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Pb2+: An endocrine disruptor in Drosophila?
Physiology & Behavior 99 (2010) 254–259
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Pb2+: An endocrine disruptor in Drosophila? Helmut V.B. Hirsch a,⁎, Debra Possidente b,1, Bernard Possidente b,1 a b
Department of Biological Sciences, The University at Albany, SUNY, 1400 Washington Avenue, Albany, NY 12222, USA Department of Biology, Skidmore College, Saratoga Springs, NY 12866, USA
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 10 September 2009 Accepted 17 September 2009 Keywords: Endocrine disruptor Pb2+ Drosophila Neural development Synaptic function Model systems
a b s t r a c t Environmental exposure to Pb2+ affects hormone-mediated responses in vertebrates. To help establish the fruit fly, Drosophila melanogaster, as a model system for studying such disruption, we describe effects of Pb2+ on hormonally regulated traits. These include duration of development, longevity, females' willingness to mate, fecundity and adult locomotor activity. Developmental Pb2+ exposure has been shown to affect gene expression in a specific region of the Drosophila genome (~122 genes) involved in lead-induced changes in adult locomotion and to affect regulation of intracellular calcium levels associated with neuronal activity at identified synapses in the larval neuromuscular junction. We suggest ways in which Drosophila could become a new model system for the study of endocrine disruptors at genetic, neural and behavioral levels of analysis, particularly by use of genomic methods. This will facilitate efforts to distinguish between behavioral effects of Pb2+ caused by direct action on neural mechanisms versus effects of Pb+ 2 on behavior mediated through endocrine disruption. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The toxicity of the heavy metal Pb2+ has been recognized for some 4000 years; nevertheless, its use increased dramatically from the beginning of the twentieth century, especially with the introduction of leaded gasoline [1], and Pb2+ is now ubiquitous in the biosphere [1,2]. Although the phase-out of leaded paint and gasoline in the 1980s produced a drop in blood lead levels in the United States population as a whole [3,4], Pb2+ exposure remains a significant public health problem [5–7] because children continue to be exposed to lead-contaminated paint [3,8,9] and soil [10,11]. Furthermore, in some countries lead-based paint is still being manufactured and sold [12,13], while industries such as e-waste recycling introduce new sources of contamination [14]. Furthermore, many adults carry Pb2+ in their skeletons [15–19] which affects their cognitive function [19,20], cardiovascular health [20,21], and renal function [20], and can be mobilized and passed on to fetuses or infants [22]. The bulk of the research on effects of chronic developmental exposure of children to Pb2+ has focused on deficits in cognitive ability [4,23–29], increases in delinquency [30–32] and in behaviors associated with Attention Deficit Hyperactivity Disorder [33], changes in activity [34] and possibly altered sensory function [35–40]. It is likely that these Pb2+-dependent effects involve synaptic changes (e.g. muscarinic modulation [41–43], changes in N-methyl-D-aspar⁎ Corresponding author. Tel.: +1 518 442 4311; fax: +1 518 442 4767. E-mail addresses: [email protected] (H.V.B. Hirsch), [email protected] (D. Possidente), [email protected] (B. Possidente). 1 Tel.: +1 518 580 5082; fax: +1 518 580 5071. 0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.09.014
tate receptor function [6,44,45], effects on voltage-gated sodium channels [46] and changes in calcium signaling), which have been shown both in vertebrates [47–52] and at identified synapses at the neuromuscular junction in third instar Drosophila larvae [53]. A second, much smaller, focus has been on Pb2+-dependent delays in the onset of sexual maturity in girls [54–57] (but see also [58]) and on reductions in testicular volume in boys [56]; acute Pb2+ exposure can also affect reproductive function in both women [59] and men [60–63]. Thus, not only does Pb2+ have direct effects on neuronal development and function, it may also have indirect ones by acting as an endocrine disruptor. Estrogens serve vital functions in brain development, affecting not only the establishment of sex differences but also acting as trophic and neuroprotective agents [64–67]. Furthermore, brain levels of estradiol, estradiol synthase and estrogen receptors are all at their peaks prenatally or during the first few days of life [65], the same period in which the majority of synaptic connections develop in the brain [68]. This raises the question of whether Pb2+ acts directly on synaptic development [53,69–71], and/or whether Pb2+-dependent synaptic effects are mediated through changes in endocrine function. What is missing are studies that look at both “cognitive” and “endocrine disruptive” effects of chronic developmental Pb2+ exposure [72], for example looking at the concurrent effects of developmental Pb2+ exposure on timing of sexual maturity, sexual function and cognitive function. Recent reviews summarize both the scope of the problem and progress made in understanding the mechanisms by which endocrine disruptors affect growth and development [73–77]. The majority of this work has focused on endocrine disruptor effects in humans or in
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rodents, although there is comparable evidence for birds, reptiles, fish and aquatic invertebrates as well (for reviews see [75,76]). As Iavicoli et al. [78] note, Pb2+ affects multiple cellular functions and endocrine mechanisms, including multiple effects on the hypothalamus– pituitary–gonadal axis, altered onset of puberty in humans and rodents, altered estrogen and LH receptor affinity, lowered serum levels of IGF-1, testosterone and estradiol, altered sperm morphology and function, inhibition of GH synthesis and LH secretion, reduced expression of the steroidogenic acute regulatory protein (StAR), increased lipid peroxidation in seminal plasma and increased production of reactive oxygen species (ROS). In this paper we summarize evidence that Drosophila, which has already shown itself to be an overlooked yet very powerful model system for the study of such neurotoxins as Pb2+ [53,70,79–81], Zn2+[82], Cd2+ [83–85] and Hg2+ [86], may also be an excellent system for studying endocrine disruptors. 2. Drosophila endocrinology Insects have true endocrine glands that are derived from epithelial tissue and that function in much the same way as vertebrate glands [87,88]. There are both circulating hormones and nuclear receptors (NRs), much like those in vertebrates [89]. The Drosophila genome codes for NRs that represent all major subclasses of human receptors and includes orthologs of key human NRs [89]. Two major hormonal systems in Drosophila involve Ecdysone (Ecy), which is a steroid, and Juvenile Hormone (JH), a sesquiterpinoid. Ecy is produced in the prothoracic glands, while JH is produced in the corpora allata in the brain [88]. The ecdysteroids of Drosophila are homologous to the cholesterol-derived steroid hormones such as estradiol, and JH shares some characteristics with retinoic acid (Fig. 1). Comparing 18 known Drosophila hormone NRs with 48 NRs identified in humans gives 16 orthologs, covering all 6 NR superfamily subtypes, including many for steroid NRs, and two for retinoic acid NRs [90]. At least 8 of the 16 human-ortholog NRs are known to be regulated by Ecy, however the one receptor known to use Ecy as a ligand (Ecdysone Receptor, or EcR), also uses the retinoic acid NR homolog (USP) in the ligand's heterodimer [87]. The EcR–USP dimer continues the signaling cascade to turn on the seven other NRs, which together control the processes of gastrulation, embryonic segmentation, oogenesis, and larval polytene chromosome puffing [90], as well as modulation of three essential molecules, alcohol dehydrogenase, urate oxidase, and glucose dehydrogenase [88]. Each peak of Ecy precedes and controls a molt. At the end of the third larval instar, JH stops being produced, and this allows the peak in Ecy to initiate pupation, massive cell death, the development of adult
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structure from the imaginal discs, and head eversion [88]. JH returns in the adult stage and regulates spermatogenesis [88], life-span [91,92], sex differences in locomotor behavior [92,93] and feeding behavior [94], as well as intensity of courtship [92,95], interacting with Ecy to promote fertility [90]. Understanding the regulation of development by Drosophila hormones and their 16 human-orthologous NRs should help elucidate control of human hormone–NR systems [96–103]. 2.1. Pb2+ effects on hormonally regulated behavior in Drosophila Chronic developmental exposure of Drosophila to Pb2+, even at low doses, affects a number of behaviors that are hormone-mediated. These include developmental delays following acute exposure (rearing Drosophila during preadult stages on medium containing 100– 2500 ppm Pb2+ increases the time to pupation [83,104,105]), and shortening in preadult development after many generations of exposure to high levels of Pb2+ [104]. Exposure to high doses of Pb2+ (2070 ppm) decreases life-span [106] while low doses (0.207–2.07 ppm) have no significant effect. As may be the case in mice [78], when Drosophila are reared from egg stages to adult days 5–7 on medium made with very low doses of Pb2+ (2–8 ppm), females are more willing to mate than controls (reared on medium made with distilled water), while exposure over the same interval to somewhat higher doses (20–50 ppm) reduces females' willingness to mate [79]. Fecundity (the number of viable offspring per female) is increased by exposure to low loses (2 ppm, again from egg stages to adult days 5–7) [79], while exposure to a range of higher doses (20 ppm [79] and 100–500 ppm [105]) does not appear to affect fecundity; at even higher doses (500–50,000 ppm) fecundity is reduced [107]. Finally, locomotor activity of adults (the distance travelled in a set interval of time) is reduced by exposure to Pb2+, but only at higher doses (50 ppm; the change is significant only for males [79]). As in vertebrates, the underlying mechanisms are poorly understood, and Drosophila provide another model system for investigating them. Note that for females' willingness to mate and fecundity the dose response curves are biphasic, that is, hormetic [108]. Furthermore, in Drosophila third instar larvae, chronic Pb2+ exposure modifies development of individual, identifiable synapses between motorneurons and the muscle fibers they innervate [70]. At the same larval synapses Pb2+ exposure also increases intracellular calcium levels and synaptic facilitation; most likely this is due to Pb2+-dependent reduction in the activity of the plasma membrane Ca2+ ATPase (PMCA), which extrudes Ca2+ from these synaptic terminals [53]. We have recently identified, by QTL analysis [109], a region (30AB) of the Drosophila second chromosome that mediates a Pb2+dependent change in locomotor behavior [80]. This region is also known to control the expression of cis and trans downstream genes in
Fig. 1. Human estradiol compared to insect ecdysone, and human retinoic acid compared to insect juvenile hormone.
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response to Pb2+ [81]. Genes in 30AB are therefore candidate genes for study of this gene expression-to-behavior link. We have focused on 122 genes extending through 30AB and half-way through the adjoining cytological regions (because of the low resolution of this technique relative to a single locus) for further analysis. Based on known gene functions and on an unbiased correlation of lead-induced changes in gene expression [81] with Pb2+-induced changes in behavior [80] we have identified a subset of these as candidate loci for the QTL. Drosophila are thus a useful model for studying behavioral, synaptic and genetic changes following chronic exposure to the neurotoxin Pb2+. 3. Pb2+ toxicity mediated by oxidative stress PbAc causes oxidative stress by direct conjugation with glutathione (GSH), a tripeptide in mM concentrations in cells that buffers against oxidative stress by forming covalent adducts with oxidized proteins and lipids. PbAc has also been proposed to induce oxidative stress by inhibiting sulfhydryl-dependent enzymes, interfering with other metals needed for antioxidant enzyme activities, and by causing the oxidation of lipids and interfering with membrane integrity and fatty acid composition (reviewed in [110]). In 1965, E.D. Wills published the first paper linking PbAc toxicity to oxidative stress, and demonstrated that lead and other metals catalyze the oxidation of unsaturated fatty acids [111]. More recently, in 1995, Yiin and Lin demonstrated that PbAc enhances oxidation of linoic, linoleic, and arachidonic acids [112]. Several other studies have demonstrated elevated lipid peroxidation or decreased antioxidant defense in tissues of PbAc exposed animals [113–116]. Because of the possibility that many of the toxic effects of PbAc are mediated by oxidative stress, Gurer and Ercal have recently proposed that antioxidants might be beneficial in the treatment of lead poisoning combined with CaNa2EGTA or succimer, the most commonly used chelating agents [110]. Any system that depends on glutathione control of oxidative stress (such as solubility of toxins, heme regulation, insecticide resistance, stress signal transduction, membrane integrity, and egg size and shape) can be studied in lead-treated Drosophila. For example, regulation of circadian rhythms by one of Drosophila's NRs (E75) has recently been suggested by the finding of a heme group in its ligand-binding domain, which should confer the ability to attract small gas molecules such as NO and CO, known to function in stress– response pathways and circadian rhythm regulation [99,102]. 4. Advantages of Drosophila for genetic analysis of lead toxicity Certainly, Drosophila can be a useful system for the genetic analysis of endocrine disruptors and their mechanisms. Advantages of Drosophila as a model system in this field include a short life cycle, economy, efficiency and a long history of research with an extensive literature as well as a full complement of genetic and genomic methods (see the FlyBase web server (http://flybase.org/) and [117] for a comprehensive genetic and genomic database on Drosophila, and the Drosophila Information Service (http://www.ou.edu/journals/dis/) for a rich database of Drosophila research and technical publications). Genetic tools available in Drosophila include (1) the existence of many mutants (e.g. Ecdysoneless [118] and Methoprene-tolerant [95]); (2) transgenic constructs in which mammalian genes are inserted into flies (e.g. [103]); and (3) quantitative trait locus mapping that allows genetic variation affecting complex traits to be mapped to specific regions of the genome [80,119–121]. Drosophila have proven to be a useful genetic model, in combination with mammalian models, for successful analysis of such complex traits as circadian rhythmicity [122] including circadian modulation of susceptibility to pesticides [123]. These approaches, combined with the most current genetic methods for analysis of complex traits [119,120,122,124–126] can facilitate efficient screening of known hormone disruptors, and
comprehensive analysis of their genetic, neural and behavioral mechanisms of action. Thackray et al. [103] have constructed transgenic Drosophila with the human estrogen receptor alpha and an estrogen responsive green fluorescent reporter gene and demonstrated a response to estrogen compounds in the food. This system has not yet been used to examine potential effects of lead on human estrogen receptor function. A Drosophila-derived cell culture assay for direct effects of ecdysteroids and their agonists and antagonists also has yet to be employed to investigate direct effects of lead as an endocrine disruptor [127]. Drosophila are also relatively amenable to genetic manipulations of target phenotypes relevant to endocrine disruption by methods such as artificial selection that would be arduous and expensive to apply even to rodent animal models. Magnusson and Ramel [128], for example, showed that genetic differences in tolerance to toxic metals including lead was genetically variable among strains, with little correlation between patterns of strain differences across different metals. They were also able to increase tolerance to mercury compounds, specifically, by 12 generations of artificial selection from a base population of four wild-type strains. Nassar [129] also used artificial selection for 25 generations of adaptation of Pb2+ exposure and found increases in fecundity and longevity, as was the case for developmental exposure to low Pb2+ levels for one generation ([79,106] and see also [104]). On a genomic level, Ruden et al. [81] have assayed genome-wide transcription levels in 75 recombinant inbred Drosophila strains raised for one generation with and without lead, identifying over 1000 transcripts with altered expression in response to developmental lead exposure. This approach presents, for example, an opportunity to screen lead-dependent changes in transcription directly for individual loci known to be involved in endocrine regulation, to conduct unbiased screens for transcripts previously unassociated with lead metabolism, and to assay the same strains for lead-induced effects on endocrine-dependent traits that may be driven by the changes in transcription. Such genetical–genomic analysis [81] can be combined with systems genetics approaches [82] as a model for genetic regulation of complex traits and genetic correlations among them. Ayroles et al. [124] for example, have identified 241 “transcriptional modules” in Drosophila representing distinct complexes of transcripts, each of which is “enriched” for specific biological functions, allowing interactions among transcripts within modules and among modules to be mapped. This type of approach should be particularly useful for analysis of complex interactions among endocrine system components, traits that they regulate, and numerous environmental factors, and for distinguishing direct endocrine effects from those that may affect the same pathways indirectly through intersecting developmental, neural and metabolic mechanisms.
5. Evolutionary and life-history studies of lead toxicity Drosophila also provide a model system for evolution of life-history strategies, including endocrine-mediated regulation of trade-offs among life-span, fecundity and timing of reproduction [130], that could be employed to investigate population responses to environmental factors such as lead as well as their potential dependence on additional toxins and environmental factors such as temperature stress [131] and nutritional factors [125]. These types of studies can provide insights into potential long-term evolutionary and ecological impacts of environmental toxins such as Pb+ 2. For example, it might be useful to assay gene expression during artificial selection for lead resistance in control and selected lines after one generation of exposure, and again after multiple generations to determine whether initial lead-induced changes in the transcriptome predict long-term constitutive changes in gene expression in response to selection for adaptation to higher lead levels.
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6. Summary Developmental Pb2+ exposure affects hormone-dependent behaviors in Drosophila, suggesting that Pb2+ is an endocrine disruptor in this species. We believe it is important, therefore, that experiments aimed at identifying the direct neural effects of Pb2+ take into consideration the possibility that Pb2+ may also alter behavior by modifying endocrine system function. Only then may we distinguish between separate pathways and mechanisms that are likely to play a role in the effects of this neurotoxin.
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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b
Pb2+: An endocrine disruptor in Drosophila? Helmut V.B. Hirsch a,⁎, Debra Possidente b,1, Bernard Possidente b,1 a b
Department of Biological Sciences, The University at Albany, SUNY, 1400 Washington Avenue, Albany, NY 12222, USA Department of Biology, Skidmore College, Saratoga Springs, NY 12866, USA
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 10 September 2009 Accepted 17 September 2009 Keywords: Endocrine disruptor Pb2+ Drosophila Neural development Synaptic function Model systems
a b s t r a c t Environmental exposure to Pb2+ affects hormone-mediated responses in vertebrates. To help establish the fruit fly, Drosophila melanogaster, as a model system for studying such disruption, we describe effects of Pb2+ on hormonally regulated traits. These include duration of development, longevity, females' willingness to mate, fecundity and adult locomotor activity. Developmental Pb2+ exposure has been shown to affect gene expression in a specific region of the Drosophila genome (~122 genes) involved in lead-induced changes in adult locomotion and to affect regulation of intracellular calcium levels associated with neuronal activity at identified synapses in the larval neuromuscular junction. We suggest ways in which Drosophila could become a new model system for the study of endocrine disruptors at genetic, neural and behavioral levels of analysis, particularly by use of genomic methods. This will facilitate efforts to distinguish between behavioral effects of Pb2+ caused by direct action on neural mechanisms versus effects of Pb+ 2 on behavior mediated through endocrine disruption. © 2009 Elsevier Inc. All rights reserved.
1. Introduction The toxicity of the heavy metal Pb2+ has been recognized for some 4000 years; nevertheless, its use increased dramatically from the beginning of the twentieth century, especially with the introduction of leaded gasoline [1], and Pb2+ is now ubiquitous in the biosphere [1,2]. Although the phase-out of leaded paint and gasoline in the 1980s produced a drop in blood lead levels in the United States population as a whole [3,4], Pb2+ exposure remains a significant public health problem [5–7] because children continue to be exposed to lead-contaminated paint [3,8,9] and soil [10,11]. Furthermore, in some countries lead-based paint is still being manufactured and sold [12,13], while industries such as e-waste recycling introduce new sources of contamination [14]. Furthermore, many adults carry Pb2+ in their skeletons [15–19] which affects their cognitive function [19,20], cardiovascular health [20,21], and renal function [20], and can be mobilized and passed on to fetuses or infants [22]. The bulk of the research on effects of chronic developmental exposure of children to Pb2+ has focused on deficits in cognitive ability [4,23–29], increases in delinquency [30–32] and in behaviors associated with Attention Deficit Hyperactivity Disorder [33], changes in activity [34] and possibly altered sensory function [35–40]. It is likely that these Pb2+-dependent effects involve synaptic changes (e.g. muscarinic modulation [41–43], changes in N-methyl-D-aspar⁎ Corresponding author. Tel.: +1 518 442 4311; fax: +1 518 442 4767. E-mail addresses: [email protected] (H.V.B. Hirsch), [email protected] (D. Possidente), [email protected] (B. Possidente). 1 Tel.: +1 518 580 5082; fax: +1 518 580 5071. 0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.09.014
tate receptor function [6,44,45], effects on voltage-gated sodium channels [46] and changes in calcium signaling), which have been shown both in vertebrates [47–52] and at identified synapses at the neuromuscular junction in third instar Drosophila larvae [53]. A second, much smaller, focus has been on Pb2+-dependent delays in the onset of sexual maturity in girls [54–57] (but see also [58]) and on reductions in testicular volume in boys [56]; acute Pb2+ exposure can also affect reproductive function in both women [59] and men [60–63]. Thus, not only does Pb2+ have direct effects on neuronal development and function, it may also have indirect ones by acting as an endocrine disruptor. Estrogens serve vital functions in brain development, affecting not only the establishment of sex differences but also acting as trophic and neuroprotective agents [64–67]. Furthermore, brain levels of estradiol, estradiol synthase and estrogen receptors are all at their peaks prenatally or during the first few days of life [65], the same period in which the majority of synaptic connections develop in the brain [68]. This raises the question of whether Pb2+ acts directly on synaptic development [53,69–71], and/or whether Pb2+-dependent synaptic effects are mediated through changes in endocrine function. What is missing are studies that look at both “cognitive” and “endocrine disruptive” effects of chronic developmental Pb2+ exposure [72], for example looking at the concurrent effects of developmental Pb2+ exposure on timing of sexual maturity, sexual function and cognitive function. Recent reviews summarize both the scope of the problem and progress made in understanding the mechanisms by which endocrine disruptors affect growth and development [73–77]. The majority of this work has focused on endocrine disruptor effects in humans or in
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rodents, although there is comparable evidence for birds, reptiles, fish and aquatic invertebrates as well (for reviews see [75,76]). As Iavicoli et al. [78] note, Pb2+ affects multiple cellular functions and endocrine mechanisms, including multiple effects on the hypothalamus– pituitary–gonadal axis, altered onset of puberty in humans and rodents, altered estrogen and LH receptor affinity, lowered serum levels of IGF-1, testosterone and estradiol, altered sperm morphology and function, inhibition of GH synthesis and LH secretion, reduced expression of the steroidogenic acute regulatory protein (StAR), increased lipid peroxidation in seminal plasma and increased production of reactive oxygen species (ROS). In this paper we summarize evidence that Drosophila, which has already shown itself to be an overlooked yet very powerful model system for the study of such neurotoxins as Pb2+ [53,70,79–81], Zn2+[82], Cd2+ [83–85] and Hg2+ [86], may also be an excellent system for studying endocrine disruptors. 2. Drosophila endocrinology Insects have true endocrine glands that are derived from epithelial tissue and that function in much the same way as vertebrate glands [87,88]. There are both circulating hormones and nuclear receptors (NRs), much like those in vertebrates [89]. The Drosophila genome codes for NRs that represent all major subclasses of human receptors and includes orthologs of key human NRs [89]. Two major hormonal systems in Drosophila involve Ecdysone (Ecy), which is a steroid, and Juvenile Hormone (JH), a sesquiterpinoid. Ecy is produced in the prothoracic glands, while JH is produced in the corpora allata in the brain [88]. The ecdysteroids of Drosophila are homologous to the cholesterol-derived steroid hormones such as estradiol, and JH shares some characteristics with retinoic acid (Fig. 1). Comparing 18 known Drosophila hormone NRs with 48 NRs identified in humans gives 16 orthologs, covering all 6 NR superfamily subtypes, including many for steroid NRs, and two for retinoic acid NRs [90]. At least 8 of the 16 human-ortholog NRs are known to be regulated by Ecy, however the one receptor known to use Ecy as a ligand (Ecdysone Receptor, or EcR), also uses the retinoic acid NR homolog (USP) in the ligand's heterodimer [87]. The EcR–USP dimer continues the signaling cascade to turn on the seven other NRs, which together control the processes of gastrulation, embryonic segmentation, oogenesis, and larval polytene chromosome puffing [90], as well as modulation of three essential molecules, alcohol dehydrogenase, urate oxidase, and glucose dehydrogenase [88]. Each peak of Ecy precedes and controls a molt. At the end of the third larval instar, JH stops being produced, and this allows the peak in Ecy to initiate pupation, massive cell death, the development of adult
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structure from the imaginal discs, and head eversion [88]. JH returns in the adult stage and regulates spermatogenesis [88], life-span [91,92], sex differences in locomotor behavior [92,93] and feeding behavior [94], as well as intensity of courtship [92,95], interacting with Ecy to promote fertility [90]. Understanding the regulation of development by Drosophila hormones and their 16 human-orthologous NRs should help elucidate control of human hormone–NR systems [96–103]. 2.1. Pb2+ effects on hormonally regulated behavior in Drosophila Chronic developmental exposure of Drosophila to Pb2+, even at low doses, affects a number of behaviors that are hormone-mediated. These include developmental delays following acute exposure (rearing Drosophila during preadult stages on medium containing 100– 2500 ppm Pb2+ increases the time to pupation [83,104,105]), and shortening in preadult development after many generations of exposure to high levels of Pb2+ [104]. Exposure to high doses of Pb2+ (2070 ppm) decreases life-span [106] while low doses (0.207–2.07 ppm) have no significant effect. As may be the case in mice [78], when Drosophila are reared from egg stages to adult days 5–7 on medium made with very low doses of Pb2+ (2–8 ppm), females are more willing to mate than controls (reared on medium made with distilled water), while exposure over the same interval to somewhat higher doses (20–50 ppm) reduces females' willingness to mate [79]. Fecundity (the number of viable offspring per female) is increased by exposure to low loses (2 ppm, again from egg stages to adult days 5–7) [79], while exposure to a range of higher doses (20 ppm [79] and 100–500 ppm [105]) does not appear to affect fecundity; at even higher doses (500–50,000 ppm) fecundity is reduced [107]. Finally, locomotor activity of adults (the distance travelled in a set interval of time) is reduced by exposure to Pb2+, but only at higher doses (50 ppm; the change is significant only for males [79]). As in vertebrates, the underlying mechanisms are poorly understood, and Drosophila provide another model system for investigating them. Note that for females' willingness to mate and fecundity the dose response curves are biphasic, that is, hormetic [108]. Furthermore, in Drosophila third instar larvae, chronic Pb2+ exposure modifies development of individual, identifiable synapses between motorneurons and the muscle fibers they innervate [70]. At the same larval synapses Pb2+ exposure also increases intracellular calcium levels and synaptic facilitation; most likely this is due to Pb2+-dependent reduction in the activity of the plasma membrane Ca2+ ATPase (PMCA), which extrudes Ca2+ from these synaptic terminals [53]. We have recently identified, by QTL analysis [109], a region (30AB) of the Drosophila second chromosome that mediates a Pb2+dependent change in locomotor behavior [80]. This region is also known to control the expression of cis and trans downstream genes in
Fig. 1. Human estradiol compared to insect ecdysone, and human retinoic acid compared to insect juvenile hormone.
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response to Pb2+ [81]. Genes in 30AB are therefore candidate genes for study of this gene expression-to-behavior link. We have focused on 122 genes extending through 30AB and half-way through the adjoining cytological regions (because of the low resolution of this technique relative to a single locus) for further analysis. Based on known gene functions and on an unbiased correlation of lead-induced changes in gene expression [81] with Pb2+-induced changes in behavior [80] we have identified a subset of these as candidate loci for the QTL. Drosophila are thus a useful model for studying behavioral, synaptic and genetic changes following chronic exposure to the neurotoxin Pb2+. 3. Pb2+ toxicity mediated by oxidative stress PbAc causes oxidative stress by direct conjugation with glutathione (GSH), a tripeptide in mM concentrations in cells that buffers against oxidative stress by forming covalent adducts with oxidized proteins and lipids. PbAc has also been proposed to induce oxidative stress by inhibiting sulfhydryl-dependent enzymes, interfering with other metals needed for antioxidant enzyme activities, and by causing the oxidation of lipids and interfering with membrane integrity and fatty acid composition (reviewed in [110]). In 1965, E.D. Wills published the first paper linking PbAc toxicity to oxidative stress, and demonstrated that lead and other metals catalyze the oxidation of unsaturated fatty acids [111]. More recently, in 1995, Yiin and Lin demonstrated that PbAc enhances oxidation of linoic, linoleic, and arachidonic acids [112]. Several other studies have demonstrated elevated lipid peroxidation or decreased antioxidant defense in tissues of PbAc exposed animals [113–116]. Because of the possibility that many of the toxic effects of PbAc are mediated by oxidative stress, Gurer and Ercal have recently proposed that antioxidants might be beneficial in the treatment of lead poisoning combined with CaNa2EGTA or succimer, the most commonly used chelating agents [110]. Any system that depends on glutathione control of oxidative stress (such as solubility of toxins, heme regulation, insecticide resistance, stress signal transduction, membrane integrity, and egg size and shape) can be studied in lead-treated Drosophila. For example, regulation of circadian rhythms by one of Drosophila's NRs (E75) has recently been suggested by the finding of a heme group in its ligand-binding domain, which should confer the ability to attract small gas molecules such as NO and CO, known to function in stress– response pathways and circadian rhythm regulation [99,102]. 4. Advantages of Drosophila for genetic analysis of lead toxicity Certainly, Drosophila can be a useful system for the genetic analysis of endocrine disruptors and their mechanisms. Advantages of Drosophila as a model system in this field include a short life cycle, economy, efficiency and a long history of research with an extensive literature as well as a full complement of genetic and genomic methods (see the FlyBase web server (http://flybase.org/) and [117] for a comprehensive genetic and genomic database on Drosophila, and the Drosophila Information Service (http://www.ou.edu/journals/dis/) for a rich database of Drosophila research and technical publications). Genetic tools available in Drosophila include (1) the existence of many mutants (e.g. Ecdysoneless [118] and Methoprene-tolerant [95]); (2) transgenic constructs in which mammalian genes are inserted into flies (e.g. [103]); and (3) quantitative trait locus mapping that allows genetic variation affecting complex traits to be mapped to specific regions of the genome [80,119–121]. Drosophila have proven to be a useful genetic model, in combination with mammalian models, for successful analysis of such complex traits as circadian rhythmicity [122] including circadian modulation of susceptibility to pesticides [123]. These approaches, combined with the most current genetic methods for analysis of complex traits [119,120,122,124–126] can facilitate efficient screening of known hormone disruptors, and
comprehensive analysis of their genetic, neural and behavioral mechanisms of action. Thackray et al. [103] have constructed transgenic Drosophila with the human estrogen receptor alpha and an estrogen responsive green fluorescent reporter gene and demonstrated a response to estrogen compounds in the food. This system has not yet been used to examine potential effects of lead on human estrogen receptor function. A Drosophila-derived cell culture assay for direct effects of ecdysteroids and their agonists and antagonists also has yet to be employed to investigate direct effects of lead as an endocrine disruptor [127]. Drosophila are also relatively amenable to genetic manipulations of target phenotypes relevant to endocrine disruption by methods such as artificial selection that would be arduous and expensive to apply even to rodent animal models. Magnusson and Ramel [128], for example, showed that genetic differences in tolerance to toxic metals including lead was genetically variable among strains, with little correlation between patterns of strain differences across different metals. They were also able to increase tolerance to mercury compounds, specifically, by 12 generations of artificial selection from a base population of four wild-type strains. Nassar [129] also used artificial selection for 25 generations of adaptation of Pb2+ exposure and found increases in fecundity and longevity, as was the case for developmental exposure to low Pb2+ levels for one generation ([79,106] and see also [104]). On a genomic level, Ruden et al. [81] have assayed genome-wide transcription levels in 75 recombinant inbred Drosophila strains raised for one generation with and without lead, identifying over 1000 transcripts with altered expression in response to developmental lead exposure. This approach presents, for example, an opportunity to screen lead-dependent changes in transcription directly for individual loci known to be involved in endocrine regulation, to conduct unbiased screens for transcripts previously unassociated with lead metabolism, and to assay the same strains for lead-induced effects on endocrine-dependent traits that may be driven by the changes in transcription. Such genetical–genomic analysis [81] can be combined with systems genetics approaches [82] as a model for genetic regulation of complex traits and genetic correlations among them. Ayroles et al. [124] for example, have identified 241 “transcriptional modules” in Drosophila representing distinct complexes of transcripts, each of which is “enriched” for specific biological functions, allowing interactions among transcripts within modules and among modules to be mapped. This type of approach should be particularly useful for analysis of complex interactions among endocrine system components, traits that they regulate, and numerous environmental factors, and for distinguishing direct endocrine effects from those that may affect the same pathways indirectly through intersecting developmental, neural and metabolic mechanisms.
5. Evolutionary and life-history studies of lead toxicity Drosophila also provide a model system for evolution of life-history strategies, including endocrine-mediated regulation of trade-offs among life-span, fecundity and timing of reproduction [130], that could be employed to investigate population responses to environmental factors such as lead as well as their potential dependence on additional toxins and environmental factors such as temperature stress [131] and nutritional factors [125]. These types of studies can provide insights into potential long-term evolutionary and ecological impacts of environmental toxins such as Pb+ 2. For example, it might be useful to assay gene expression during artificial selection for lead resistance in control and selected lines after one generation of exposure, and again after multiple generations to determine whether initial lead-induced changes in the transcriptome predict long-term constitutive changes in gene expression in response to selection for adaptation to higher lead levels.
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6. Summary Developmental Pb2+ exposure affects hormone-dependent behaviors in Drosophila, suggesting that Pb2+ is an endocrine disruptor in this species. We believe it is important, therefore, that experiments aimed at identifying the direct neural effects of Pb2+ take into consideration the possibility that Pb2+ may also alter behavior by modifying endocrine system function. Only then may we distinguish between separate pathways and mechanisms that are likely to play a role in the effects of this neurotoxin.
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