Ecotoxicology (2007) 16:15–28 DOI 10.1007/s10646-006-0108-y
Endocrine disruption in nematodes: effects and mechanisms Sebastian Ho¨ss Æ Lennart Weltje
Accepted: 4 October 2006 / Published online: 13 January 2007 Springer Science+Business Media, LLC 2007
Abstract This paper reviews the current knowledge on endocrine disruption in nematodes. These organisms have received little attention in the field of ecotoxicology, in spite of their important role in aquatic ecosystems. Research on endocrine regulation and disruption in nematodes, especially the more recent studies, concentrate mainly on one species, Caenorhabditis elegans. Although an endocrine system is not known in nematodes, there is evidence that many processes are regulated via hormonal pathways. As vertebrate hormones, such as steroids, may have endocrine functions in nematodes as well, endocrine disrupting chemicals (EDCs) defined for vertebrates may also be able to influence nematodes. The studies that are reviewed here, and own data showed that potential EDCs can affect nematodes on all organizational levels, from molecules to communities. It is concluded that nematodes, notably its prominent species C. elegans, are a promising organism group for the development of biomonitoring tools, provided that more mechanistic evidence is gathered on hormonal processes within these animals.
S. Ho¨ss (&) Ecossa, Thierschstrasse 43, D-80538 Munich, Germany e-mail:
[email protected] L. Weltje Department of Ecology and Evolution—Ecotoxicology, J.W. Goethe University, Siesmayerstrasse 70, D-60323 Frankfurt am Main, Germany Present Address: L. Weltje Agricultural Center, Ecotoxicology, BASF AG, Carl-Bosch-Strasse 64, D-67117 Limburgerhof, Germany
Keywords Caenorhabditis elegans Endocrine disruption Gene expression Nematodes Reproduction Review
Introduction Nematodes are important representatives of the aquatic benthos, however, they are often neglected in ecological and ecotoxicological studies. Reasons for this are their small body size (they belong to the meiofauna: 42–500 lm; Fenchel 1978) requiring specialists for handling them in the laboratory, as well as substantial problems with the culturing of most nematodes species, making routine experimental studies difficult. Despite these problems, a number of nematode species served as test organisms in the field of ecotoxicology, including also studies on endocrine disruption (for reviews see, e.g., Neher 2001; Ho¨ss et al. 2006). The most famous nematode is Caenorhabditis elegans. Since several decades, C. elegans has been exploited as model organism for developmental biology, genetics and medical sciences, making it one of the best investigated multicellular organisms (Riddle et al. 1997). Especially the comprehensive work that has been done on sequencing the genome of C. elegans (C. elegans Sequencing Consortium 1998) attracted many scientists to study the regulation of body functions and the disruption of these functions on a genomic and proteomic level. The majority of studies on endocrine regulation and disruption in nematodes were also conducted with C. elegans as test organism. C. elegans is a free-living soil nematode, but now and again it appears in aquatic habitats (Hirschmann 1952; Zullini 1988). Also, the natural habitat of C. elegans is
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comparable to the habitat of freshwater nematodes, both living in a more or less water-filled interstitial space between particles. Furthermore, C. elegans can be cultured in liquid medium, which allows an aqueous exposure to chemicals. This paper gives an overview of studies dealing with substances that might influence or disrupt the endocrine regulation of certain processes in nematodes. Effects of such potential endocrine disrupting chemicals (EDCs) on molecular, organismic and community levels were considered to discuss the broader risk of these chemicals for simple aquatic invertebrates and the suitability of nematodes as sentinels for endocrine disruption in aquatic ecosystems.
Nematodes in aquatic ecosystems Ecological relevance Nematodes are usually the most dominant metazoans within benthic communities, in freshwater and marine sediments (Heip et al. 1985; Traunspurger 2002) and in soils (Yeates 1981). They can reach densities in freshwater habitats up to 11.4 million per m2 (Michiels and Traunspurger 2005). Free-living nematodes occupy many different trophic levels with species feeding on bacteria, algae, fungi and higher plants, as well as omnivorous and predatory species (Yeates et al. 1993; Traunspurger 1997). Moreover, nematodes play a major role as parasites in insects, fish and mammals (Poinar 1975; Lee 2002). As a food source for macroinvertebrates and small fish, nematodes link various trophic levels (Coull et al. 1995; Beier et al. 2004). Nematodes are able to stimulate bacterial activity in freshwater sediments (Traunspurger et al. 1997), thus also influencing benthic nutrient cycling. In terrestrial habitats, nematodes can account for up to 20% of the nitrogen mineralization (Beare 1997). Therefore, it is beyond doubt that this organism group plays an important role in benthic food webs. Exposure pathways Many hormones or suspected EDCs are hydrophobic substances with the affinity to bind to particles and thus may accumulate in sediments (Bennie 1999; Yu et al. 2004). Therefore, benthic organisms can be exposed to higher concentrations of hydrophobic chemicals than pelagic organisms. However, if a chemical is bioavailable (and thus potentially effective) for an organism is not only dependent on the properties of the chemical, but also on the uptake route relevant for the respective
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organism. Nematodes live in the interstitial space between the sediment particles, in close contact to the pore water. Substances dissolved in pore water may be taken up by nematodes through diffusive uptake via the skin. Depending on the feeding type, nematodes ingest living and dead particles from different sediment compartments, which may be loaded with hydrophobic chemicals. During digestion, these chemicals may become bioavailable again (Leppa¨nen and Kukkonen 1998). Via this exposure pathway sediment-bound chemicals might pose a risk to nematodes as well.
Endocrine regulation in nematodes In the context of endocrine disruption, it is important to examine the endocrine system of nematodes. Effects of chemicals on nematodes can only unequivocally be explained as endocrine disruption, if the underlying mechanism of these effects is known. To achieve this, knowledge about the hormonal regulation of processes is required. This includes knowledge about hormone metabolism and synthesis, hormone receptors and signaling pathways that are responsible for processes such as molting, reproduction, cellular metabolism and homeostasis in nematodes. Studies on nematodes suggest that this primordial organism group already regulates many processes via endocrine pathways. Various aspects of nematode endocrinology have been reviewed elsewhere (Davey 1988; Fleming 1997; Chitwood 1999; Spindler and Spindler-Barth 2000). Nuclear receptors Nematodes posses a high number of nuclear receptor (NR) genes: 270 in C. elegans, compared to 21 in Drosophila and approximately 50 in humans (Maglich et al. 2001). NRs are a large family of transcriptional regulators, members of which mediate hormonal signaling in vertebrates and insects (Mangelsdorf et al. 1995). Although the majority of nematode NRs belongs to the orphan class of NRs (without an identified ligand), there is evidence that sterol metabolites serve as ligands to NRs (Motola et al. 2006). The NR3 subfamily, which contains the vertebrate steroid receptors, is not present in nematodes. Still, little is known about the biological functions of NRs in nematodes. It is suggested that NRs of C. elegans are involved in the regulation of important developmental processes such as molting (Gissendanner et al. 2004), reproductive development (Antebi et al. 2000) and sex determination (Carmi et al. 1998).
Endocrine disruption in nematodes
Role of sterols Studies on the function of steroids in nematodes also indicate hormonal regulation. Nematodes (like insects) require exogenous dietary sterols for their development (Hieb and Rothstein 1968). While absolutely necessary for nematode development, sterols are required only in very low amounts (Matyash et al. 2004), suggesting that the primary role resides in signaling, rather than being structural components for cell membranes (Kurzchalia and Ward 2003). Mammalian sex steroids have been isolated from lipid extracts of parasitic (Lee et al. 1989), and free-living nematodes (Lee et al. 1990), although it was not proven, that the steroids originated from the nematodes, rather than from the host or medium components. Nematodes are able to metabolize sterols, although it was not possible until recently to identify a distinct hormonal function of the sterol metabolites (Chitwood 1999). Only recently Motola et al. (2006) were able to identify two 3-keto-steroids as ligands for a NR (DAF-12), that regulates dauer formation and reproduction in C. elegans (see below). Moreover, steroid/thyroid hormone receptor genes have been reported for C. elegans (Kostrouch et al. 2005), and the presence of estrogen-binding proteins was demonstrated for parasitic (Kiser et al. 1986) and free-living nematodes (Hood et al. 2000). A further study, that used vitellogenin as biomarker, the prototypical endpoint for estrogen response in all vertebrates (except mammals), showed a dose-dependent vitellogenin synthesis and the upregulation of vitellogenin genes (vit-2 and vit-6) in the presence of estrogens (Custodia et al. 2001). Regulation of molting First evidence of endocrine regulation in nematodes was the description of neurosecretory cells in nematodes (Gersch and Scheffel 1958; Davey 1966). For the parasitic species Phocanema decipiens, the function of neurosecretory cells could be related to endocrine regulation of molting (Davey and Kan 1968). The apparent similarity of the physiological and anatomical changes that occur in nematodes during their life cycle to the known endocrine control of superficially identical processes that occur in arthropods (the ecdysozoan scenario merges arthropods and nematodes in one phylogenetic clade of molting animals; Aguinaldo et al. 1997), stimulated scientists since the early 1970s to investigate the effects of arthropod hormones, such as juvenile hormones (JH) and ecdysone, on nematode growth, development and reproduction (Dropkin et al.
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1971). However, these relatively unspecific endpoints are not suitable to identify endocrine activity of these hormones in nematodes. Ecdysteroids were found in extracts of the nematode species, Panagrellus redivivus, Aphelenchus avenae and Haemonchus contortus (Dennis 1977), in eggs and embryos of Ascaris suum and H. contortus (Fleming 1987, 1993), and in the reproductive tract of A. suum and Dirofilaria immitis (Fleming 1985a; Cleator et al. 1987), while b-ecdysone-binding proteins could not be detected in these species (Dennis 1977). Davies and Fischer (1994) also detected ecdysteroids in A. avenae by using an insect pupation bioassay. The same authors could stimulate molting in A. avenae specimens, which were prevented from molting by ligating them, with externally applied 20-hydroxyecdysone, however, at very high concentrations (i.e., 2.0 mM). At present, there is no evidence that nematodes are able to convert sterols into ecdysteroids. For instance, C. elegans failed to produce ecdysteroids from radiolabeled cholesterol (Chitwood and Feldlaufer 1990). Although an endocrine trigger for nematode molting has not been identified up to now, there is evidence that steroid hormones, though probably not ecdysteroids, are involved in this process (Frand et al. 2005). First, molting of C. elegans requires cholesterol, the biosynthetic precursor of steroid hormones, as well as a protein (LRP-1), that might serve as a receptor for the sterol endocytosis from the growth medium (Yochem et al. 1999). Secondly, two nuclear hormone receptors, NHR-23 and NHR-25, orthologous, respectively, to the ecdysone-responsive gene products DHR3 and Ftz-F1 of Drosophila melanogaster, are required for molting and development in C. elegans (Gissendanner and Sluder 2000; Kostrouchova et al. 2001). Studies on the function of conserved NR genes, which are orthologs of the NR genes that function during molting and metamorphosis in insects (‘ecdysone cascade’ NRs), confirm their role during the continuous growth and dauer molts in C. elegans (Gissendanner et al. 2004). Regulation of reproductive development and life span The choice for reproductive development of C. elegans is regulated by the environment. Under favorable conditions, C. elegans develops through four larval stages (L1–L4) to reproductive hermaphrodites within 3 days, with a total life span of approximately three weeks. Under unfavorable conditions, particularly lack of food, C. elegans is able to choose an alternative development strategy, forming so called dauer larvae, which are sexual immature but
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tolerant to harsh conditions (Riddle and Albert 1997). The pheromone daumone, a fatty acid derivative, is a key regulator of these chemosensory processes (Schackwitz et al. 1996; Jeong et al. 2005). The underlying processes require organism-wide coordination, indicating complex endocrine regulation. Molecular, genetic and cellular analysis of dauer formation gene loci (daf) reveal a network of conserved neuroendocrine pathways. The discovery that daf-9 encodes for a cytochrome P450 (DAF-9; related to steroid metabolizing enzymes, Gerisch et al. 2001) and daf-12 for a nuclear hormone receptor (DAF-12, Antebi et al. 2000) provides further evidence for hormonal signaling by lipophilic molecules in C. elegans. DAF-9 is regulated nonautonomously by environmental signals via insulin/IGF-1 and TGFb signaling pathways (Ren et al. 1996; Schackwitz et al. 1996; Kimura et al. 1997). It functions as a steroid metabolizing enzyme (3-keto-sterol-26-monooxygenase) that catalyzes the production of ligands (3-keto-cholestenoic acids) for DAF-12, a NR that regulates dauer formation and reproduction (Motola et al. 2006). There is evidence that the regulation of reproductive development is governed by hormones in multiple steps, as also other NRs were shown to be involved in hormone production. As an example, DAF-36, a Rieske-like oxygenase, is expressed primarily within the intestine and works early in the hormone biosynthetic pathway (Rottiers et al. 2006). Matyash et al. (2004) demonstrated that a sterolderived hormone (called gamravali: molecular formula not identified yet) regulates the decision to enter diapause. Gamravali derives from cholesterol and promotes reproduction and prevents dauer larvae formation by inhibiting the NR DAF-12. Lee et al. (2005) could show that a cholesterol producing transgenic C. elegans lived longer than the wild-type strain, due to enhanced stress resistance. However, it was not clear if the extended life span was a result of a biophysical interaction in membranes or of endocrine regulation through cholesterol metabolites.
Endocrine disruption in nematodes Although the role of hormones in nematodes is not completely elucidated, it is likely that endocrine disruption through xenobiotics can occur. This section gives an overview of studies on endocrine effects of xenobiotics on nematodes, including studies on effects of known vertebrate or arthropod EDCs, that match typical symptoms of endocrine disruption (see also Table 1).
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Arthropod hormones Indications of endocrine disruption were first found for parasitic nematodes. In Trichinella spiralis molting was inhibited by the insect JH mimics farnesol and farnesyl ethyl ether, indicating an inhibition of an endocrine mechanism that governs nematode development (Meerovitch 1965; Shanta and Meerovitch 1970). Furthermore, JH and its analogs inhibited molting (Davey 1971; Gibb and Fisher 1989) and egg hatching (Rogers 1978) in certain stages of parasitic nematodes. For sugarbeet nematode larvae, Heterodera schachtii, hypodermal and gonadal growth abnormalities were induced in the presence of a steroid androgen and insect JH mimics (Johnson and Viglierchio 1970). The JH antagonist precocene II had lethal effects on the larvae and adults of two parasitic nematodes (Spindler et al. 1986). For the parasitic nematode, D. immitis, Warbrick et al. (1993) found a stimulation of premature molting in the presence of 20-hydroxyecdysone, while molting was significantly inhibited by the ecdysone agonist azadirachtin. Also for free-living nematodes (Caenorhabditis briggsae), effects of JH analogs could be observed (Hansen and Buecher 1971). In contrast, effects of mammalian and insect hormones on the free-living nematodes C. elegans and P. redivivus showed no endocrine specificity, as effects on growth and development occurred at rather high concentrations (most effective compound at 10 lM) and no correlation between molecular structure and growth inhibition was found (Dropkin et al. 1971). Fodor et al. (1982) found distinct effects of the JH antagonist precocene II on C. elegans, resulting in mortality and miniaturized adults, with effects being more pronounced when exposure took place during larval development (Fig. 1). The finding that these effects could be partly compensated by the JH analog methoprene (Fig. 1), led the authors to assume that JH play an important role in nematode development, which can be disrupted by antagonists, such as precocene II. These results could be confirmed by a study using Caenorhabditis remanei and various precocene analogs (Fodor and Timar 1989; Fodor et al. 1989). Only the effect of the precocene analogs with a precocene-like activity in nematodes or an antiallatal activity in insects could be compensated for via simultaneous exposure to the JH analog methoprene. Disruption of hormone metabolism It is known, that substances such as azasteroids and structurally related long-chain alkylamines are able to disrupt sterol metabolism in nematodes (Chitwood
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Table 1 Effects of potential EDCs on free-living nematodes (Caenorhabditis elegans, unless otherwise noted); (›) = increase; (fl) = decrease; (0) = no effect Chemical
Tested concentrations (M)
Studied parameter
Reference
10–10–10–6 0.510–6–510–6 10–10–10–6 10–8–10–7 10–10–10–6 10–9–10–5 310–9–210–7 a 10–8–10–7 210–10–210–7
Growth (fl); reproduction (0) Fecundity (fl) Growth (0); reproduction (0) Reproduction (fl); males (›) Germ cell number (fl) Vitellogenin expression (fl) Growth (›)d Reproduction (fl) Growth (›); reproduction (fl)
Weltje et al. (2003) Tominaga et al. (2003b) Weltje et al. (2003) Tominaga et al. (2002) Hoshi et al. (2003) Custodia et al. (2001) Thong and Webster (1971) Tominaga et al. (2002) Ho¨ss et al. (2001)
10–10–10–6 10–10–10–6 Not indicated
Growth (fl); reproduction (›) Growth (fl); reproduction (0) Vitellogenin mRNA (›)
Weltje et al. (2003) Weltje et al. (2003) Kohra et al. (1999)
Toxaphene
10–10–10–6 10–10–10–5 10–9–10–7 Not indicated 10–6–10–3 2.510–10 0.510–6–510–6 510–6–710–5 210–6 10–9–10–5 510–7–510–6 10–11–10–6 10–7 410–9–210–7 a 10–7–10–3 10–10–10–6 210–8–210–6 210–7 –210–6 10–10–10–6 210–8–210–6 10–9–10–5 10–7 810–5–810–4 2.510–10
Growth (0); reproduction (›) Germ cell number (›) Fecundity (fl); egg hatching (fl) Vitellogenin mRNA (›) Gene expression (›/fl) Estrogen binding (fl); fecundity (fl)e Fecundity (fl) Fecundity (fl); nuclear aberrations Gene expression (›/fl) Vitellogenin expression (›) Fecundity (fl) Germ cell number (›) Gene expression (›/fl) Growth (›)d Growth (›); reproduction (fl) Growth (0); reproduction (0) Reproduction (fl); vulva abnormalities Growth (›); reproduction (›) Growth (fl); reproduction (›) Reproduction (fl); vulva abnormalities Vitellogenin expression (›) Gene expression (›/fl) Growth (fl) Estrogen binding (fl); fecundity (fl)e
Weltje et al. (2003) Hoshi et al. (2003) Tominaga et al. (2003a) Kohra et al. (1999) Novillo et al. (2005) Hood et al. (2000) Tominaga et al. (2003b) Goldstein (1986) Reichert and Menzel (2005) Custodia et al. (2001) Tominaga et al. (2003b) Hoshi et al. (2003) Novillo et al. (2005) Thong and Webster (1971) Ho¨ss et al. (2001) Weltje et al. (2003) Tominaga et al. (2002) Ho¨ss et al. (2002) Weltje et al. (2003) Tominaga et al. (2002) Custodia et al. (2001) Novillo et al. (2005) Dropkin et al. (1971) Hood et al. (2000)
Antiestrogen Tamoxifen
10–10–10–6
Reproduction (›); growth (fl)
Weltje et al. (2003)
10–5–510–3 10–5–510–3 10–5–410–3 310–6–310–4 710–5
Motility (fl), population growth (fl) Motility (fl), population growth (fl) Motility (fl); population growth (fl) Motility (fl); reproduction (fl) Growth (fl); egg deposition (fl)f
Lozano et al. (1984) Lozano et al. (1984) Lozano et al. (1984) Chitwood et al. (1984) Bottjer et al. (1985)
10–9–10–5
Vitellogenin expression (›)
Custodia et al. (2001)
510–3 10–10–10–6 910–5–910–4 210–4–710–4
Egg hatching (fl)g Growth (0); reproduction (›) Growth; reproduction (›) Reproduction (fl)f
Rogers (1978) Weltje et al. (2003) Dropkin et al. (1971) Hansen and Buecher (1971)
Growth (›/fl); reproduction (›) Survival (fl); growth (fl) Survival (fl); growth (fl)h
Weltje et al. (2003) Fodor et al. (1982) Fodor et al. (1989)
Androgen Fenarimol 17a-Methyltestosterone Tributyltin Testosterone Triphenyltin Trenbolone Antiandrogen Cyproterone acetate Vinclozolin Estrogen Benzylbutylphthalate Bisphenol A
Cadmium Dieldrin Diethylstilbestrol
17b-Estradiol
17a-Ethinylestradiol p-Nonylphenol n-Octylphenol Progesterone
Disruptor of sterol metabolism Alkylamide (C12) Alkylamines (C12–C16) Alkylphosphonate (C12) 25–Azacoprostane Hormone precursor Cholesterol JH analog Dichlorofarnesoate Methoprene Various JH analogs JH antagonist Precocene II Precocene analogs
b b
10–10–10–6 4.510–4–4.510–3 1.510–4–4.510–3
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20 Table 1 continued Chemical Molting hormone analog 20–hydroxyecdysone Molting hormone antagonist Luteolin
Tested concentrations (M)
Studied parameter
Reference
210–7–210–3 10–10–10–6
molting (›)g Growth (0); reproduction (›)
Davies and Fischer (1994) Weltje et al. (2003)
10–10–10–6
Growth (fl); reproduction (›)
Weltje et al. (2003)
Growth (fl), reproduction (fl)
Dropkin et al. (1971)
Mammalian sex hormone analogs Various steroids 810–5–810–4 a
c
mol/petri dish
b
assuming a mean molecular weight of 280 g/mol
c
assuming a mean molecular weight of 300 g/mol
d
Cephalobus sp.
e
Panagrellus redivivus
f
Caenorhabditis briggsae
g
Aphelenchus avenae
h
Caenorhabditis remanei
1999). 25-Azacoprostane hydrochloride inhibited delta 24-sterol reductase in C. elegans, resulting in an accumulation of delta 24-sterols (96% of the total nematode sterol). In inhibitor-untreated nematodes these sterols could only be detected in trace quantities, while 7-dehydrocholesterol, cholesterol and lathosterol were found predominantly (Chitwood et al. 1984). The same azasteroid was shown to strongly inhibit growth, reproduction and egg deposition in C. elegans and Fig. 1 Mortality rate (upper graphs) and body length (lower graphs) of Caenorhabditis elegans exposed to different concentrations of the juvenile hormone (JH) antagonist precocene II in absence and presence of the JH analog methoprene on nematode growth medium agar plates; exposure started at the stage that is indicated (adults; L3: third larval stage, 24 h after hatching; L2: second larval stage, 14 h after hatching); data from Fodor et al. (1982)
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C. briggsae, but not in P. redivivus (Chitwood et al. 1984; Bottjer et al. 1985). Similar effects on delta 24sterol reductase, as well as on nematode growth and reproduction, occurred in the presence of the structurally simpler aliphatic amines, but to a lesser extent than in the presence of 25-azacoprostane (Lozano et al. 1984). As these inhibitors are also able to affect ecdysteroid metabolism in insects (Svoboda et al. 1972), similar enzymes which regulate steroid metab-
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olism in nematodes may be affected by these compounds, and induce changes in nematode growth and reproduction. Vitellogenin expression The expression of vitellogenin (yolk protein) is a typical endpoint for estrogen response in nonmammalian vertebrates. Although the role of vitellogenin in nematodes is not identical to that in vertebrates, it is involved in the cholesterol transport to the oocyte (Matyash et al. 2001), similar to the function of the low density lipoprotein pathway in vertebrates. It could be shown that the expression of yolk protein can be enhanced in the presence of exogenous cholesterol and progesterone (Custodia et al. 2001). The same study showed a decrease of the expression of two yolk proteins (YP-88, YP-170) at low concentrations of 17b-estradiol (E2) and testosterone (1.0 nM), and a dose-dependent increase of these yolk proteins at higher E2 concentrations (0.1 and 10 lM; Fig. 2). DNA microarray analysis revealed that vitellogenin genes (vit) were upregulated in the presence of E2 (10 lM) and progesterone (0.1 lM). Similarly, vitellogenin mRNA levels increased dose-dependently in the presence of E2, and also the vertebrate endocrine disruptors bisphenol A (BPA, estrogen) and vinclozolin (antiandrogen) enhanced vitellogenin mRNA levels (Kohra et al. 1999).
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an affinity for estrogen-binding sites of these chemicals in P. redivivus. Accompanying studies on effects on nematode growth and fecundity showed that fecundity was inhibited by these chemicals (25 nM), while growth was not affected. Similar results were found for the parasitic nematode Nippostrongylus brasiliensis. Binding of radiolabeled testosterone, progesterone and E2 was inhibited by respective competitive hormone analogs (Majundar et al. 1987). Moreover, these authors found partial inhibition of in vivo development of N. brasiliensis by selected steroid analogs.
Effects of potential EDCs on nematodes A number of studies, including laboratory single species tests, as well as microcosm community studies, investigated effects of vertebrate or arthropod EDCs on nematodes, without proving a mechanism of endocrine disruption behind them. Such identified potential risks of suspected EDCs for nematodes may prove to be ecologically relevant. Below, these empirical studies are discussed on basis of the evidence for endocrine disruption from studies with a more mechanistic approach (discussed above). Effects on single species
Using a radioimmunoassay, Hood et al. (2000) investigated the effects of vertebrate xeno-estrogens, such as nonylphenol, dieldrin and toxaphene, on E2 binding by homogenates of the nematode P. redivivus, which were thought to contain estrogen-binding proteins. Dieldrin, toxaphene and a mixture of dieldrin and nonylphenol (25 nM) significantly inhibited E2 binding, indicating
Hoshi et al. (2003) counted the number of germ cells in the uterus of C. elegans after exposure to E2, BPA and tributyltin (TBT). While in the presence of the estrogenic compounds E2 and BPA the number of germ cells increased significantly at concentrations as low as 0.1 and 1.0 nM, significantly lower numbers of germ cells were observed after exposure to 1.0 nM of the androgenic compound TBT (Fig. 3). Although this endpoint is not specific for endocrine disruption, it is able to show direct effects on reproductive organs, which might be controlled by hormones. Expression of genes that are
Fig. 2 Relative band intensity of the Caenorhabditis elegans yolk proteins YP-88 (left) and YP-170 (right), obtained with Western blot analysis after exposure to 0.001, 0.1 and 10 lM estradiol
(E2). Asterisks indicate significant difference from the control (ANOVA, Newman–Keuls, P < 0.01); reprinted from Custodia et al. (2001) with kind permission of Blackwell Publishing
Inhibition of estrogen binding
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involved in germ cell development (mes-4; Garvin et al. 1998), can be upregulated by cholesterol at a concentration of 1.0 nM (Novillo et al. 2005). Own studies on the effects of several vertebrate and arthropod EDCs on growth and reproduction of C. elegans revealed interesting results, as effects of some substances on nematode reproduction were stimulating, while effects on growth were mostly inhibitory (Figs. 4, 5, 6; Weltje et al. 2003). Stimulating effects on the reproduction of C. elegans occurred in the presence of the xeno-estrogen benzylbutylphthalate and, more obviously, with 4-n-octylphenol (Fig. 4). Reproduction stimulation was also seen in experiments with the antiestrogen tamoxifen, the antiandrogen cyproterone acetate (Fig. 5) and four different insect hormone analogs or antagonists (methoprene: JH analog; precocene II: JH antagonist; 20-hydroxyecdysone: ecdysone analog; luteolin: ecdysone antagonist/anties-
Fig. 3 The relative percentage of germ cells (RPG, mean ± standard deviation; n = 5) of Caenorhabditis elegans exposed for 6 days to 17b-estradiol (A), bisphenol A (B) and tributyltin chloride (C) on agar plates with E. coli OP50 as food source; control: solvent control (ethanol: 0.3% v/v); RPG in control worms was taken as 100%; double asterisks indicate significant difference from the control (ANOVA, Dunnett’s test, P < 0.01); reprinted from Hoshi et al. (2003) with kind permission of the Japanese Society of Veterinary Science
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trogen; Fig. 6). Often this stimulation of reproduction was observed at lower concentrations (0.1–100 nM) and disappeared at higher concentrations (10– 1,000 nM). Nematode growth was inhibited when exposed to the estrogen 4-n-octylphenol, the antiestrogen tamoxifen, the androgen fenarimol, and the antiandrogens vinclozolin and cyproterone acetate (Figs. 4, 5). Effects of the insect hormone antagonists precocene II and luteolin, which also acts as an antiestrogen, were more pronounced (Fig. 6). Luteolin inhibited nematode growth at concentrations of 0.1–10 nM, an effect that disappeared at higher concentrations. The JH antagonist precocene II stimulated growth at low concentrations (0.1 and 1.0 nM), inhibited growth at medium concentrations (10 nM) and had no effect at higher concentrations (100 and 1,000 nM). Enhancement of reproduction is not a typical response to a toxic substance. Thus, mechanisms, such as endocrine activity, may have caused the observed effects in C. elegans. Acute effects of estrogens on the survival of C. elegans (LC50 for E2 >1 mM, for BPA >1 mM, for nonylphenol 33 lM; Ura et al. 2002) lie several orders of magnitude higher than the concentration levels for sublethal effects (Weltje et al. 2003). A study with the estrogenic compound 4-nonylphenol also showed a concentration-dependent stimulation of reproduction and growth in C. elegans, with first effects at 0.2 and 0.3 lM, respectively (Ho¨ss et al. 2002). Tominaga et al. (2002) also found effects of alkylphenols on the reproduction of C. elegans, however, nonylphenol and octylphenol significantly reduced nematode reproduction at concentrations of 0.02–2.0 lM. For the freeliving nematode Cephalobus sp. an increase in growth was observed in the presence of E2 (Thong and Webster 1971). These ambiguous results do not allow a mechanistic interpretation. Also, hormesis (a stimulating effect of toxic substances at low concentrations; Calabrese et al. 1987) might be an explanation for the stimulation of growth and reproduction. The effects of the estrogen 17a-ethinylestradiol (EE2) on C. elegans are ambiguous as well. While EE2 inhibited reproduction and stimulated growth of the nematodes at concentrations of 0.8 and 1.7 nM in an earlier study (Ho¨ss et al. 2001), we did not observe an effect of EE2 in the later study (Fig. 4; 0.1–1,000 nM). The androgens 17a-methyltestosterone and fenarimol did not show an effect up to 1.0 lM (Weltje et al. 2003), but in the study of Tominaga et al. (2003b) a concentration of 5.0 lM methyltestosterone significantly reduced nematode fecundity. The androgen trenbolone showed inhibiting effects on reproduction (0.2 nM) and stimulating effects on growth (20 nM) of
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Fig. 4 Number of offspring per test organism (reproduction) and body length (lm) of Caenorhabditis elegans after 96 h aqueous exposure to estrogens (EE2 17a-ethinylestradiol, BBP benzylbutylphthalate, OP 4-n-octylphenol) and an antiestrogen (TAM tamoxifen) at nominal concentrations of 0.1–1,000 nM;
control: solvent control (1% ethanol in M9-buffer), asterisks indicate significant difference from the control (ANOVA, Dunnett’s test, P < 0.05); tests were largely carried out according to Ho¨ss et al. (2002)
Fig. 5 Number of offspring per test organism (reproduction) and body length (lm) of Caenorhabditis elegans after 96 h aqueous exposure to androgens (TES 17a-methyltestosterone, FEN fenarimol) and antiandrogens (VIN vinclozolin, CPA cyproterone acetate) at nominal concentrations of 0.1–1,000 nM;
control: solvent control (1% ethanol in M9-buffer), asterisks indicate significant difference from the control (ANOVA, Dunnett’s test, P < 0.05); tests were largely carried out according to Ho¨ss et al. (2002)
C. elegans (Ho¨ss et al. 2001). Testosterone was shown to increase the growth of the free-living species Cephalobus sp. (Thong and Webster 1971) and reproduction of the parasitic species N. brasiliensis (Swanson et al. 1984). Both antiandrogens vinclozolin and cyproterone acetate inhibited nematode growth (Fig. 5), but only cyproterone acetate stimulated
reproduction. The potential of androgens (e.g., testosterone) to decrease vitellogenin expression (Custodia et al. 2001) and of antiandrogens (e.g., vinclozolin) to increase vitellogenin mRNA levels (Kohra et al. 1999) suggests that the observed effects of androgenic and antiandrogenic substances on nematode growth and reproduction could be related to endocrine activity.
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Fig. 6 Number of offspring per test organism (reproduction) and body length (lm) of Caenorhabditis elegans after 96 h aqueous exposure to a juvenile hormone (JH) analog (MET methoprene), a JH antagonist (PRE precocene II), an ecdysone analog (ECDY 20-hydroxyecdysone) and an ecdysone antagonist/antiestrogen
(LUT luteolin) at nominal concentrations of 0.1–1,000 nM; control: solvent control (1% ethanol in M9-buffer), asterisks indicate significant difference from the control (ANOVA, Dunnett’s test, P < 0.05); tests were largely carried out according to Ho¨ss et al. (2002)
Regarding the effects of insect hormone analogs and antagonists, it is obvious that only the antagonists precocene II (JH antagonist) and luteolin (ecdysone antagonist) inhibited growth of C. elegans (Fig. 6). The growth inhibiting potential of precocene II was already described by Fodor et al. (1982, 1989). They found an interaction between the JH antagonist and the JH analog methoprene, as methoprene was able to partly antagonize the inhibiting effect of precocene II, indicating JH disruption by precocene II (Fig. 1). The arthropod hormone 20-hydroxyecdysone and the JH analog methoprene showed a stimulating effect on reproduction of C. elegans, already at very low concentrations (1.0 and 0.1 mM, respectively; Fig. 6). However, the stimulating effect disappeared or decreased at higher concentrations (Fig. 6). Fleming (1985b) observed a similar dose–response curve for effects of 20-hydroxyecdysone on growth of the parasitic species A. suum (stimulation at 0.1 nM; maximal effect at 10 nM).
ment of tolerance to a chemical stressor in organisms that comprise multiple generations within the exposure period can be taken into account. Of course, investigation on higher ecological levels cannot distinguish between specific modes of action, such as endocrine disruption, cyto- or genotoxicity. This information must be provided by specialized assays. However, only through the combination of studies on molecular or single species level with higher-tier studies in model ecosystems or in the field, the risk of chemicals, including EDCs, for aquatic ecosystems can be adequately estimated. Only few studies exist that investigated the effects of EDCs on aquatic nematode communities. Austen and McEvoy (1997) found effects of the androgenic chemical TBT on nematode species composition in microcosms that had been filled with natural sediments from different locations. First effects occurred at nominal sediment concentrations of 0.73 mg TBT/kg dry sediment. Effects were more pronounced in a sandy sediment compared to muddy sediments, which was explained by the higher TBT bioavailability in the sandy sediment. Moreover, effects could only be detected by multivariate techniques, such as multidimensional scaling ordination, and not by using univariate measures, such as total abundance or number of species. In a more recent study, Schratzberger et al. (2002) confirmed the potential of TBT to disturb estuarine nematode communities. The results showed that the response of the nematode species depended
Effects on nematode community structure Studies on the effects of suspected EDCs on a community level are important, as they consider more realistic conditions than laboratory assays on a molecular or single species level do. Studies in model ecosystems, micro- or mesocosms, integrate interactions between trophic levels, thus also consider indirect effects of chemicals. Moreover, the develop-
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not only on the level of TBT contamination (1.0 and 10 mg TBT/kg dry sediment) but also on the exposure duration (4 and 8 weeks) and the sediment spiking method. A microcosm study on freshwater nematode communities showed that the estrogenic chemical 4-nonylphenol was able to alter the species composition as well (Ho¨ss et al. 2004). Nonylphenol was applied to seven microcosms over a period of 6 weeks, reaching maximal sediment concentrations of 0.30–3.37 mg/kg dry weight. Nematode community structure in those treatments was compared to four controls over a period of 15 weeks. Species composition was analyzed using a multivariate method (Principle Response Curves, PRC; van den Brink and ter Braak 1999). The PRC analysis showed a nonylphenol-induced change in the species composition over a period of 7 weeks, from the end of the application until the end of the experiment. Also, the species distribution over the different feeding types and life history strategists was affected in the highest dosed treatment. However, these effects occurred only within the last three weeks of the study. Nematode abundance and diversity indices were not affected by nonylphenol throughout the entire experiment.
Conclusion and future prospects The studies discussed in this paper show that EDCs, defined as such for vertebrate and arthropod systems, can affect nematodes on all organizational levels, from molecules to communities. While molecular (i.e., genome and proteome) studies on nematodes indicate that various chemicals are able to disrupt endocrine mediated processes in nematodes, studies on single species, populations and communities show that these effects can become ecologically relevant for these organisms. Nematodes are important members of aquatic and terrestrial food webs. Thus, an impairment of this organism group is likely to have consequences for the functioning of the whole ecosystem. The nematode C. elegans is an excellent model organism for research on and assessment of endocrine disruption. The comprehensive knowledge of the genome of C. elegans allows for the development of new methods to detect disruptions of specific functions, such as hormonal regulation. The combination of tests on different organizational levels, such as genome, proteome, organ, organism and population level, allows to assess endocrine disruption on the basis of specific modes of action and ecologically relevant parameters. The use of specific gene chips
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enables the detection of endocrine disruption on a genomic level (Novillo et al. 2005; Reichert and Menzel 2005). The information that certain genes, which are involved in hormonal regulation are up- or downregulated can be a good indicator for the bioavailability of EDCs. Whether however, the genomic response is translated into a relevant organismic function, such as growth or reproduction, can only be assessed by additional whole organism bioassays. As a genomic response is usually detected much earlier than effects on growth or reproduction of the organism, these chips might also function as an early warning system. As C. elegans can be held in sediments, soils and aqueous media, endocrine disruption can be assessed in environmental samples with unknown quantity and quality of pollution (e.g., sediments, soils, solid and aqueous waste, pore water, elutriates, extracts). Moreover, specially designed mutant (knock-out) strains, with specific dysfunctional genes and therefore lacking certain functions, may be used as targeted bioindicators (Watanabe et al. 2005). To increase our understanding of causal relationships between effects on nematodes at the molecular level, which may identify endocrine disruption, and those on whole organisms and communities, a greater scientific effort is necessary. All in all, nematodes possess excellent qualifications as bioindicators for a holistic approach to assess endocrine disruption in various types of ecosystems.
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