Hum. Reprod. Advance Access published online on September 11, 2007
Human Reproduction, doi:10.1093/humrep/dem256
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Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations
1 Department of Obstetrics and Gynaecology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK 2 Department of Medical Genetics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK 3 Medical Microbiology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK 4 School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, UK 5 Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111, USA 6 School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, UK
7 Correspondence address. Tel: +44 1224 552633; Fax: +44 1224 684880; E-mail: p.a.fowler{at}abdn.ac.uk
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Abstract |
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BACKGROUND: Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis.
METHODS: Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10–1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated.
RESULTS: Exposure to dieldrin reduced LH-induced testosterone secretion (P < 0.05) and tissue protein concentrations of LH receptor and steroid acute regulatory protein (P < 0.05). Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals.
CONCLUSIONS: Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.
Key words: human/fetal/testis/pesticide/endocrine disruption
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Introduction |
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There has been considerable interest in apparently rising human reproductive defects, especially in the male, as a result of exposure to environmental chemicals, including endocrine disrupting compounds (Safe, 2004
; Sharpe and Irvine, 2004; Vidaeff and Sever, 2005; Maffini et al., 2006; Skakkebaek et al., 2006). Male reproductive dysfunction has been characterized as (i) increased testicular cancer rates, (ii) increased rates of urinogenital malformation, including cryptorchidism and hypospadias and (iii) falling sperm counts. These symptoms have been linked together as testicular dysgenesis syndrome and this is thought to originate during fetal and/or neonatal life (Skakkebaek et al., 2001). Although environment and lifestyle are key determinants in many human diseases, links between specific chemicals and human testis dysgenesis syndrome are not directly proven (Hughes, 2001; Vidaeff and Sever, 2005; Sharpe, 2006). Nevertheless, the hypothesis is well supported by associative data (Sharpe and Irvine, 2004). In contrast, the animal data is generally persuasive (Sweeney, 2002; Paul et al., 2005), but species differences in gestational ontogeny (timing, duration of developmental events) and metabolism indicate that it is important to strengthen the human database to enable a rational approach to this field.
The widely used insecticide dieldrin, the major metabolite of aldrin, is a well-known endocrine disrupting compound and is a persistent organic pollutant since it is extremely toxic, difficult to metabolize and poorly biodegradable, making it environmentally persistent. Although both compounds have been widely restricted or banned since the 1970s, they remain at around 8 p.p.b. (20 nM or 8 µg/l) in human blood, adipose and other tissues (Jorgenson, 2001; Poon et al., 2005). Dieldrin has anti-androgenic effects (Andersen et al., 2002) and activates extracellular-regulated kinases in cells expressing estrogen receptors (ER) (Bulayeva and Watson, 2004). In addition, dieldrin increases ER
expression in the presence of estradiol (E2) (Grunfeld and Bonefeld-Jorgensen, 2004), amplifying its estrogenic effects. Dieldrin also stimulates apoptosis by inducing mitochondrial depolarization and caspase-3 expression (Kitazawa et al., 2003). We therefore selected dieldrin as a model endocrine disrupting compound because it accumulates in humans (being widely detectable in adipose tissue and serum), demonstrates both oestrogenic and anti-androgenic effects, has the ability to increase ER expression and activates a wide range of intracellular mechanisms. This is typical of many endocrine disrupting compounds (Tabb and Blumberg, 2006).
One major problem with investigations of endocrine disruption revolves around the doses of chemicals to be employed, and below we detail the rationale for the doses of dieldrin we used. A recent study reported women’s adipose dieldrin concentrations 14 times higher than in their blood (1.21 p.p.b. or 3.025 nM or 1.21 µg/l serum) (Botella et al., 2004), reflecting its lipophilic nature and bio-accumulation through the food chain. Since significant maternal fat mobilization occurs during the third trimester of pregnancy and lactation [dieldrin concentrations in breast milk are around 50 p.p.b. (125 nM or 50 µg/l] (Harris et al., 1999)), the human fetus and neonate are potentially exposed to the mother’s lifetime legacy of lipophilic contaminants during the second developmentally critical period of elevated testosterone around 2 months post-partum. A complicating factor is variation in exposure levels in non-adipose tissues and the extent of endocrine disrupting compound transmission from mother to fetus. For example, testicular organochlorine accumulation is much lower than in liver or epididymis, at only 1–5% of levels seen in adipose tissue (Cooke et al., 2001). Therefore, the developmental impact of endocrine disrupting compounds on the fetus may be influenced by storage capacities of specific fetal organs. Although matched persistent organic pollutant data is almost non-existent for the human mother and fetus, we can extrapolate from data separately published by Botella et al. (2004), Cooke et al. (2001) and others. Given that adipose tissue in women contains up to 40 nM dieldrin (17 p.p.b./17 µg/l), if the testes contained 5% of this, testicular cells would be exposed to up to 2 nM dieldrin (0.85 p.p.b./0.85 µg/l). Thus, a reasonable estimate of the dieldrin content of the second trimester human fetal testis, prior to fat mobilization, would be 1 pM–5 nM. We therefore selected two dieldrin doses: 1 nM (10–9 M) and 1 pM (10–12 M) dieldrin (equivalent to 0.3809 µg/l or 0.4 p.p.b. and 380.9 pg/l or 0.0004 p.p.b., respectively), which bracketed the lower end of the range of dieldrin concentrations measured in the environment, food and peripheral blood of women and fell within estimated concentrations in the fetal testis.
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Subjects and Methods |
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Tissue collection and culture
Informed consent was obtained from women undergoing medical termination [using a standard combined mifepristone and misoprostol protocol (Ashok et al., 2004
)] of normally progressing pregnancies. Gestational stage was accurately established by the measurement of crown-rump length at the time of ultrasound investigation prior to terminations as part of routine clinical practice. Morphologically normal fetuses were collected between 13 and 19 weeks of gestation (Grampian Health Board and University of Aberdeen Joint Ethical Committee, Project No 2470, D.R.A.) (Murray et al., 2000). Testes were cut into explants <0.5 mm2 and transferred into 24-well culture plates (one explant/well) containing 500 µl of DME/F12 (Sigma-Aldrich Company Ltd, Poole, Dorset, UK) supplemented with 10% human adult male serum, 17 mM NaCl, 5.0 µg insulin/ml and 20 µg gentamycin and penstrep/ml. Explants were incubated at 37°C in 5% CO2 with gentle shaking to aid diffusion of waste gases. Blood and released proteins were removed by 30 min shaking in serum-free DME/F12. Explants were then incubated with supplemented DME/F12 for 4 h; this served as a baseline for hormone production allowing normalization of explant secretory capacity (see Statistical analysis section) prior to the addition of dieldrin/gonadotrophin(s). This compensated for the fact that Leydig cells are not uniformly distributed in the testis, which normalization against explant weight would not have adjusted for. As fetuses became available at irregular intervals, numbers were built up as follows: for each experiment, the testes were dissected free of connective tissue and epididymis and sliced into between 24 and 48 explants, depending upon testis size. For each experiment, each treatment condition was applied to between two and four explants (up to four wells/treatment/fetus).
Optimization of culture duration
In order to test the effects of dieldrin on the human fetal testis, an in vitro model that retained cell inter-connectivity was required. The determination of optimal culture duration was based on the immunohistochemical detection of critical markers of testis development and function. Testis explants from four fetuses were cultured in supplemented DME/F12 for 0 h (ex vivo control), 2, 8, 24 and 48 h prior to fixation in Bouin’s (Sigma-Aldrich Company Ltd) and standard processing into wax.
Determination of maximally steroidogenic gonadotrophin doses
In the fetal and neonatal human (unlike the fetal rodent), gonadotrophin-dependent testosterone secretion is an essential component of developmental signalling. Therefore, it was critical to establish optimum treatments to stimulate testosterone secretion in vitro. Testis explants from nine fetuses were cultured for 24 h with 0–1000 IU LH/l (AFP4395A, NHPP, <0.001% FSH), both alone and combined with either 1900 IU recombinant human FSH/l (NHPP) or 12 µg GnRH/l (Intervet UK Ltd, Cambridge, Cambs, UK).
Investigation of the effects of dieldrin
In order to establish likely ‘real-life’ effects of dieldrin, two doses of this pesticide were used which encompassed the high and low limits of likely circulating concentrations of dieldrin in women. In addition, because it was not known whether high doses of LH might overcome any effects of dieldrin, a range of LH doses were used. Therefore, testis explants from 10 fetuses were cultured for 24 h with 0–1000 IU LH/l (AFP4395A, NHPP, <0.001% FSH), both with and without 10–9 or 10–12 M dieldrin (equivalent to 0.3809 µg/l or 0.4 p.p.b. and 380.9 pg/l or 0.0004 p.p.b., respectively) (Sigma-Aldrich Company Ltd). A stock solution of dieldrin at 10–3 M in dimethylsulphoxide (DMSO) was used to prepare the treatments. Either the dieldrin in DMSO or DMSO alone were diluted with culture medium as appropriate to ensure that all wells received the same total concentration of DMSO (10–6 M) to control for vehicle effects. The testis explants were then divided between fixation for immunostaining or snap freezing for proteomic analysis.
Immunolocalization of marker proteins
Testis explants were Bouins fixed and processed into wax. Sections (5 µm) were used for the immunolocalization [see Murray et al., (2000) for detailed methods] of: proliferation cell nuclear antigen (PCNA: Novocastra Laboratories Ltd, Newcastle), proto-oncogene products (c-myc, bax: Santa Cruz Biotechnology Inc., Heidelberg, Germany, and bcl-2: DAKO Ltd, High Wycomb, UK), apoptosis [single stranded DNA (ssDNA): Alexis Biochemicals, Nottingham], androgen receptor (AR: Novocastra), the steroidogenic enzyme cytochrome P450c17 and the cholesterol transporter steroid acute regulatory protein (StAR) [supplied by Professor Ian Mason, University of Edinburgh). In addition, Wnt-2b (1:10 dilution, rabbit polyclonal: Zymed Laboratories, South San Francisco, CA, USA) was immunolocalized using the same methods. Non-specific mouse or rabbit immunoglobulin G (IgG) at the same concentrations as the primary antibodies served as negative controls. Immunostained sections were assessed by R.G.L. and T.J.M., independently, blinded to treatments, using six fields of view per slide and scored using an arbitrary scoring system (for either immunostaining or necrosis) of 0–3, where 0 = zero staining/no necrosis and 3 = intense staining/intense necrosis, normalized relative to explants receiving no treatments.
2-Dimensional gel electrophoresis and mass spectroscopy
Testis explants were pooled from the respective fetuses and replicates and sonicated at 100 mg wet weight:100 µl lysis buffer and proteomic analysis performed as previously described (Smith et al., 2005; Fowler et al., 2007). Briefly, 70 µg of protein from pooled testis explants were electrophoresed using pH 3–10 immobilized pH gradient strips, then 7 x 8 cm, 10–15% gradient, polyacrylamide gels and stained with Coomassie blue. Phoretix-2D software (NonLinear Dynamics Ltd, Newcastle-upon-Tyne, UK) was used to identify differentially expressed protein spots. Normalization was performed by expressing spot volumes as a percentage of total spot volume for each gel separately, reducing potential error from variations in gel loading. A subset of the most abundant, most differentially expressed, protein spots were processed for mass spectroscopy. MASCOT [http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2 & SEARCH=PMF] and MS-Fit [http://128.40.158.151/ucsfhtml3.4/msfit.htm] were used to interrogate The National Center for Biotechnology Information (NCBI) database, with the masses of the tryptic peptides, to identify the proteins.
1-Dimensional gel electrophoresis and western blot
Pooled testis explants were processed using PAGEprep kits (Perbio Science UK Limited, Tattenhall, Cheshire, UK), electrophoresed (15 µg/lane) and western blot performed as previously described (Lea et al., 2005). Primary antibodies were diluted in phosphate-buffered saline: (i) LH receptor (LHR) 1:200, (ii) -actin 1:5000 (AbCam Ltd., Cambridge, UK). Western blot images were analysed using Phoretix-1D (NonLinear Dynamics Ltd), which calculates band volumes. For each lane separately, band volumes of interest were normalized by division by band volumes for -actin in the same lanes in order to ensure that variations in protein loading did not skew the data. Values were then expressed relative to controls.
Hormone assays
At the end of each culture period, the media were collected and assayed for testosterone (the key endocrine signal for masculinization and marker for Leydig cell activity) and anti-Mullerian hormone (AMH, critical for regression of the Mullerian ducts and a marker of Sertoli cell activity). Testosterone: DELFIA kits (Perkin-Elmer Life Sciences, Cambridge, UK); detection limit of 0.3 nmol/l; intra- and inter-assay coefficients of variation 8.6 and 3.2%, respectively. AMH: a sandwich immunoassay (Al-Qahtani et al., 2005); detection limit 0.078 ng/ml; intra- and inter-assay coefficients of variation 4.0 and 3.6%, respectively. Where necessary, culture medium samples were diluted 1:10 with unused culture medium to bring values within the standard curves. Typically, the 2–3 mg explants secreted 10–30 nmol testosterone/l [similar to results for culture of adult human testis explants (Roulet et al., 2006)] and rose to 30–100 nmol testosterone/l following LH treatment. The use of the commercial assay with culture medium was checked by serially diluting both standards and samples in culture medium. Both continued to show good parallelism with the assay buffer-diluted standard curves.
Statistical analysis
In order to accurately quantify hormone output, the explants were cultured for 4 h and the medium collected prior to the addition of treatments for a baseline measure, fresh medium was added and explants were cultured for a further 24 h after which the medium was collected again. Hormone assays were performed on both sets of media and the values of the 24 h incubations were divided by the values for the 4 h incubations and then expressed as percentages of controls. This process resulted in hormone output being normalized to the secretory capacity of each explant, avoiding problems of accurately determining explant weights after culture and obviating issues with differential cell-type make-up of different explants cut from various parts of the testes. All endocrine data for each fetus were analysed separately, and the means for replicate wells then combined with the replicate means of the same treatments for other fetuses. Since the data were not normally distributed, differences between treatments were tested using two-way analysis of variance (ANOVA) and Bonferroni–Dunn post hoc tests following log-transformation of the data (Statview 5: abacus Concepts Inc., Berkley, CA, USA). Results are presented as mean ± SEM where appropriate.
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Results |
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Optimizing testis explant culture duration
Morphological analysis revealed that acceptable tissue structure was maintained after 24 h in culture (Fig. 1). The testis cords and interstitial areas were well preserved and the peritubular myoid cells (PMCs) were distinguishable surrounding the testis cords. PCNA immunopositive cells were found predominantly within the testis cords and immunoreactivity was also observed in the PMCs and in the interstitium (Fig. 1a). Bcl-2 remained specifically immunolocalized to the interstitial area and PMCs (Fig. 1b) and c-myc showed extensive immunoreactivity in the interstitial area (Fig. 1c). Immunoreactivity for the key steroidogenic enzyme, P450c17, was faint but consistently localized to the Leydig cells (Fig. 1d). The cultured tissues were also exposed to antibody for bax and AR, both of which exhibited the staining pattern previously reported for non-cultured tissues (Murray et al., 2000
: data not shown). Tissues cultured for 48 h showed deterioration in tissue morphology and disrupted immunostaining profiles for a majority of markers tested (data not shown). On the basis of these data, the culture period selected for subsequent experiments was 24 h.
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Optimizing gonadotrophin doses to stimulate testosterone secretion
Testosterone values, corrected for dilution during assay, typically ranged between 10 and 30 nmol/l depending on explant size and, possibly, the numbers of Leydig cells contained within the explants. Peak LH-stimulated testosterone levels were typically 30–100 nmol/l. Although LH administration was highly effective at stimulating testosterone secretion at 10–1000 IU/l (Fig. 2a), the addition of either FSH (Fig. 2b) or GnRH did not significantly potentiate testosterone secretion (Fig. 2c). On the basis of these data, doses of 10–1000 IU LH/l were selected to investigate the effects of dieldrin.
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Effects of dieldrin on testis explant endocrinology
Strikingly, none of the morphological indices, scoring for necrosis, or expression of immunohistological markers for proliferation (PCNA) and apoptosis (ssDNA: not shown, Bcl-2, Bax), showed any significant effects of in vitro exposure to dieldrin, indicating that it had no direct toxicological effects on the testis explants (data not shown). In cultures that included dieldrin-exposure, the control LH dose–response curve (Fig. 3a) was very similar to that seen in the gonadotrophin dose–response experiment (Fig. 2a), demonstrating a consistent physiological response in vitro. Only 100 IU LH/l, 10–9 M dieldrin significantly reduced LH-induced testosterone secretion at (Fig. 3a). Surprisingly, 10–12 M dieldrin significantly reduced testosterone secretion at 10, 100, 1000 IU LH/l (Fig. 3a). Neither dose of dieldrin significantly altered constitutive testosterone or AMH secretion (Fig. 3b), the latter also remaining unaffected by any dose of LH. This confirms the validity of using AMH production as a specific marker for Sertoli cell activity.
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Effects of dieldrin on testis explant proteome
LH administration altered the expression of 81 protein spots (27 increased
2-fold, 54 decreased 2-fold), but the two doses of dieldrin, in combination with LH, had quite different effects. The 10–12 M dose resulted in a larger number of spots down-regulated (62 decreased 2-fold, only 16 increased 2-fold) compared with the LH control, whereas the 10–9 M dose resulted in a larger number of spots showing up-regulation (76 increased 2-fold, only 19 decreased 2-fold). A subset of five of the more abundant spots showing treatment-specific alterations in expression, and three spots showing no change between treatments (Fig. 4) were positively identified by mass spectroscopic peptide mass mapping (Table 1). An interesting trend was the reversal of the effects of LH by co-administration of dieldrin with LH (e.g. Table 1). This would suggest that the Leydig cells were particularly sensitive to dieldrin.
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Effects of dieldrin on the Leydig cell
In order to investigate the apparent targeting of the Leydig cell by dieldrin, we determined the effects of dieldrin on StAR immunoexpression and observed an up to 30% decrease (Fig. 5a). Since LH-induced testosterone secretion was so dramatically reduced by dieldrin, LHR expression was determined by Western blot. Figure 5b illustrates that 10–12 M dieldrin combined with LH, reversed the LH-induced increase in LHR protein expression. However, dieldrin alone appeared to stimulate LHR expression by an, as yet undetermined, mechanism. In addition, one protein spot down-regulated by 10–12 M dieldrin in combination with LH, compared with LH alone (spot number 4, Table 1), was identified as Wnt-2b. To follow-up on this finding, Wnt-2b immunoreactivity was localized in two normal 14 week gestation human fetal testes. We found that Wnt-2b expression was predominately located to the Leydig and Sertoli cells (Fig. 5c). Some cells, including Leydig cells, showed areas of very intense, almost granular, staining. Interestingly, both the PMCs and germ cells were Wnt-2b negative.
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Discussion |
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Our findings demonstrate that the fetal human testis can be disrupted by environmental concentrations of a pesticide, dieldrin. The fact that this can occur during a critical period of androgen secretion and Leydig cell proliferation, and at pesticide concentrations well below those commonly observed in women’s circulations is critical. Although the exposure was in vitro, our short-term exposure model answers one of the fundamental questions: Whether or not the fetal human testis really is exquisitely sensitive to endocrine disruption? This sensitivity has been emphasized in recent reviews, such as (Delbes et al., 2006
). The culture of the human fetal testis needs to be performed very carefully to ensure the collection of reproducible data and to demonstrate LH-responsiveness, but this has been achieved by other groups [e.g. (Huhtaniemi et al., 1977)], most recently Hallmark et al. (2007). Both the latter papers and 2006 publications (Lambrot et al., 2006a,b) also found consistent responsiveness to LH/hCG and that short-term culture was optimal compared with longer-term cultures. This indicates that our fetal human testis explant cultures were performing in a predictable manner. Combined with observations of high levels of organochlorine pesticides reaching the placental cord and fetus, therefore not being removed or metabolized completely by the placenta, e.g. (van der Ven et al., 1992), we can conclude that our findings are biologically relevant.
Since testosterone is a Leydig cell product and AMH is only produced by the Sertoli cell (Franke et al., 2004), this aspect of Sertoli cell function was not directly affected by dieldrin following short-term exposure. However, it is likely that long-term exposure would have more marked effects on the Sertoli cell, as seen in real-life exposure models such as the sheep model used by Paul et al. (2005). In the latter study, in utero exposure to processed sewage sludge fertilizer, to investigate effects of real-life gestational challenge to a mixture of environmental chemicals at low concentrations, demonstrated significant defects in both Sertoli and Leydig cell development. Two further aspects deserve consideration: first, the concept of ‘windows of sensitivity’, as demonstrated by observations that hypospadias can occur in isolation from abnormalities in endocrine function (Rey et al., 2005), and secondly, the role of other hormones and signalling molecules, such as FSH which stimulates AMH secretion from Sertoli cells (Lukas-Croisier et al., 2003). Addressing these issues would require considerable research and should be pursued in future studies.
The use of short-term culture has the potential criticism that more extreme effects might have been observed in testes exposed to dieldrin for a longer period, particularly as the fetus in utero will be exposed to environmental chemicals throughout development. However, it is our view that the short-term culture system was optimal because culture-induced changes in organ function are observed relatively rapidly, even in the adult human testis (Roulet et al., 2006). In addition, since our studies have shown the loss of several critical testicular markers, such as P450c17 and PCNA, and signs of deteriorating organ morphology at 48 h, the 24 h culture period was most likely to yield physiologically valid data. As part of the process to maximize the validity of our approach, we used LH instead of hCG to stimulate the explants because of the known importance of fetal pituitary LH production in masculinization in primates, unlike the situation in rodent.
An additional point that cannot be answered in the present study due to ethical constraints and limited fetal tissue size is the extant concentrations of dieldrin and other endocrine disrupting compounds, in the fetus (especially the testis) and the maternal circulation. Inevitably, our model will include variable existing levels of environmental chemicals. In future studies, the fetal liver should be used to determine potential toxicant concentrations, although studies in a sheep model of low-level exposure have shown considerable tissue-specific variation in concentrations of endocrine disrupting compounds (Paul et al., 2005; Rhind et al., 2005). Notwithstanding this imponderable, low doses of dieldrin clearly affected the fetal testis in vitro, although some of the variability in responses could well have been due to prior exposure to dieldrin and other endocrine disrupting compounds.
The mechanism(s) by which such low concentrations of dieldrin were acting is not clear. Our data suggest that dieldrin affected LHR expression but not that of StAR. However, the estrogenic and anti-androgenic activity of dieldrin is indicative of more than one mode of action, and, as outlined in the Introduction section, there is evidence of altered ER expression and dieldrin sensitive intracellular mechanisms (Wozniak et al., 2005). Although dieldrin does have estrogenic activity, and this is significant at doses as low as 1 µmol (Lemaire et al., 2006), there is little in vitro evidence for direct estrogenic effects at the 1 pmol lowest dose we used. A potential problem with many of the published in vitro studies, however, is that when environmental chemicals, such as pesticides, are tested, their effects in the presence of physiological concentrations of E2 (which are high during the second trimester) are not investigated and the environmental chemicals are simply compared directly with E2. However, dieldrin, and other environmental chemicals, significantly induces mast cell degranulation at doses as low as 1 pmol, with non-standard dose–response curves, especially in the presence of E2 (Narita et al., 2007). These findings are very similar to our data. It is recognized that multiple mechanisms of action probably underpin many effects of environmental chemicals. It is pertinent therefore that in terms of inhibition of the GABA receptor the IC50 point for dieldrin was 0.2 µmol in vitro (Vale et al., 2003).
It is highly relevant that, in terms of studies on dieldrin, and indeed, other environmental chemicals, species differences must be considered. For instance, in a recent study, mono and di(n-butyl) phthalate reduced hCG-induced testosterone from rat fetal testis explants, but not from those of the human, despite highly significant effects of hCG which gave a 300–400% increase in testosterone (Hallmark et al., 2007). In addition, our preliminary data showing an effect of 1 pmol dieldrin/l, but not 0.01 pmol/l, on human fetal testis explant testosterone secretion (Fowler et al., 2003) demonstrates that the disruptive effect of dieldrin is lost at a dose lower than 1 pmol/l. We can safely conclude from these and other studies that our findings of a low dose effect with evidence of a non-standard dose–response are unlikely to be due to study artefacts. The fact that AMH secretion was not significantly altered in our study also supports both the robustness of the model (i.e. the data are not representative of methodological artefacts) and the suggestion that the Leydig cell is particularly vulnerable.
Analysis of the 2-dimensional gels suggests considerable effects of dieldrin on the testis proteome that was, interestingly, similar in magnitude to that of LH. Unfortunately, detailed mapping of these effects requires more protein than we had, but our findings nevertheless suggest widespread effects on intra-cellular protein expression. The data also suggest that dieldrin reverses some of the effects of LH on the testis proteome. This could have serious consequences for the human fetus in which LH is important, unlike the pituitary-independence of gonadal development in the rodent (Klonisch et al., 2004) and confirms the importance of studies using the human fetus rather than reliance on rodent models only.
The positively identified proteins that were affected by dieldrin (Table 1) included IRAS (nischarin), which increased following exposure to dieldrin. IRAS inhibits cell migration (Alahari et al., 2004) and exerts anti-apoptotic effects (Dontenwill et al., 2003). Dieldrin increased tropomyosin isoform 3 (TPM-3) expression. In non-muscle cells, TPM-3 has been implicated in stabilizing actin filaments in the cytoskeleton and TPM expression alters during carcinogenesis (Pawlak et al., 2004). It is possible that these effects would increase the incidence of abnormal cell development. The zinc-finger protein KOX31 disappeared following dieldrin treatment, with potentially serious consequences since its function as a regulator of transcription would suggest that it might be developmentally important. The homologous gene in drosophila embryos regulates a secreted protein (Reuter et al., 1996) and if true in the human, could have effects on paracrine signalling in the developing testis. The three proteins that were unchanged across the treatments were fundamental to normal cell function: UCHL1, is involved in processing ubiquitin precursors and is important in germ cell apoptosis and spermatogenesis (Wang et al., 2006). The peroxiredoxins, Prx2 and Prx6, protect cells from oxidative stress and are involved in cell signalling and differentiation, as well as being putative tumour suppressors (Immenschuh and Baumgart-Vogt, 2005).
The 3-fold reduction in Wnt-2b following exposure to dieldrin was particularly interesting since this member of the Wnt signalling family is expressed in the fetal testis (Katoh et al., 1996) and is important in determining cell fate during development (Katoh et al., 1996). Although Wnt-2b is important for ovarian signalling (Ricken et al., 2002), its specific role in testis development is not known. If reduced Wnt-2b was accompanied by impaired migration and differentiation of Leydig precursor cells, then one obvious outcome would be reduced testosterone production and impaired androgen signalling.
In conclusion, short-term in vitro exposure of the fetal human testis to a model endocrine disrupting compound, dieldrin, at doses up to 1000-fold lower than circulating levels in women, significantly affects endocrine and developmental proteins in the Leydig cell and antagonizes LH action. This suggests that very low-level exposure to endocrine disrupting compounds could have the potential to disrupt a range of endocrine and other cellular systems in the developing fetus. Therefore, complacency about the fact that exposure to many endocrine disrupting compounds is only at relatively low levels in the human is probably misplaced. In addition, we have tested just one compound, whereas the mother and fetus are in real life exposed to hundreds of xenotoxins at the same time. Hence, data on significant effects of a single compound may be a very cautious underestimation of the real life situation.
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NHS Grampian Endowments and Dr James Alexander Mearns Trust to P.A.F. (PI) and some co-authors.
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The impartial help of the staff of the Pregnancy Counselling Service was essential for the collection of fetal testes. We thank Ms F. MacGregor, M. Fraser, P. Cunningham, Ms E. Argo, E. Stewart and Mr I. Davidson (University of Aberdeen) and Ms L. Hannah (Rowett Research Institute) for their expert technical assistance and Dr A. Schofield, Ms R. Munyariwa, R. Rehan and the IMS Histology Facility for help with the Wnt-2b immunohistochemistry. Dr E. Brown assisted with preliminary experiments leading to this study. We are grateful to Dr A. Parlow, NHPP, for human gonadotrophins. Profs J. Strauss III (University of Pennsylvania Medical Centre, Philadelphia, USA) and J. Wilamasena (University of Tennessee College of Veterinary Medicine, Knoxville, USA) kindly supplied the StAR and LHR antibodies, respectively. We thank Prof J.I. Mason (University of Edinburgh) for supplying the P450c17 antibody. We are grateful to Prof I Huhtaniemi (Imperial College, London, UK) and Prof R.M. Sharpe (MRC Human Reproductive Sciences Unit, Edinburgh, UK) for their advice.
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References |
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Submitted on May 25, 2007; resubmitted on June 28, 2007; accepted on July 11, 2007.
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