Environment International 32 (2006) 749 – 757
www.elsevier.com/locate/envint
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 c , Patrick W. Bradley b , Tannia Gracia b , Zdenek Ďuriš d ,
Ivona Horká d , Ivan Holoubek a , John P. Giesy b,e
b
a
RECETOX — Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, CZ62500 Brno, Czech Republic
Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Department of Zoology, Michigan State University,
East Lansing, MI 48824, USA
c
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, 17βHSD1, 17βHSD4, CYP21, 3βHSD2, 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 3βHSD2 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
⁎ Corresponding author.
E-mail address: blaha@recetox.muni.cz (L. Bláha).
0160-4120/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi10.1016/j.envint.2006.03.011
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;
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L. Bláha et al. / Environment International 32 (2006) 749–757
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 screening 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 OstravaKarvina 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 particular), and the endangered species of the narrow-cawed crayfish
Pontastacus (syn. Astacus) leptodactylus (Decapoda, Crustacea) 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
Cholesterol
CYP11A
Pregnenolone
3β -HSD
Progesterone
CYP21
11-Deoxycorticosterone
CYP11B2
Corticosterone
CYP11B2
CYP17
17α-OHPregnenolone
3β -HSD
CYP17
Androstene
-dione
CYP21
17β -HSD
11-Deoxycortisol
CYP11B1
Cortisol
Zona
fasciculata
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 anthropogenic 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, KarvinaDoly). 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.
DHEA
3β -HSD
CYP17
17α-OHProgesterone
Aldosterone
Zona
glomerulosa
CYP17
of intersex individuals (18% of more than 1000 adult femalelike 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, 17βHSD1, 17βHSD4, CYP21,
3βHSD2, HMGR, StAR; Fig. 1) were measured by quantitative
real-time polymerase chain reaction (Q-PCR) using the H295R
cell bioassay.
Testosterone
CYP19
17β-Estradiol
Zona
reticularis
Fig. 1. Schematic presentation of steroid hormone synthesis pathways in H295R
cells. Depicted are pathways including eight steroidogenic enzymes (CYP11A,
CYP11B2, CYP17, CYP19, 17βHSD1, 17βHSD4, CYP21B2, 3βHSD2) and
other two proteins involved in synthesis and transport of cholesterol (HMGR —
3-hydroxy-3-methylgluatryl coenzyme A reductase, StAR — steroidogenic
acute regulatory protein). The localization of particular pathways within the
human adult adrenal cortex is indicated. Adapted from Hilscherova et al. (2004).
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 acidtreated 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.
L. Bláha et al. / Environment International 32 (2006) 749–757
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 dichloromethane: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 noncytotoxic 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/μL. 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 betaactin 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). Quantification of PCR products was accomplished by use of a comparison
method of target mRNA concentration to an endogenous control (betaactin) used. The Ct values (the first cycle at which the fluorescence
significantly increase above the defined background level) were determined 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.
751
Table 1
Levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls
(PCBs) and organochlorine pesticides (OCPs), and TCDD equivalents in study
sediments [ng/g d.w.]
Pilnok Ref.
Literature
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]
anthracene
Chrysene
Benzo[b]
fluoranthene
Benzo[k]
fluoranthene
Benzo[a]pyrene
Indeno[1,2,3-cd]
pyrene
Dibenzo[a,h]
anthracene
Benzo[ghi]
perylene
Sum of 16 PAHs
2588
533
1473
1994
4242
259
1128
1016
518
564
46
83
225
1226
224
1470
1120
638
1602
880
1305
780
248
603
661
238
710
494
173
127
868
460
PCB 52
PCB 66 + 95
PCB 90 + 101
PCB 118
PCB 126 + 129 +
178
PCB 153
PCB 138
PCB 180
Sum of PCBs
3.88
3.76
2.84
0.80
2.37
bLOD
bLOD
1.70
bLOD
bLOD
1.74
1.93
1.38
18.7
1.57
2.11
1.31
6.70
228
bLOD
529
71.6
9.38
bLOD
197
bLOD
1.72
9.36
51.7
891
2.38
4.16
bLOD
2213 1.8–53.1 (Sum of DDT) [2]
α-HCH
β-HCH
γ-HCH
Heptachlor
epoxide
DDE
DDD
Methoxychlor
Sum of OCPs
18 420 10 075 3500–61 700 [1], 600–13 200 [2]
9–85 [1], 2.6–37.8 [2]
Mass-balance TEQs [ng/g d.w.]
PCB contrib. [3] 0.237 bLOD
PAH contrib. [4] 0.914 1.459
Sum of TEQs
1.151 1.459
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 °C) 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).
Bioassay TCDD-EQs [ng/g d.w.]
Crude-24h
123.4 50.7
Acid-treat-24h
10.3
1.18
Crude-72h
13.5
21.0
Acid-treat-72h
6.90
0.11
1.9–23 ng/g d.w. [1], 1.5–7.8 ng/g d.w. [2]
Comparison with the values previously reported in the Czech Republic sediments
is provided — [1] Hilscherova et al. (2001); [2] Vondráček et al. (2001). Massbalance 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]. (bLOD — below the limit of detection).
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L. Bláha et al. / Environment International 32 (2006) 749–757
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™ (Packard, Meriden, CT, USA) and were exposed
1.0
1.0
0.5
0.5
Pr.Blank
Cells
0.0
0.025 / 0.25 / 2.5
Pilnok
0.025 0.25 2.5
Ref. sed.
CYP11A
0.025 / 0.25 / 2.5
Pilnok
0.025 0.25 2.5
Ref. sed.
3β
βHSD2
CYP17
1.5
1.0
1.0
1.0
0.5
0.5
0.5
0.0
0.0
0.0
Pilnok
0.025 0.25 2.5
Pr.Blank
Cells
0.025 / 0.25 / 2.5
0.025 / 0.25 / 2.5
Pilnok
Ref. sed.
CYP21
Cells
1.5
DMSO
1.5
Pr.Blank
2.0
Cells
2.0
DMSO
2.0
0.025 0.25 2.5
Ref. sed.
0.025 / 0.25 / 2.5
Pilnok
0.025 0.25 2.5
Ref. sed.
CYP11B2
15.0
2.0
12.0
1.5
9.0
1.0
6.0
0.5
3.0
Pilnok
0.025 0.25 2.5
Ref. sed.
17βHSD4
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
Ref. sed.
Pr.Blank
Pilnok
0.025 0.25 2.5
DMSO
Pr.Blank
Cells
DMSO
0.025 / 0.25 / 2.5
Cells
0.0
0.0
0.0
0.025 / 0.25 / 2.5
Pilnok
0.025 0.25 2.5
Ref. sed.
DMSO
17βHSD1
0.025 / 0.25 / 2.5
Pr.Blank
Ref. sed.
Cells
0.025 0.25 2.5
Pr.Blank
Pilnok
Cells
Pr.Blank
Cells
0.025 / 0.25 / 2.5
DMSO
0.0
0.0
DMSO
Gene expression (fold induction - relative to solvent control, DMSO)
DMSO
0.0
Pr.Blank
1.5
DMSO
1.5
Pr.Blank
2.0
Cells
HMGR
2.0
DMSO
StAR
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: 2,3,7,8tetrachlorodibenzo-p-dioxin (AhR) and 17β-estradiol (ER).
0.025 / 0.25 / 2.5
Pilnok
0.025 0.25 2.5
Ref. sed.
Fig. 2. Effects of sediment extracts on the expression of steroidogenesis genes in H295R cells as determined with quantitative real-time PCR. H295R cells cultured in
6-well plates were exposed to crude extracts of Pilnok and Reference sediments at three concentrations (0.025, 0.25 and 2.5 mg sediment d.w./mL) for 24 h. Total RNA
was extracted, reverse-transcribed and quantified with real-time PCR. Data are expressed as means +/− standard deviations of 3 replicate exposures analyzed in
triplicates (data were normalized to the expression of beta-actin and expressed as fold induction relative to appropriate solvent control; DMSO fold induction = 1).
L. Bláha et al. / Environment International 32 (2006) 749–757
3βHSD2
2.0
1.5
*
1.0
*
0.5
Pilnok
AcidTreated
Crude
AcidTreated
Crude
DMSO
0.0
Cells
Ref. sed.
CYP21
2.0
1.5
*
1.0
*
0.5
Pilnok
AcidTreated
Crude
AcidTreated
Crude
DMSO
0.0
Ref. sed.
CYP11B2
15.0
*
12.0
*
*
AcidTreated
*
6.0
Crude
9.0
3.0
AcidTreated
Crude
0.0
DMSO
The instrumental analyses (Table 1) revealed relatively high
concentrations of PAHs in both Pilnok Pond (18 μg ∑PAHs 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 evaluated 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 N 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
3βHSD2 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, 3βHSD2, CYP21) revealed trends similar to those observed 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
Cells
3. Results
Cells
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).
prolonged 72 h exposures resulted in a decrease of the induction potencies. 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, 2,3,7,8TCDD and TCDD equivalents (TCDD-EQs) were estimated (Hilscherova 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 substantial contribution of acid labile compounds (particularly PAHs) to the
observed AhR-modulated responses (Table 1) as also reported previously (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
Gene expression (fold induction - relative to solvent control, DMSO)
2.7. Statistical analyses
753
Pilnok
Ref. sed.
Fig. 3. Effects of crude extracts and sulfuric acid-treated samples on the expression
of selected steroidogenic genes in H295R cells. (For legend see Fig. 2; asterisks
indicate significant difference from solvent control, DMSO, ANOVA + Dunnet's
test P b 0.05).
754
L. Bláha et al. / Environment International 32 (2006) 749–757
TCDD-24h
TCDD-72h
Crude-24h
Crude-72h
Acid-Treat-24h
Acid-Treat-72h
100
A
4. Discussion
10
1
100
B
Reference
(sed. No. 2)
10
1
1x10-8
1x10-7
ug TCDD/mL
1x10-6
1
10
100
1000
ug sediment dw/mL
Fig. 4. Concentration–induction curves of AhR dependent luciferase in H4IIEluc cell bioassays after exposure to sediment extracts of Pilnok (A) and
Reference sediments (B), and standard 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD). The cells were exposed for 24 h (filled symbols) and 72 h (empty
symbols). Effects of crude extracts (diamonds) and sulfuric acid-treated samples
(triangles) are compared. The data represent means of 3 replicates, error bars are
not presented to keep the presentation clear (coefficient of variance did not
exceed 20%).
the crude sample (13.5 ng/g d.w.) after acid treatment. Lesser AhRmediated 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 10fold 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 recorded at concentrations around 2.5 mg d.w./mL (Figs. 2 and 3), maximum stimulation of EROD in H295R cells as well as maximal
inductions of AhR-dependent luciferase in H4IIE-luc bioassay occurred at concentrations significantly lower (0.025–0.1 mg d.w./mL,
Figs. 4 and 5).
Instrumental identification and quantification of known AhRinducing 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
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). Therefore, we focused on characterization of the major organic contaminants 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)
AhR-dependent luciferase / fold-induction [log]
Pilnok (sed. No. 1)
H4IIE-luc cells (Machala et al., 2001). Resulting TEQs were
1.15 ng TCDD/g d.w. for Pilnok Pond sediment (contribution of
PCBs ∼ 0.24 ng/g, PAHs ∼ 0.91 ng/g), and 1.46 for Reference sediment
(PCBs b LOD, PAHs ∼ 1.46 ng/g).
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)
Fig. 5. Concentration–induction curves of ethoxyresorufin-O-deethylase
(EROD) activity by sediment extracts in H295R cells after a 24 h in 24-well
plates. Mean values +/− standard deviation of 3 replicates.
L. Bláha et al. / Environment International 32 (2006) 749–757
(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 antiestrogenic 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 concentrations 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 concentrations of PCBs at both localities and minor contribution to
E2-24h
E2-72h
ER-dependent luciferase
fold-induction
5.0
4.5
Pilnok-24h
Pilnok-72h
Ref.sed.-24h
Ref.sed.-72h
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
1x10
-6
1x10
-5
1x10
ug/mL
17β-estradiol
-4
1
10
100
ug dw/mL
Sediment
Fig. 6. Concentration–induction curves of ER-dependent luciferase in MVLN
cell bioassay after exposure to crude extracts of Pilnok and Reference sediments,
and 17β-estradiol. The data represent means of 3 replicates, for clarity error bars
are not presented (coefficients of variance did not exceed 20%).
755
overall TEQs was confirmed (Table 1), other persistent AhRactive 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 3βHSD2 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 specific 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 3βHSD2, 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)
756
L. Bláha et al. / Environment International 32 (2006) 749–757
who observed significant up-regulation of both 3βHSD2 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 betanaphthoflavone (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 between the effects of prototypical single compounds and natural
mixtures were demonstrated also in placental explants (Augustowska 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 upregulate 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 AhRdependent 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 (upregulation of CYP11B2 and down-regulations of CYP21 and
3βHSD2) 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 confirmations 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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