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Alteration of steroidogenesis in H295R cells by organic sediment contaminants and relationships to other endocrine disrupting effects
Luděk Bláha a,*, Klára Hilscherová a, Edita Mazurová ª, Markus Hecker b, Paul D. Jones b, John L. Newsted ℃, Patrick W. Bradley b, Tannia Gracia b, Zdenek Ďuriš d, Ivona Horká ª, Ivan Holoubek ª, John P. Giesy b,e
ª RECETOX - Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, CZ62500 Brno, Czech Republic b Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Department of Zoology, Michigan State University, East Lansing, MI 48824, USA ENTRIX Inc., 4295 Okemos Rd., Okemos, MI 48864, USA d Department of Biology and Ecology, University of Ostrava, Ostrava, Czech Republic e Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China
Received 15 December 2005; accepted 20 March 2006 Available online 2 May 2006
Abstract
A novel bioassay with the human adrenocortical carcinoma cell line H295R can be used to screen for endocrine disrupting chemicals that affect the expression of genes important in steroidogenesis. This assay was employed to study the effects of organic contaminants associated with the freshwater pond sediments collected in the Ostrava-Karvina region, Czech Republic. The modulation of ten major genes involved in the synthesis of steroid hormones (CYP11A, CYP11B2, CYP17, CYP19, 17BHSD1, 17BHSD4, CYP21, 3BHSD2, HMGR, StAR) after exposure of H295R cells to sediment extracts was investigated using quantitative real-time polymerase chain reaction (PCR). Crude sediment extracts, containing high concentrations of polycyclic aromatic hydrocarbons (PAHs) and moderate amounts of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) significantly stimulated expression of the CYP11B2 gene (up to 10-fold induction), and suppressed expression of 3BHSD2 and CYP21 genes. A similar pattern was observed with the extracts after treatment with concentrated sulfuric acid to remove labile chemicals (including PAHs) leaving only persistent PCBs, OCPs and potentially PCDD/Fs. Comparison of the results with other mechanistically based bioassays (arylhydrocarbon receptor, AhR, mediated responses in H4IIE-luc cells, and estrogen receptor mediated effects in MVLN cells) revealed significant endocrine disrupting potencies of organic contaminants present in the sediments (most likely antiestrogenicity). Pronounced effects were observed particularly in sediment extracts from the Pilnok Pond which harbors an unusual intersexual population of the narrow-cawed crayfish Pontastacus leptodactylus (Decapoda, Crustacea). This pilot study provided the first experimental evidence of the wider application of the H295R bioassay for screening complex environmental samples, and the results support the hypothesis of chemical-induced endocrine disruption in intersexual crayfish.
@ 2006 Elsevier Ltd. All rights reserved.
Keywords: Estrogen receptor; ER; Arylhydrocarbon receptor; AhR; Dioxin; Coal sediment; Crayfish; Pontastacus (= Astacus) leptodactylus; Intersex; Steroidogenesis; H295R
1. Introduction
Chemical-induced endocrine disruption is of increasing concern worldwide (Sumpter and Johnson, 2005). Several
receptor-mediated mechanisms have been investigated in detail including modulations mediated via the estrogen or androgen receptors (ER, AR), or the cross-talk of these receptors with the arylhydrocarbon receptor, AhR (Jana et al., 1999; Tan et al., 2002). However, several studies have demonstrated that some xenobiotics exert their effects on endocrine systems via other mechanisms, such as disrupting production of crucial steroid hormones or steroidogenic enzymes (Connor et al., 1996;
* Corresponding author. E-mail address: blaha@recetox.muni.cz (L. Bláha).
Sanderson et al., 2000). Recently, a new bioassay with the human H295R cell line was developed for the quantitative evaluation of xenobiotic effects on the expression of genes involved in steroidogenesis (Hilscherova et al., 2004, Zhang et al., 2005). H295R cells express all the key enzymes involved in the synthesis of steroid hormones (Fig. 1; Gazdar et al., 1990), and the assay has been successfully used for the characterization of effects of model chemicals, individual contaminants and pesticides (Hilscherova et al., 2004, Zhang et al., 2005, Sanderson et al., 2000). Here we present the results of the first application of the new H295R bioassay for scree- ning of complex contaminated matrices in this case sediment extracts.
Freshwater and marine sediments are known to accumulate and retain many pollutants released by human activities, and have been shown to reflect environmental risks at particular localities and areas. In the present study, we employed a H295R bioassay for the investigation of sediments from the Ostrava- Karvina region in the Czech Republic. In spite of a long history of black coal mining and heavy industry in this area, there is a surprising lack of information on the potential environmental effects of these practices. Several ponds in the area have been used as sludge lagoons for deposition of waste coal dust and cinder from the steel industry. Basic parameters of water quality in these ponds supplied by underground springs remained relatively stable (oxygen content and transparency in particu- lar), and the endangered species of the narrow-cawed crayfish Pontastacus (syn. Astacus) leptodactylus (Decapoda, Crusta- cea) live in these reservoirs. However, an abnormal population of the crayfish has been observed at a single specific locality (Pilnok Pond). This population shows a greatly increased ratio
StAR
HMGR
100
100
Cholesterol
CYP11A Pregnenolone
CYP17
17a-OH-
CYP17
Pregnenolone
DHEA
3B-HSD
CYP17
38-HSD
3B-HSD
Progesterone
17a-OH-
CYP17
Androstene
CYP21
Progesterone
-dione
11-Deoxy- corticosterone
CYP21
17B-HSD|
CYP11B2
11-Deoxycortisol
Testosterone
Corticosterone
CYP11B1
CYP19
CYP11B2
Cortisol
17ß-Estradiol
Aldosterone
Zona glomerulosa
Zona fasciculata
Zona reticularis
of intersex individuals (18% of more than 1000 adult female- like specimens; Ďuriš et al., unpublished results).
Our research focused on a detailed characterization of the Pilnok Pond sediments and the sediments from the Reference site in an attempt to determine a possible chemical cause of the occurrence of the unique intersexual crayfish population. Parallel to the instrumental identification and quantification of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs), a series of in vitro bioassays for studies of endocrine disruption potencies was employed. Estrogenicity was assessed using the MVLN cell bioassay that has a reporter luciferase reporter gene under the control of the ER (Hilscherova et al., 2002). Additionally, we investigated the role of AhR using two bioassays: (i) H4IIE-luc cells with the luciferase reporter gene under the control of the AhR (Hilscherova et al., 2000), and (ii) ethoxyresorufin-O-deethylase activity (EROD) in H295R cells (Sanderson et al., 2001). Finally, the effects of sediment extracts on the expression of ten steroidogenic genes (CYP11A, CYP11B2, CYP17, CYP19,17BHSD1, 17BHSD4, CYP21, 3ßHSD2, HMGR, StAR; Fig. 1) were measured by quantitative real-time polymerase chain reaction (Q-PCR) using the H295R cell bioassay.
2. Materials and methods
2.1. Sediment samples
Sediments were collected from two freshwater ponds in the Ostrava-Karvina region in the Czech Republic. The sampling locations were similar with respect to geology, geomorphology and anthropo- genic impacts, but differed by biological observations in the field. While abnormal intersexual animals of the narrow-cawed crayfish occur in the Pilnok Pond, “normal” heterosexual animals of this species live in the reference location (land depression near Mir mine, Karvina- Doly). Several individual sediment subsamples were collected at each location and were pooled. Sediments were allowed to dry at room temperature and they were then ground and sieved through a 2 mm mesh before further processing.
2.2. Extract preparation
Dried and sieved sediments (10 g) were Soxhlet extracted for 20 h with dichloromethane and hexane (3:1 v/v, 400 mL), free sulfur was removed by copper treatment. The extracts were concentrated initially by rotary evaporation and then by a gentle stream of nitrogen. The final extract was then divided into two portions for either chemical analysis or bioassay testing. To evaluate the contribution of labile and stable (persistent) compounds in the tested samples, portions of the extracts were further treated by repeated liquid/liquid extraction with concentrated sulfuric acid (96% H2SO4, 1:5 v/v acid/extract ratio). Extraction with sulfuric acid removed labile compounds including PAHs leaving only persistent chemicals such as PCBs, OCPs and PCDD/Fs (Hilscherova et al., 2000). The organic phase of the acid- treated extracts was retained, dried by passing through anhydrous Na2SO4 and was then concentrated under the stream of nitrogen. The solvent in the subsamples intended for bioassays was replaced with dimethylsulfoxide (DMSO). A procedural blank was prepared and analyzed in parallel with the sediment extractions.
2.3. Instrumental analyses
Before the instrumental analyses of contaminants, the crude extracts were purified by passage through 10 g of activated florisil (60-100 mesh size; Sigma, St. Louis, MO; packed in a glass columns of 10 mm diameter, washed with 50 mL of hexane). Samples were eluted sequentially with 200 mL of hexane followed by 200 mL of dichlo- romethane:hexane (1:2), and concentrated. Concentrations of PAHs, PCBs and OCPs were quantified using a Hewlett Packard 5890 series II gas chromatograph equipped with 5972 series mass spectrometer detector by methods described elsewhere (Khim et al., 2001).
2.4. H295R cell bioassay
The H295R human adrenocortical carcinoma cell line was obtained from the American Type Culture Collection, Manassas, VA, USA, and the cells were cultured as previously described (Hilscherova et al., 2004). For the experiments, the cells were seeded into 6-well plates and exposed for 24 h to various concentrations of test sediment extracts, procedural blank or solvent (DMSO). To assure that gene modulations (inhibitions in particular) in the H295R bioassay were not a result of cytotoxic effects, viability of the cells was carefully checked with a conventional MTT bioassay (Mosmann, 1983), and only the non- cytotoxic doses were evaluated. Maximum solvent concentration during exposure was 0.1% v/v, non-treated cells served as a negative control. After 24 h exposure, RNA from the H295R cells was isolated using the SV Total RNA Isolation System (Promega, Madison, WI, USA) following the manufacturer’s procedure. Isolated RNA was quantified using a RiboGreen(R) RNA Quantitation Kit (Molecular Probes, Eugene, OR, USA) and samples were diluted to a final concentration of 50 ng RNA/uL. cDNA was prepared from 500 ng of RNA using the Superscript II First-Strand cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s procedure. The resulting cDNA was diluted by 50 times, and the amount of cDNA for ten target genes plus the endogenous gene beta- actin was quantified by real-time PCR using a Smart Cycler System (Cepheid, Sunnyvale, CA, USA) with SYBR® Green RT-PCR Core Reagents (Applied Biosystems, Foster City, CA, USA). The thermal cycling reaction conditions, primer sequences and concentrations have been described in detail previously (Hilscherova et al., 2004). Quan- tification of PCR products was accomplished by use of a comparison method of target mRNA concentration to an endogenous control (beta- actin) used. The Ct values (the first cycle at which the fluorescence significantly increase above the defined background level) were deter- mined for each reaction and normalized for Ct value of beta-actin. The differences between the sediment sample treatments and the solvent control were expressed as fold induction (FI) for each particular gene (FI=1 for the solvent control). Gene expression was measured in triplicate for each cDNA sample, and each extract exposure was repeated three times.
2.5. EROD assay in H295R cells
Ethoxyresorufin-O-deethylase (EROD) activities in H295R cells were determined using a method of Burke and Mayer (1974) as modified by Sanderson et al. (2001). H295R cells grown in 24-well plates were exposed to extracts, blank and solvent alone for 24 h. After washing with pre-warmed (37 ℃) phosphate-buffered saline (PBS), cells were further incubated with 7-ethoxyresorufin (25 µM, Sigma, St. Louis, MO, USA) and the kinetics of resorufin formation (linear within 60 min) was measured with Cytofluor microplate reader (Millipore, Billerica, MA, USA).
| Pilnok | Ref. | Literature | |
|---|---|---|---|
| Naphthalene | 2588 | 564 | |
| Acenaphthylene | 533 | 46 | |
| Acenaphthene | 1473 | 83 | |
| Fluorene | 1994 | 225 | |
| Phenanthrene | 4242 | 1226 | |
| Anthracene | 259 | 224 | |
| Fluoranthene | 1128 | 1470 | |
| Pyrene | 1016 | 1120 | |
| Benzo[a] | 518 | 638 | |
| anthracene | |||
| Chrysene | 1602 | 1305 | |
| Benzo[b] | 880 | 780 | |
| fluoranthene | |||
| Benzo[k] | 248 | 603 | |
| fluoranthene | |||
| Benzo[a]pyrene | 661 | 710 | |
| Indeno[1,2,3-cd] | 238 | 494 | |
| pyrene | |||
| Dibenzo[a,h] | 173 | 127 | |
| anthracene | |||
| Benzo[ghi] | 868 | 460 | |
| perylene | |||
| Sum of 16 PAHs | 18420 | 10075 | 3500-61 700 [1], 600-13 200 [2] |
| PCB 52 | 3.88 | <LOD | |
| PCB 66+95 | 3.76 | <LOD | |
| PCB 90+101 | 2.84 | 1.70 | |
| PCB 118 | 0.80 | <LOD | |
| PCB 126+129+ | 2.37 | <LOD | |
| 178 | |||
| PCB 153 | 1.74 | 1.57 | |
| PCB 138 | 1.93 | 2.11 | |
| PCB 180 | 1.38 | 1.31 | |
| Sum of PCBs | 18.7 | 6.70 | 9-85 [1], 2.6-37.8 [2] |
| a-HCH | 228 | 9.38 | |
| B-HCH | <LOD | <LOD | |
| y-HCH | 529 | 197 | |
| Heptachlor | 71.6 | <LOD | |
| epoxide | |||
| DDE | 1.72 | 2.38 | |
| DDD | 9.36 | 4.16 | |
| Methoxychlor | 51.7 | <LOD | |
| Sum of OCPs | 891 | 2213 | 1.8-53.1 (Sum of DDT) [2] |
| Mass-balance TEQs [ng/g d.w.] | |||
| PCB contrib. [3] | 0.237 | <LOD | |
| PAH contrib. [4] | 0.914 | 1.459 | |
| Sum of TEQs | 1.151 | 1.459 | |
| Bioassay TCDD-EQs [ng/g d.w.] | |||
| Crude-24h | 123.4 | 50.7 | 1.9-23 ng/g d.w. [1], 1.5-7.8 ng/g d.w. [2] |
| Acid-treat-24h | 10.3 | 1.18 | |
| Crude-72h | 13.5 | 21.0 | |
| Acid-treat-72h | 6.90 | 0.11 | |
Comparison with the values previously reported in the Czech Republic sediments is provided - [1] Hilscherova et al. (2001); [2] Vondráček et al. (2001). Mass- balance calculated TEQs for PCBs are based on WHO TEFs ([3] - Van den Berg et al., 1998), contribution of PAHs to TEQs used relative potencies suggested by Machala et al. (2001) [4]. (<LOD - below the limit of detection).
2.6. H4IIE-luc and MVLN bioassays
The potencies of the samples to induce AhR- and ER-mediated effects were determined with H4IIE-luc and MVLN bioassays, respectively. Procedural details for these luciferase reporter gene based assays have been described elsewhere (Hilscherova et al., 2002, 2001). In brief, cells (H4IIE-luc or MVLN) were seeded in 96-well culture ViewPlates™M (Packard, Meriden, CT, USA) and were exposed
to dilutions of sediment extracts for 24 and 72 h in triplicate. The amount of AhR- (ER-) induced luciferase was quantified using the LucLite(R) Reporter Gene Assay System (Perkin Elmer, Netherlands). After the initial range-finding experiments, full concentration- response curves for induction of AhR- and ER-mediated responses were generated in triplicate. The effects of sediment samples were related to the luciferase induction by the reference compounds: 2,3,7,8- tetrachlorodibenzo-p-dioxin (AhR) and 17B-estradiol (ER).
StAR
HMGR
2.0
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
Cells
Gene expression (fold induction - relative to solvent control, DMSO)
DMSO
Pr.Blank
0.0
0.025/0.25/2.5
0.025 0.25 2.5 Ref. sed.
Cells
DMSO
Pr.Blank
0.025 / 0.25 / 2.5
0.025 0.25 2.5
Pilnok
Pilnok
Ref. sed.
CYP11A
CYP17
3₿HSD2
2.0
2.0
2.0
1.5
1.5
1.5
1.0
1.0
1.0
0.5
0.5
0.5
0.0
0.0
0.0
Cells
DMSO
Pr.Blank
Cells
DMSO
Pr. Blank
Cells
DMSO
Pr.Blank
0.025/0.25/2.5
0.025 0.25 2.5
0.025 / 0.25/ 2.5
0.025 0.25 2.5
0.025/0.25/ 2.5
Pilnok
Ref. sed.
Pilnok
Ref. sed.
Pilnok
0.025 0.25 2.5 Ref. sed.
CYP21
CYP11B2
2.0
15.0
1.5
12.0
9.0
1.0
6.0
0.5
3.0
0.0
LA
0.0
Cells
DMSO
Pr.Blank
Cells
DMSO
Pr.Blank
0.025/0.25/2.5
0.025 0.25 2.5
0.025/0.25/2.5
Pilnok
0.025 0.25 2.5 Ref. sed.
Pilnok
Ref. sed.
17₿HSD1
17₿HSD4
CYP19
2.0
2.0
2.0
1.5
1.5
1.5
1.0
1.0
1.0
0.5
0.5
0.5
0.0
0.0
Cells
DMSO
Pr.Blank
Cells
DMSO
Pr. Blank
0.0
0.025 / 0.25/2.5
0.025 0.25 2.5
0.025/0.25/2.5
0.025 0.25 2.5
Cells
DMSO
Pr.Blank
0.025 / 0.25 / 2.5
0.025 0.25 2.5
Pilnok
Ref. sed.
Pilnok
Ref. sed.
Pilnok
Ref. sed.
2.7. Statistical analyses
Results of repeated experiments are expressed as the mean value ±standard deviation. Differences between the experimental variants were evaluated by ANOVA followed by Dunnet’s test, P-values less than 0.05 were considered statistically significant. The EC50 values (H4IIE-luc, MVLN, MTT-assay) were estimated using least-squares regressions derived for the log-linear portion of the full concentration- response curves. TCDD equivalents based on the H4IIE-luc bioassay (TCDD-EQs) were calculated using the effect-equivalency approach by comparing the EC25 value of the TCDD standard calibration with the concentration of tested sample inducing the same bioassay response as the EC25 of TCDD (ECEQ) (Hilscherova et al., 2000).
3. Results
The instrumental analyses (Table 1) revealed relatively high concentrations of PAHs in both Pilnok Pond (18 µg EPAHs per g sediment dry weight) and in the sediment from the Reference location (10 µg/g d.w.). Concentrations of other analyzed persistent pollutants (PCBs and OCPs) were relatively low (Table 1).
The viability of the cells treated with sediment extracts was eva- luated with the MTT assay prior to application of specific bioassays, and only the non-cytotoxic doses of sediment extracts were used to eliminate possible non-specific effects during necrotic or apoptic cell processes (≤2.5 mg sediment d.w./mL).
The H295R bioassay for screening of effects on expression of steroidogenic genes has been proposed as a tool for the screening of chemicals as well as complex mixtures and environmental samples (Hilscherova et al., 2004). In our study, the expression of 10 major genes was studied in response to the organic contaminants present in the sediment extracts. There were no significant differences between the blank (non-treated cells), solvent controls and the procedural blank for any of the genes (ANOVA+Dunnet’s test, P>0.05). Significant effects on the expression pattern were observed after exposure to sediment extracts (Fig. 2). The most significant change was the 10-fold up-regulation of the CYP11B2 gene after exposures to both Pilnok and Reference sediments at two of the concentrations tested (0.25 and 2.5 mg d.w./mL). In addition, significant down-regulations of the 3BHSD2 and CYP21 genes were observed (Fig. 2).
To further investigate the role of different classes of contaminants, the sediment extracts were treated with concentrated sulfuric acid to remove reactive and labile contaminants (such as PAHs, phthalate esters etc.) while chlorinated persistent chemicals such as PCBs, OCPs or PCDD/Fs remain preserved in the organic extract (Hilscherova et al., 2000). Quantitative PCR for the expression of selected genes (CYP11B2, 3BHSD2, CYP21) revealed trends similar to those ob- served with crude extracts (Fig. 3).
The effects of sediment extracts in other bioassays were further assessed to study possible relationships between modulation of steroidogenesis in H295R cells and other mechanisms of endocrine disruption (Hudson et al., 1987; Sugawara et al., 2001). None of the tested samples significantly induced ER-dependent luciferase in the reporter gene bioassay with MVLN cells (Fig. 6). In contrast, both sediment samples significantly induced AhR-modulated reporter luciferase activity in the H4IIE-luc cells (Fig. 4A,B). The maximum level of induction occurred at doses of 0.1 mg sediment d.w./mL. Sulfuric acid-treated samples containing only persistent chemicals caused less pronounced effects (triangle symbols in Fig. 4). The concentration-induction curves of AhR-dependent luciferase in H4IIE-luc cells varied with the exposure time and the samples tested. For the crude extracts (diamond symbols in Fig. 4) more pronounced effects were observed after 24 h, while
prolonged 72 h exposures resulted in a decrease of the induction poten- cies. Different patterns were observed with sulfuric acid-treated samples and the reference compound TCDD with maximum responses after 72 h (Fig. 4; triangles and circles, respectively).
To compare the AhR-mediated responses of different samples, the effects were related to that caused by the reference standard, 2,3,7,8- TCDD and TCDD equivalents (TCDD-EQs) were estimated (Hilscher- ova et al., 2001). Calculated TCDD-EQs as well as the previously published values for other sediment samples from the Czech Republic are listed in Table 1. TCDD-EQs for the crude extracts of sediments from both Pilnok Pond and the Reference location were within a similar range. Significantly less pronounced effects were observed in the samples treated with sulfuric acid. This observation indicates a subs- tantial contribution of acid labile compounds (particularly PAHs) to the observed AhR-modulated responses (Table 1) as also reported pre- viously (Houtman et al., 2004, Klamer et al., 2005). The short-term 24 h TCDD-EQs values for the acid-treated extracts were 10.31 and 1.18 ng/g d.w. in sediments from Pilnok Pond and Reference site, respectively. The prolonged 72 h TCDD-EQ of acid-treated extract from Pilnok Pond sediment (6.9 ng/g d.w.) was comparable with that of
3฿HSD2
2.0
1.5
I
1.0
*
Gene expression (fold induction - relative to solvent control, DMSO)
*
T
0.5
I
0.0
Cells
DMSO
Crude
Acid-
Treated
Crude
Acid-
Treated
Pilnok
Ref. sed.
CYP21
2.0
1.5
1.0
T
T
*
*
0.5
T
0.0
Cells
DMSO
Crude
Acid-
Treated
Crude
Acid-
Treated
Pilnok
Ref. sed.
CYP11B2
15.0
*
12.0
9.0
6.0
*
*
*
T
3.0
I
0.0
Cells
DMSO
Crude
Acid-
Treated
Crude
Acid-
Treated
Pilnok
Ref. sed.
100
TCDD-24h
A
O- TCDD-72h
Pilnok (sed. No. 1)
Crude-24h
Crude-72h
+- - Acid-Treat-24h
AhR-dependent luciferase / fold-induction [log]
- - - Acid-Treat-72h
10
1
100
B
Reference (sed. No. 2)
10
1
1×10-8
1x10-7 1x106
1
10
100
1000
ug TCDD/mL
ug sediment dw/mL
the crude sample (13.5 ng/g d.w.) after acid treatment. Lesser AhR- mediated activity was observed in the acid-treated extract of the Reference sediment (0.112 ng/g d.w., Table 1).
Similarly to the inductions of AhR-dependent reporter luciferase in H4IIE-luc cells, EROD activity was significantly elevated in H295R cells exposed to both sediment extracts (Fig. 5). Pilnok Pond sediment contained significantly greater concentrations of CYP1A inducing xenobiotics with maximum effects observed at concentrations 0.025-0.1 mg d.w./mL. Maximum effects of the Reference sediment were observed at about 10- fold greater concentrations (1 mg d.w./mL, Fig. 5). Decreases in EROD observed at greater concentrations were not related to cellular toxicity (no cytotoxic effects were observed up to 2.5 mg d.w./mL as discussed above), and might result from nonspecific inhibition of EROD enzymatic activity by complex sediment extracts (Murk et al., 1996).
Effective concentrations varied significantly among the different bioassays. While the maximum effects on steroidogenesis were recor- ded at concentrations around 2.5 mg d.w./mL (Figs. 2 and 3), maxi- mum stimulation of EROD in H295R cells as well as maximal inductions of AhR-dependent luciferase in H4IIE-luc bioassay oc- curred at concentrations significantly lower (0.025-0.1 mg d.w./mL, Figs. 4 and 5).
Instrumental identification and quantification of known AhR- inducing compounds, such as PAHs and PCBs, allowed mass balance calculations of chemical toxic equivalents (TEQs; Table 1). The WHO TCDD Equivalency Factors for PCBs were used (Van den Berg et al., 1998) and the Induction Equivalency Factors for PAHs derived with
H4IIE-luc cells (Machala et al., 2001). Resulting TEQs were
1.15 ng TCDD/g d.w. for Pilnok Pond sediment (contribution of
PCBs0.24 ng/g, PAHs0.91 ng/g), and 1.46 for Reference sediment
(PCBs<LOD, PAHs~1.46 ng/g).
4. Discussion
In the present study, we demonstrate an approach that combined both instrumental and bioanalytical techniques to elucidate the potential causes of intersex population of the narrow-cawed crayfish in Pilnok Pond (Ostrava-Karvina region, Czech Republic). Endocrine disruption caused by chemical contamination is one of the major current environmental issues and natural occurrence and/or laboratory induction of intersex in decapod crustaceans have been previously documented (Rudolph, 1995; Pinn et al., 2001). However, to the best of our knowledge, such highly pronounced impairment of secondary sexual characteristics (i.e. development of male-type gonopods in 18% of female-like specimens) had not been previously reported for the endangered European species of P. leptodactylus.
Several causes of intersex development in crustaceans have been recognized including photoperiod changes, parasitism or chemically induced effects (Depledge and Billinghurst, 1999; Ladewig et al., 2002). In the large decapod crustaceans, the androgenic gland is considered to play a major role in sexual differentiation (Sagi et al., 1997). Although mechanisms of hormonal regulation among invertebrates and vertebrates differ significantly, several studies show multiple similarities, and the general vulnerability of animal endocrine systems to adverse effects of common classes of environmental pollutants such as PAHs or PCBs (LaFont, 2000; Oberdorster et al., 1999). There- fore, we focused on characterization of the major organic con- taminants present in sediments and employed a series of available in vitro bioanalytical techniques specifically designed to screen for the endocrine disrupting potential of chemicals.
Modulation of ER-mediated activities by chemicals (“xenoestrogenicity”) is one of the most extensively studied endocrine disrupting mechanisms so far proposed (Gray et al., 1997). In our study we employed a luciferase reporter gene bioassay with MVLN cells for studies of ER-mediated effects
EROD (pmol/min/mg protein)
6.0
5.5
Pilnok
5.0
Ref.sed.
4.5
4.0
3.5
3.0
2.5
2.0
1.5
0.1
1
10
100
1000
10000
Sediment Extract (ug d.w./mL)
(Hilscherova et al., 2002) but we observed no significant inductions of ER-dependent luciferase in the presence of the sediment extracts being studied (Fig. 6). Our observations indicate either minor concentrations of compounds directly activating ER, and/or simultaneous manifestation of antiestro- genic effects of other organic chemicals present in the sediment extracts (Hilscherova et al., 2002). The hypothesis that antiestrogenic effects dominate other potential receptor based responses in the sediment extracts is supported by the known antiestrogenic effects of many PAHs (Arcaro et al., 1999; Chaloupka et al., 1992) that were detected in high concentra- tions in the study sediments (Table 1). Also, significant inductions of AhR-dependent luciferase in the H4IIE-luc bioassay and EROD in the H295R cells (Figs. 4 and 5) reflect high levels of AhR ligands that are generally considered to be antiestrogens (Safe et al., 1998).
Significant shifts in the concentration-response curves for AhR activations (Figs. 4 and 5) as well as changes in TEQ values (Table 1) were observed at different exposure times (24 vs. 72 h) and the TEQs also differed for the crude and acid-treated extracts. It has been shown previously that the prolonged exposure periods (72 h) reflects predominantly the effects of persistent chlorinated dioxin-like chemicals (particularly PCBs and PCDD/Fs) while other AhR-activating pollutants (such as PAHs that were removed by sulfuric acid in our extracts) elicited stronger inductions only at shorter (6-24 h) exposures (Hilscherova et al., 2001; Vondráček et al., 2001). Removal of labile PAHs by cellular metabolism during prolonged exposures has been suggested to contribute to these differences (Machala et al., 2001). Surprisingly, 72 h exposures to both crude and sulfuric acid-treated Pilnok Pond sediment extracts resulted in relatively similar TEQs (13.5 and 6.9 ng TCDD equivalents per g d.w., respectively; Table 1). On the other hand, highly significant differences were observed for the extracts of Reference sediment (21 vs. 0.12 ng/g d.w., Table 1). These findings indicate a substantial difference between both study locations, i.e. increased concentrations of persistent chlorinated AhR-inducing compounds in Pilnok Pond. Because instrumental analyses showed only minor or negligible concen- trations of PCBs at both localities and minor contribution to
5.0
E2-24h
-+-Pilnok-24h -Ref.sed .- 24h
ER-dependent luciferase
4.5
-0-E2-72h
-+ — Pilnok-72h
4- Ref.sed .- 72h
fold-induction
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
1×10-6
1×10-5
1×10-4
1
10
100
ug/mL
ug dw/mL
17ß-estradiol
Sediment
overall TEQs was confirmed (Table 1), other persistent AhR- active chemicals such as polychlorinated dioxins and furans (PCDD/Fs) are likely to be present in the Pilnok sediment at elevated concentrations.
Analytical results as well as the observations from mechanistic bioassays for ER and AhR modulations indicate the presence of a range of antiestrogenic chemicals, particularly in Pilnok Pond sediment samples, that might adversely affect normal development of female characteristics. This conclusion corresponds to the field observations from the Pilnok Pond, where most of the intersexual P. leptodactylus specimens seem to be functional females, however, developing male secondary characteristics - gonopods.
In addition to the effects of endocrine disrupters mediated via ER or AhR, other mechanisms such as modulation of steroid hormone synthesis are of particular concern (Connor et al., 1996; Sanderson et al., 2000). Some chemicals have been found to significantly affect steroidogenesis in vivo and in vitro (Harvey and Everett, 2003), and the recently developed bioassay using H295R cells has been proposed for wider screening of endocrine disruption potencies (Hilscherova et al., 2004; Zhang et al., 2005). To the best of our knowledge, this report is the first study to focus on the modulation of steroidogenesis by complex environmental mixtures (sediment extracts). Our study of the expression of 10 major steroidogenic genes revealed both significant up-regulation of CYP11B2 and down-regulations of 3ßHSD2 and CYP21 by crude organic sediment extracts as well as acid-treated samples. More pronounced effects on steroidogenesis were generally observed in the extracts of Pilnok Pond sediment. Based on our observations, both labile organic contaminants including PAHs (present in the crude sample but removed by sulfuric acid treatment) and persistent chlorinated chemicals (dominating the samples after removal of labile compounds) contributed to the observed modulations of steroidogenic genes in H295R cells.
The observed effects might significantly unbalance the synthesis of steroid hormones and result in substantially enhanced synthesis of aldosterone related to up-regulated CYP11B2. Our observations partially correspond to the recent study of Li et al. (2004) which showed a significant increase in the basal expression of CYP11B2 gene along with elevated production of aldosterone in H295R cells after exposure to prototypical coplanar PCB126 (Li et al., 2004). Since CYP11B enzymes are important in the adrenal steroid biosynthesis (Bureik et al., 2002), their modulations could significantly alter various physiological processes controlled by cortisol and aldosterone in vivo. Another important role of the CYP11B enzyme class in the endocrine toxicity of PAHs was revealed by Lindhe et al. (2002). These authors have observed selective CYP11B1-catalysed binding of prototypical 7,12-dimethylbenz[a ]anthracene (DMBA) in specif- ic cells in rat adrenal cortex resulting in selective apoplexy and massive necroses.
In addition to the effects of sediment extracts on CYP11B2 expression, significant suppressions of another two genes (CYP21 and 3ßHSD2, Figs. 2 and 3) might further imbalance the substrate pools available for the synthesis of sex steroid hormones (Fig. 1). The inhibitions observed in our study, however, do not fully correspond to findings of Li et al. (2004)
who observed significant up-regulation of both 3BHSD2 and CYP21 after exposure to pure PCB126.
The steroidogenic acute regulatory protein (StAR, Fig. 1) is considered a rate limiting factor in steroid hormone production. Increased activity of the StAR gene promoter in mouse Y-1 adrenal tumor cells has been observed in the presence of low concentrations (up to 1 µM) of model AhR ligand beta- naphthoflavone (Sugawara et al., 2001). We did not observe any significant changes in the expression of the StAR gene in H295R cells exposed to sediment extracts. However, this finding corresponds to other observations of Sugawara et al. (2001), who reported biphasic responses of the StAR gene promoter with significant suppressions (even below the baseline levels) at high beta-naphthoflavone concentrations. Apparent differences be- tween the effects of prototypical single compounds and natural mixtures were demonstrated also in placental explants (Augus- towska et al., 2003). These authors have observed a two-fold suppression in estradiol production after exposure to prototypical 2,3,7,8-TCDD but an apparent increase in estradiol production after exposure to environmental mixtures of 17 PCDDs and PCDFs. Taken together, the effects of pure chemicals and naturally occurring mixtures on the expression and activities of steroidogenic proteins might significantly differ. As these phenomena are only poorly characterized so far, their elucidation will require further research.
Expression of another important steroidogenic enzyme CYP19, which catalyses the key aromatization of the androgen testosterone to estradiol, is known to be affected by environmental chemicals. An important herbicide atrazine has been shown to up- regulate CYP19 levels (Sanderson et al., 2002), while other pesticides such as lindane or bisphenol-A showed no effect on CYP19 mRNA levels (Nativelle-Serpentini et al., 2003). Also the effects of AhR ligands such as TCDD and diindolylmethane on CYP19 expression have been studied in H295R cells (Sanderson et al., 2001). While both chemicals significantly induced AhR- dependent CYP1A1 and CYP1B1 genes, only diindolylmethane (a weak ligand of AhR known to act as an antiestrogen; Safe et al., 1998) up-regulated CYP19, while TCDD had no significant effect. Correspondingly, there were no significant modulations of CYP19 in our study with complex organic sediment extracts containing high levels of AhR-active contaminants.
In conclusion, the synthesis of steroid hormones is one of the crucial processes in endocrine regulation. It consists of a complex network of sensitively regulated steps and it may be affected by different endocrine disrupting chemicals including PCBs (Li et al., 2004), pesticides (Sanderson et al., 2002) or phthalate esters (Nakajin et al., 2001). To the best of our knowledge, our pilot study is the first revealing significant endocrine disrupting potencies of complex environmental samples (sediment extracts) on expression of steroidogenic genes in the novel H295R cell bioassay. Significant effects (up- regulation of CYP11B2 and down-regulations of CYP21 and 3ßHSD2) were induced by both labile and persistent sediment contaminants, and were apparently more pronounced in the sediment from Pilnok Pond. Additional chemical analyses and bioassays of ER- and AhR-modulating potencies indicate substantially elevated concentrations of persistent PCDD/Fs in
the Pilnok locality, known to act as antiestrogens (Safe et al., 1998). Our study thus might partially explain the development of male sexual characteristics in females of the narrow-cawed crayfish P. leptodactylus in the Pilnok Pond. Since documented, modulation of steroidogenesis appears to be dose-dependent (Sugawara et al., 2001), and there are apparent differences in the effects of pure chemicals and mixtures (Augustowska et al., 2003). Therefore, further research into the endocrine disrupting potencies should focus not only on individual contaminants but also on characterization of effects of naturally occurring complex mixtures. Additionally, experimental in vivo con- firmations of effects observed in vitro are required to improve our understanding of chemically induced endocrine disruption.
Acknowledgements
Research was supported in part by USEPA, ORD Service Center/NHEERL (Contract GS-10F-0041L) and Grant Agency of the Czech Republic (grant No. 525/05/P160). Support from Ministry of Education of the Czech Republic is also highly acknowledged (travel grant 1K04006 awarded to L.B. and VZ0021622412 grant to RECETOX, Masaryk University in Brno).
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