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Pesticides: multiple mechanisms of demasculinization
Molecular and Cellular Endocrinology 126 (1997) 1 – 5
At the Cutting Edge
Pesticides: multiple mechanisms of demasculinization Gerald A. LeBlanc*, Lisa J. Bain, Vickie S. Wilson Department of Toxicology, Box 7633, North Carolina State Uni6ersity, Raleigh, NC 27695 -7633, USA Received 28 August 1996; accepted 20 October 1996
Abstract Many pesticides are known to produce reproductive and developmental effects in chronically-exposed non-target organisms, including humans. Recent evidence suggests that demasculinization may be an important mechanism responsible for some of these effects. Some pesticides have been shown to interact with the androgen receptor and to act as antagonists, while others have been shown to interact with the estrogen receptor and function as estrogens both in vitro and in vivo. Many pesticides can also lower serum androgen levels by altering rates of synthesis or metabolism. Given the ubiquity of pesticides in the environment and the multiple mechanisms whereby they can elicit demasculinizing effects, synergy between such compounds may produce clinical endocrine dysfunction at current human exposure levels. Copyright © 1997 Elsevier Science Ireland Ltd. Keywords: Pesticides; Demasculinization; Feminization; Endocrine; Steroid hormones; Receptors; Biotransformation; Synergism
1. Introduction Pesticide efficacy reflects in their high level acute toxicity for target organisms compared with nontarget organisms. There is, however, an increasing body of evidence suggesting that chronic pesticide exposure may have deleterious effects on humans. Historically, the central endpoint in evaluating risks to humans of chronic pesticide exposure has been carcinogenicity [1]. More recently, numerous laboratory and epidemiological studies have shown the importance of noncancer endpoints in chronic toxicity assessments (e.g. [2 – 4]). Accordingly, increasing emphasis is currently placed on endpoints such as neural-behavioral toxicity, developmental toxicity and reproductive toxicity during risk assessments by regulatory agencies [1,5]. The endocrine system as a target site of pesticide toxicity can manifest neuro-behavioral, developmental * Corresponding author. Tel.: +1 919 5157404; fax: + 1 919 5157169; email: [email protected]
or reproductive consequences, particularly in terms of pesticide-induced effects on steroid hormone function. Steroid hormones are involved with both primary sex determination and neonatal development [6], as well as the acquisition and maintenance of secondary sex characteristics in adults [7]. Steroid hormones also program the brain- and neuro-endocrine system prenatally or neonatally, depending upon the species [8], resulting in gender dimorphism in both behavioral and metabolic aspects of reproduction [8,9]. Accordingly, pesticides that alter endocrine function either by acting as hormone agonists/antagonists or altering endogenous steroid hormone levels may severely affect biochemical, developmental and behavioral events vital to reproductive success. The present paper reviews recent evidence suggesting that pesticides can elicit endocrine-disrupting effects via several mechanisms which may operate synergistically to produce clinical manifestations of demasculinization.
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2. Receptor Interactions Endocrine disruption by xenobiotics acting as steroid hormone agonists or antagonists has been best described for organochlorine pesticides. o,p%-DDT has a variety of endocrine-related effects on reproductive organs and processes including inhibition of testicular growth and the development of secondary sex characteristics in males [10] and increased uterine weight [11], precocious puberty and persistent estrus in females [12] and reduced fertility [13]. o,p%-DDT can act as an estrogen agonist [11], a property presumed responsible for many of its adverse effects on reproduction [14]. More recently, anti-androgenic properties of the DDT metabolite p,p’ -DDE have been demonstrated that may also contribute to the reproductive effects of DDT [15]. The demise of raptor populations described during the 1960’s (e.g. [16]) and the recently-described demasculization of alligators in Lake Apopka, Florida [17] have been ascribed to the endocrine-disrupting effects of DDT and related organochlorine pesticides. Further, epidemiological studies have linked incidence of breast cancer with blood DDE levels [18], suggesting that environmental endocrine-active agents may cause hormone-related disease in the human population [19]. Other pesticides shown to have estrogenic, or anti-androgenic properties include chlordecone [20], dieldrin [21,22], vinclozolin [23], endosulfan [21], toxaphene [21], and linuron [24]. A human environmental impact has been suggested for chlordecone, in that chemical factory workers exposed to this pesticide for prolonged periods of time experienced a high incidence of impaired testicular function [25].
3. Hormone Modulation Some pesticides have been shown to alter normal endocrine function by inhibiting steroid hormone biosynthesis. For example, the imidazole fungicide ketoconazole has been shown to inhibit several steroidogenic enzymes resulting in impaired testosterone biosynthesis [26 – 29], and a marked reduction in serum testosterone levels [28,30,31]. Pesticides can also disrupt androgen homeostasis by increasing the rate of androgen metabolism and elimination. Such perturbations in steroid hormone biotransformation have been causally implicated in the reproductive failure of mammals following exposure to a variety of halogenated hydrocarbon pesticides and other similarly-acting xenobiotics [32]. Many pesticides induce hepatic monooxygenase and UDP-glucuronosyltransferase enzymes (e.g. [33,34]), leading to increased pesticide clearance. However, many of these enzymes are also responsible for androgen metabolism [35,36] and their induction may thus also lead to increased clearance of these hormones
[36,37]. Chronic exposure of rats to dosages of chlordane producing plasma pesticide levels comparable to those in the U.S. population, reduced plasma testosterone levels and altered sexually dimorphic functions and behaviour [38]. This effect of chlordane was ascribed, at least in part, to its ability to alter steroid-metabolizing enzymes. Though chlordane is no longer used in the US, its environmental persistence and propensity to bioaccumulate has resulted in continued human exposure [39]. Preliminary experiments conducted in our laboratory revealed that exposure of mice to pesticides such as chlordecone and endosulfan also significantly increased the catabolic metabolism of testosterone. There is clear precedence for reproductive dysfunction in humans following exposure to chemicals, such as anticancer drugs, that alter steroid hormone biotransformation processes. Cyclophosphamide markedly alters the testosterone metabolic profile of treated rats [40]. Acrolein, an oxidative metabolite of both cyclophosphamide and allylamine biocides [41], contributes to the overall effects of cyclophosphamide on steroid metabolism [40]. Since cyclophosphamide is a reproductive toxicant in humans and can render patients undergoing therapy azoospermic [42], similar effects may be seen for the pesticidal allylamine derivatives. Cisplatin demasculinizes hepatic steroid hormone metabolism in rats [43]. It also alters hormonal profiles of male patients undergoing therapy with the drug [44], which may contribute to their development of gynecomastia and galactorrhea [45–49]. Thus, disruption of steroid hormone biotransformation can alter hormone homeostasis and result in reproductive dysfunction. Pesticides can also indirectly affect steroid hormone levels. Pesticides representing chlorophenols, chlorophenoxy acids, organochlorines, and quinones have been shown to alter circulating thyroid hormone (T4) levels and/or thyroid gland function. These effects have been ascribed largely to increases in T4 metabolism via induction of hepatic UDP-glucuronosyltranferase enzymes [50] and to displacement of T4 from plasma binding-proteins [51]. We and others have shown that reductions in circulating T4 levels can alter hepatic androgen metabolism by compromising the catalytic activity of hepatic cytochrome P450 monooxygenases [52–54]. These enzymes require electrons shuttled from NADPH to the enzyme by the flavoprotein NADPHcytochrome P450 reductase (P450 reductase), which is under the regulatory control of T4 in the rat [52]. Reduction in circulating T4 levels lowers P450 reductase and reduces P450-mediated androgen metabolism, so that pesticides causing a reduction of circulating thyroxine levels might be predicted to affect androgen homeostasis.
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masculinization of females in terms of their adult steroid metabolic capabilities [58–60]. Perinatal or neonatal exposure of pesticides including DDT [12,13], chlordecone [20,61–63], chlordane [38] and atrazine [64] have all been shown to affect steroid hormone-related developmental parameters.
5. Conclusions
Fig. 1. Mechanisms by which pesticides may elicit demasculinization by a different mode of action resulting in synergistic responses.
4. Developmental Effects Prenatal and neonatal life stages may be particularly sensitive to demasculinization by pesticides. Sexual differentiation of the gonads and accessory reproductive organs occurs largely during prenatal development and to a lesser degree during the neonatal period and steroid hormones play a decisive role in establishing gender during these periods [6]. Perturbations in steroid hormone homeostasis during these periods can result in pseudohermaphroditic conditions or other abnormalities of the reproductive system producing reproductive dysfunction [7,55]. Noteworthy is the role of steroid 5a-reductase in male differentiation. Deficiency in steroid 5a-reductase causes a pseudohermaphroditic condition in males characterized by ambiguous external genitalia at birth [7]. Mature males deficient in this enzyme, which is responsible for the conversion of testosterone to dihydrotestosterone, have reduced facial and body hair, no temporal hair recession and a non-palpable prostate. We are not aware of any published data regarding the susceptibility of steroid 5a-reductase to inhibition by pesticides. However, the therapeutic inhibition of this enzyme is increasingly used in the treatment of prostate cancer, and individuals undergoing such treatment may have enhanced sensitivity to the demasculinizing effects of some pesticides. Steroid hormones are also important during prenatal and neonatal development in establishing gender dimorphism in the brain and in the imprinting of neurobehavioral and neuroendocrine components of reproduction. One consequence of this imprinting is the gender-specific expression of steroid hormone-metabolizing enzymes in the adult [35,56,57]. Perturbations in neonatal hypothalamic imprinting by steroid hormones can result in demasculinization of males or
Clearly, many pesticides have the ability to alter steroid hormone-dependent processes due to hormone receptor agonist/antagonist properties and/or their ability to alter steroid hormone homeostasis. Are such properties of consequence to the human population? Analyses of high risk populations have indicated that pesticide exposure among humans is associated with reproductive abnormalities [25], birth abnormalities [65], and skewed sex-ratios for birth defects [65]. There is evidence that pesticides can affect endocrine processes at the high end of normal human exposure. The general populace is exposed to significantly lower levels of pesticides. However, the plethora of pesticides to which the general population is exposed [66– 68] provides a potential for multiple and diverse effects on endocrine processes. Synergistic effects of these compounds on endocrine function must be considered when assessing potential and actual hazards posed to the general population. For example, recent studies [69] have shown that mildly or non-estrogenic pesticides can function synergistically with estrogenicity potencies of 150–1500 times that of the individual pesticides. Simultaneous exposure to DDT, endosulfan and propiconazole may produce profoundly synergistic effects on androgen function. The DDT metabolite p,p%-DDE is anti-androgenic; o,p-DDT, perhaps in synergy with endosulfan [69], is estrogenic; DDT and endosulfan can both enhance endogenous androgen elimination and propiconazole can inhibit de novo testosterone synthesis. The combined effects of these compounds on testosterone homeostasis would likely far exceed the sum of the individual effects (Fig. 1). Reports of dysfunction of the male reproductive system abound and include reduced sperm counts and quality [70–72]. Whether such effects are related to exposure to pesticides and related compounds remains an enigma. However, the potential consequences of such effects on human development and fertility, the hardship imposed upon affected individuals and the medical costs associated with treatment of such abnormalities warrants continued and expanded research into the cellular and molecular targets of such compounds, as well as into the pathological consequences of such insults to the endocrine system.
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Acknowledgements This work was supported by grants from the National Institutes of Health (1F32-CA08259, P01 ES00044) and the US Air Force Office of Scientific Research, Air Force Systems Command Grant (F49620-94-1-0266).
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At the Cutting Edge
Pesticides: multiple mechanisms of demasculinization Gerald A. LeBlanc*, Lisa J. Bain, Vickie S. Wilson Department of Toxicology, Box 7633, North Carolina State Uni6ersity, Raleigh, NC 27695 -7633, USA Received 28 August 1996; accepted 20 October 1996
Abstract Many pesticides are known to produce reproductive and developmental effects in chronically-exposed non-target organisms, including humans. Recent evidence suggests that demasculinization may be an important mechanism responsible for some of these effects. Some pesticides have been shown to interact with the androgen receptor and to act as antagonists, while others have been shown to interact with the estrogen receptor and function as estrogens both in vitro and in vivo. Many pesticides can also lower serum androgen levels by altering rates of synthesis or metabolism. Given the ubiquity of pesticides in the environment and the multiple mechanisms whereby they can elicit demasculinizing effects, synergy between such compounds may produce clinical endocrine dysfunction at current human exposure levels. Copyright © 1997 Elsevier Science Ireland Ltd. Keywords: Pesticides; Demasculinization; Feminization; Endocrine; Steroid hormones; Receptors; Biotransformation; Synergism
1. Introduction Pesticide efficacy reflects in their high level acute toxicity for target organisms compared with nontarget organisms. There is, however, an increasing body of evidence suggesting that chronic pesticide exposure may have deleterious effects on humans. Historically, the central endpoint in evaluating risks to humans of chronic pesticide exposure has been carcinogenicity [1]. More recently, numerous laboratory and epidemiological studies have shown the importance of noncancer endpoints in chronic toxicity assessments (e.g. [2 – 4]). Accordingly, increasing emphasis is currently placed on endpoints such as neural-behavioral toxicity, developmental toxicity and reproductive toxicity during risk assessments by regulatory agencies [1,5]. The endocrine system as a target site of pesticide toxicity can manifest neuro-behavioral, developmental * Corresponding author. Tel.: +1 919 5157404; fax: + 1 919 5157169; email: [email protected]
or reproductive consequences, particularly in terms of pesticide-induced effects on steroid hormone function. Steroid hormones are involved with both primary sex determination and neonatal development [6], as well as the acquisition and maintenance of secondary sex characteristics in adults [7]. Steroid hormones also program the brain- and neuro-endocrine system prenatally or neonatally, depending upon the species [8], resulting in gender dimorphism in both behavioral and metabolic aspects of reproduction [8,9]. Accordingly, pesticides that alter endocrine function either by acting as hormone agonists/antagonists or altering endogenous steroid hormone levels may severely affect biochemical, developmental and behavioral events vital to reproductive success. The present paper reviews recent evidence suggesting that pesticides can elicit endocrine-disrupting effects via several mechanisms which may operate synergistically to produce clinical manifestations of demasculinization.
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2. Receptor Interactions Endocrine disruption by xenobiotics acting as steroid hormone agonists or antagonists has been best described for organochlorine pesticides. o,p%-DDT has a variety of endocrine-related effects on reproductive organs and processes including inhibition of testicular growth and the development of secondary sex characteristics in males [10] and increased uterine weight [11], precocious puberty and persistent estrus in females [12] and reduced fertility [13]. o,p%-DDT can act as an estrogen agonist [11], a property presumed responsible for many of its adverse effects on reproduction [14]. More recently, anti-androgenic properties of the DDT metabolite p,p’ -DDE have been demonstrated that may also contribute to the reproductive effects of DDT [15]. The demise of raptor populations described during the 1960’s (e.g. [16]) and the recently-described demasculization of alligators in Lake Apopka, Florida [17] have been ascribed to the endocrine-disrupting effects of DDT and related organochlorine pesticides. Further, epidemiological studies have linked incidence of breast cancer with blood DDE levels [18], suggesting that environmental endocrine-active agents may cause hormone-related disease in the human population [19]. Other pesticides shown to have estrogenic, or anti-androgenic properties include chlordecone [20], dieldrin [21,22], vinclozolin [23], endosulfan [21], toxaphene [21], and linuron [24]. A human environmental impact has been suggested for chlordecone, in that chemical factory workers exposed to this pesticide for prolonged periods of time experienced a high incidence of impaired testicular function [25].
3. Hormone Modulation Some pesticides have been shown to alter normal endocrine function by inhibiting steroid hormone biosynthesis. For example, the imidazole fungicide ketoconazole has been shown to inhibit several steroidogenic enzymes resulting in impaired testosterone biosynthesis [26 – 29], and a marked reduction in serum testosterone levels [28,30,31]. Pesticides can also disrupt androgen homeostasis by increasing the rate of androgen metabolism and elimination. Such perturbations in steroid hormone biotransformation have been causally implicated in the reproductive failure of mammals following exposure to a variety of halogenated hydrocarbon pesticides and other similarly-acting xenobiotics [32]. Many pesticides induce hepatic monooxygenase and UDP-glucuronosyltransferase enzymes (e.g. [33,34]), leading to increased pesticide clearance. However, many of these enzymes are also responsible for androgen metabolism [35,36] and their induction may thus also lead to increased clearance of these hormones
[36,37]. Chronic exposure of rats to dosages of chlordane producing plasma pesticide levels comparable to those in the U.S. population, reduced plasma testosterone levels and altered sexually dimorphic functions and behaviour [38]. This effect of chlordane was ascribed, at least in part, to its ability to alter steroid-metabolizing enzymes. Though chlordane is no longer used in the US, its environmental persistence and propensity to bioaccumulate has resulted in continued human exposure [39]. Preliminary experiments conducted in our laboratory revealed that exposure of mice to pesticides such as chlordecone and endosulfan also significantly increased the catabolic metabolism of testosterone. There is clear precedence for reproductive dysfunction in humans following exposure to chemicals, such as anticancer drugs, that alter steroid hormone biotransformation processes. Cyclophosphamide markedly alters the testosterone metabolic profile of treated rats [40]. Acrolein, an oxidative metabolite of both cyclophosphamide and allylamine biocides [41], contributes to the overall effects of cyclophosphamide on steroid metabolism [40]. Since cyclophosphamide is a reproductive toxicant in humans and can render patients undergoing therapy azoospermic [42], similar effects may be seen for the pesticidal allylamine derivatives. Cisplatin demasculinizes hepatic steroid hormone metabolism in rats [43]. It also alters hormonal profiles of male patients undergoing therapy with the drug [44], which may contribute to their development of gynecomastia and galactorrhea [45–49]. Thus, disruption of steroid hormone biotransformation can alter hormone homeostasis and result in reproductive dysfunction. Pesticides can also indirectly affect steroid hormone levels. Pesticides representing chlorophenols, chlorophenoxy acids, organochlorines, and quinones have been shown to alter circulating thyroid hormone (T4) levels and/or thyroid gland function. These effects have been ascribed largely to increases in T4 metabolism via induction of hepatic UDP-glucuronosyltranferase enzymes [50] and to displacement of T4 from plasma binding-proteins [51]. We and others have shown that reductions in circulating T4 levels can alter hepatic androgen metabolism by compromising the catalytic activity of hepatic cytochrome P450 monooxygenases [52–54]. These enzymes require electrons shuttled from NADPH to the enzyme by the flavoprotein NADPHcytochrome P450 reductase (P450 reductase), which is under the regulatory control of T4 in the rat [52]. Reduction in circulating T4 levels lowers P450 reductase and reduces P450-mediated androgen metabolism, so that pesticides causing a reduction of circulating thyroxine levels might be predicted to affect androgen homeostasis.
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masculinization of females in terms of their adult steroid metabolic capabilities [58–60]. Perinatal or neonatal exposure of pesticides including DDT [12,13], chlordecone [20,61–63], chlordane [38] and atrazine [64] have all been shown to affect steroid hormone-related developmental parameters.
5. Conclusions
Fig. 1. Mechanisms by which pesticides may elicit demasculinization by a different mode of action resulting in synergistic responses.
4. Developmental Effects Prenatal and neonatal life stages may be particularly sensitive to demasculinization by pesticides. Sexual differentiation of the gonads and accessory reproductive organs occurs largely during prenatal development and to a lesser degree during the neonatal period and steroid hormones play a decisive role in establishing gender during these periods [6]. Perturbations in steroid hormone homeostasis during these periods can result in pseudohermaphroditic conditions or other abnormalities of the reproductive system producing reproductive dysfunction [7,55]. Noteworthy is the role of steroid 5a-reductase in male differentiation. Deficiency in steroid 5a-reductase causes a pseudohermaphroditic condition in males characterized by ambiguous external genitalia at birth [7]. Mature males deficient in this enzyme, which is responsible for the conversion of testosterone to dihydrotestosterone, have reduced facial and body hair, no temporal hair recession and a non-palpable prostate. We are not aware of any published data regarding the susceptibility of steroid 5a-reductase to inhibition by pesticides. However, the therapeutic inhibition of this enzyme is increasingly used in the treatment of prostate cancer, and individuals undergoing such treatment may have enhanced sensitivity to the demasculinizing effects of some pesticides. Steroid hormones are also important during prenatal and neonatal development in establishing gender dimorphism in the brain and in the imprinting of neurobehavioral and neuroendocrine components of reproduction. One consequence of this imprinting is the gender-specific expression of steroid hormone-metabolizing enzymes in the adult [35,56,57]. Perturbations in neonatal hypothalamic imprinting by steroid hormones can result in demasculinization of males or
Clearly, many pesticides have the ability to alter steroid hormone-dependent processes due to hormone receptor agonist/antagonist properties and/or their ability to alter steroid hormone homeostasis. Are such properties of consequence to the human population? Analyses of high risk populations have indicated that pesticide exposure among humans is associated with reproductive abnormalities [25], birth abnormalities [65], and skewed sex-ratios for birth defects [65]. There is evidence that pesticides can affect endocrine processes at the high end of normal human exposure. The general populace is exposed to significantly lower levels of pesticides. However, the plethora of pesticides to which the general population is exposed [66– 68] provides a potential for multiple and diverse effects on endocrine processes. Synergistic effects of these compounds on endocrine function must be considered when assessing potential and actual hazards posed to the general population. For example, recent studies [69] have shown that mildly or non-estrogenic pesticides can function synergistically with estrogenicity potencies of 150–1500 times that of the individual pesticides. Simultaneous exposure to DDT, endosulfan and propiconazole may produce profoundly synergistic effects on androgen function. The DDT metabolite p,p%-DDE is anti-androgenic; o,p-DDT, perhaps in synergy with endosulfan [69], is estrogenic; DDT and endosulfan can both enhance endogenous androgen elimination and propiconazole can inhibit de novo testosterone synthesis. The combined effects of these compounds on testosterone homeostasis would likely far exceed the sum of the individual effects (Fig. 1). Reports of dysfunction of the male reproductive system abound and include reduced sperm counts and quality [70–72]. Whether such effects are related to exposure to pesticides and related compounds remains an enigma. However, the potential consequences of such effects on human development and fertility, the hardship imposed upon affected individuals and the medical costs associated with treatment of such abnormalities warrants continued and expanded research into the cellular and molecular targets of such compounds, as well as into the pathological consequences of such insults to the endocrine system.
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Acknowledgements This work was supported by grants from the National Institutes of Health (1F32-CA08259, P01 ES00044) and the US Air Force Office of Scientific Research, Air Force Systems Command Grant (F49620-94-1-0266).
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