest science.
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FOOTNOTES |
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1 From the Symposium Integrative Biology: A Symposium Honoring George A. Bartholomew presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 5–9 January 2004, at New Orleans, Lousiana.
2 E-mail: tyrone{at}socrates.Berkeley.EDU
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