Toxicology and Applied Pharmacology 217 (2006) 114 – 124
www.elsevier.com/locate/ytaap
Human adrenocarcinoma (H295R) cells for rapid in vitro determination of
effects on steroidogenesis: Hormone production
Markus Hecker a,⁎, John L. Newsted b , Margaret B. Murphy a,c , Eric B. Higley a ,
Paul D. Jones a , Rudolf Wu c , John P. Giesy a,c,d
a
Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824, USA
b
ENTRIX, Inc., Okemos, MI 48864, USA
c
Centre for Coastal Pollution and Conservation, Department of Biology and Chemistry, City University of Hong Kong,
Kowloon, Hong Kong, SAR, China
d
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
Received 16 March 2006; revised 24 July 2006; accepted 25 July 2006
Available online 29 July 2006
Abstract
To identify and prioritize chemicals that may alter steroidogenesis, an in vitro screening assay based on measuring alterations in hormone
production was developed using the H295R human adrenocortical carcinoma cell line. Previous studies indicated that this cell line was useful to
screen for effects on gene expression of steroidogenic enzymes. This study extended that work to measure the integrated response on production of
testosterone (T), estradiol (E2), and progesterone/pregnenolone (P) using an ELISA. Under optimized culture and experimental conditions, the
basal release of P, T and E2 into the medium was 7.0 ± 1.2 ng/ml, 1.6 ± 0.4 ng/ml, and 0.51 ± 0.13 ng/ml, respectively. Model chemicals with
different modes of action on steroidogenic systems were tested. Exposure to forskolin resulted in dose-dependent increases in all three hormones
with the greatest relative increase being observed for E2. This differed from cells exposed to prochloraz or ketoconazole where P concentrations
increased while T and E2 concentrations decreased in a dose-dependent manner. In cells exposed to fadrozole, E2 decreased in a dose-dependent
manner while T and P only decreased at the greatest dose tested. Aminoglutethimide decreased P and E2 concentrations but increased T
concentrations. Vinclozolin reduced both P and T but resulted in a slight increase in E2. The alteration in the patterns of hormone production in the
H295R assay was consistent with the modes of action of the chemicals and was also consistent with observed effects of these chemicals in animal
models. Based on these results, the H295R in vitro system has potential for high throughput screening to not only characterize the effects of
chemicals on endocrine systems but also to prioritize chemicals for additional testing.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Endocrine disruption; Chemical screening; Steroid hormones; Steroidogenesis
Introduction
Recently, studies have suggested links between the exposure
to natural and man-made substances in the environment and
alterations in endocrine and reproductive systems in wildlife
and humans (Kavlock et al., 1996; Ankley et al., 1998). To
address emerging concerns that such substances may alter
endocrine system function, the US EPA has developed and
⁎ Corresponding author. 218c NFST Building, Aquatic Toxicology Laboratory, National Food Safety and Toxicology Center, Michigan State University,
East Lansing, MI 48824, USA. Fax: +1 517 432 2310.
E-mail address: heckerm@msu.edu (M. Hecker).
0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2006.07.007
implemented an endocrine disruptor-screening program (EDSP)
as mandated by the Food Quality Protection Act of 1996. The
focus of this multi-tiered program has been first to develop in
vitro and in vivo assays to identify and classify substances
relative to their potential interaction with endocrine systems
(Tier I) and then to develop concentration–response relationships in animal models (Tier II) (EDSTAC, 1998).
Much of the effort to establish Tier I assays has been focused on
the development and validation of in vitro estrogen receptor (ER)
and androgen receptor (AR) binding assays (Villeneuve et al.,
1998) or on the development and characterization of transcriptional activation assays with ER and AR in stably transfected cell
lines (Wilson et al., 2002, 2004). However, there are a number of
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
non-receptor-mediated processes that may also alter endocrine
function (Sanderson et al., 2002). For example, compounds can
modulate the enzymes involved in the production, transformation,
or elimination of steroid hormones, and thus may alter the absolute
and relative concentrations of hormones in blood and tissues
(Ohno et al., 2002; Hilscherova et al., 2004). These non-receptormediated effects are often exerted indirectly via alterations of
common signal transduction pathways (Rainey et al., 1993; Zhoa
et al., 1998). Currently there are additional efforts underway to
establish and validate a series of in vitro and in vivo test systems
such as microsomal aromatase assays, pubertal male and female
rat assays, the fish reproductive screen, and the frog thyroid assay
that attempt to address some of the issues arising from nonreceptor-mediated endocrine disruption. In this context, a rodent
minced-testis assay was proposed as the Tier I screen to detect
chemicals with potential to disrupt steroid hormone production
(EDSTAC, 1998). However, questions have been raised about the
utility of gonad explant assays. In particular, rodent-based testis
and ovary explant assays were found to yield high rates of false
positive/negative responses (Powlin et al., 1998). Also, in testis
explant assays, it is not possible to descriminate between
cytotoxicity of hormone-producing cells and other cell types.
Finally, there has been increasing criticism regarding the use of
animals in Tier I screening assays. As a consequence, there is need
for less variable and more reliable in vitro test systems as
alternatives to these tissue explant assays. One cell line that has
been shown to be a useful in vitro model for studying
steroidogenic pathways and processes is the human H295R
adrenocarcinoma cell line (Hilscherova et al., 2004; Zhang et al.,
2005).
The H295R cell line has been shown to express all the key
enzymes of steroidogenesis (Gazdar et al., 1990; Rainey et al.,
1993; Staels et al., 1993). H295R cells have physiological
characteristics of zonally undifferentiated human fetal adrenal
cells and have the ability to produce the steroid hormones found
in the adult adrenal cortex (Gazdar et al., 1990; Staels et al.,
1993; Harvey and Everett, 2003). As well as permitting the
measurement of hormone production, another advantage of the
H295R cell bioassay is that it can be used to evaluate effects of
chemicals on gene expression and the enzymatic activities of
steroidogenic genes (Hilscherova et al., 2004; Sanderson et al.,
2000; Zhang et al., 2005). Thus, the H295R cell line holds
promise as a substitute for gonad explant assays within the
tiered endocrine disruptor-screening and testing framework, and
as an alternative to explant assays for research applications.
In order to validate the capacities of the H295R cells to identify
effects on sex steroid production, the cells were exposed to six
model chemicals, chosen based on their known effects on steroid
metabolism and steroidogenic gene expression. The chemicals
tested were prochloraz, ketoconazole, fadrozole, aminogluthetimide, forskolin, and vinclozolin. Prochloraz is an agricultural
imidiazol fungicide that inhibits a CYP enzyme involved in
ergosterol synthesis, but has also been reported to inhibit other
CYP enzymes, and to act as a potent aromatase inhibitor (Mason
et al., 1987; Laignelet et al., 1989; Troesken et al., 2006).
Ketoconazole is a pharmaceutical imidozole fungicide that has
been shown to alter the activity of several cytochrome P450s
115
involved in steroid synthesis (e.g. CYP11A, CYP17, and CYP19)
(Kan et al., 1985; Johansson et al., 2002; Hilscherova et al., 2004).
Fadrozole is a non-steroidal reversible and competitive inhibitor
of aromatase as well as of CYP11B-associated enzymatic
activities (Steele et al., 1987; Muller-Vieira et al., 2005).
Aminoglutethimide is an aromatase inhibitor that was also
reported to interact with several other steroidogenic enzymes
such as StAR and 17β-HSD (Johansson et al., 2002; Hilscherova
et al., 2004). Forskolin is a general inducer of steroidogenesis that
acts via the activation of cAMP pathways (Hilscherova et al.,
2004). Vinclozolin is a chlorinated fungicide that has been shown
to act as an antagonist of the androgen receptor (Dai et al., 2001;
Kelce et al., 1997; Sanderson et al., 2000).
The aim of the current study was to develop and standardize
an in vitro Tier 1 screening assay using the H295R cell line to
prioritize chemicals that act to alter hormone production.
Specifically, the goals of this study were to: (1) optimize culture
and experimental assay conditions; (2) characterize the basal
production of testosterone, estradiol and progesterone; (3)
develop dose–response relationships for six mode chemicals;
and (4) compare possible effects of these model chemicals to
available in vivo data.
Materials and methods
Test chemicals. Forskolin (FOR), vinclozolin (VIN), ketoconazole (KET), and
aminoglutethimide (AMG) were obtained from Sigma Chemical Co. (St. Louis,
MO, USA). Prochloraz (PRO) was purchased from Aldrich (St. Louis, MO, USA).
Fadrazole (FAD) was obtained from Novartis Pharma AG (Basel, CH).
Cell culture. The H295R human adrenocortical carcinoma cell line was
obtained from the American Type Culture Collection (ATCC CRL-2128; ATCC,
Manassas, VA, USA) and was grown in 100 × 20 mm Petri Dishes with 12.5 ml
of supplemented medium at 37 °C with a 5% CO2 atmosphere as described
previously (Hilscherova et al., 2004). Briefly, the cells were grown in a 1:1
mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient
mixture (DMEM/F12) (Sigma D-2906; Sigma, St. Louis, MO, USA)
supplemented with 1.2 g/l Na2CO3, 5 ml/l of ITS+ Premix (BD Bioscience;
354352), and 12.5 ml/l of BD Nu-Serum (BD Bioscience; 355100) unless
otherwise specified.
Experimental design. All experiments were conducted in 24-well cell culture
plates (COSTAR, Bucks, UK). One ml of cell suspension was added to each well
and the cells were allowed to attach for 24 h. After the attachment period, the
medium was changed and the experiment was initiated. The effect of cell
confluency on steroid hormone production was evaluated at two different seeding
densities, 30% (∼ 200,000 cells/ml) and 70% (∼ 470,000 cells/ml) confluency.
To examine the possible influence of hormones and other substances in the cell
culture medium on hormone production, the medium was stripped with dextrancoated charcoal prior to test initiation and the results compared to normal
supplemented medium. In the time course experiment, H295R cells were
cultured under standard conditions and basal hormone production was
determined in medium collected at 3, 6, 12, 24, 48, and 72 h.
For chemical tests, cells were exposed for 48 h in 24-well plates. DMSO was
used as carrier solvent and did not exceed 0.1% v/v. Test plates included six
chemical concentrations, a solvent control (SC), and a blank control (CTR), in
triplicate. At the end of each experiment, the culture medium was transferred to
an Eppendorf tube and stored at − 80 °C prior to analysis. Four separate tests
were conducted for each model chemical.
Prior to measuring hormone concentrations, cell viability was evaluated in
CTR, SC, and the three greatest exposure test chemical concentrations of each
chemical with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide) bioassay (Mosman, 1983). Cytotoxic chemical concentrations were
not included in the hormone concentration measurements.
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M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
Table 1
Quality performance criteria for determining progesterone (P), testosterone (T), and estradiol (E2) in the medium of H295R cells
Detection limit (pg/ml)
Assay
d
S-LOQ
e
Linearity
(fold medium dilution)
Within-assay CV
(%) a (mean ± SD)
Between-plate CV
(%) b (mean ± SD)
P
7.8
<100
10–250
11 ± 10
8±5
T
3.9
<50
10–250
14 ± 9
18 ± 11
E2
7.8
<8
1–25
10 ± 7
8±7
Accuracy c
y = 1.0362 x
r2 = 0.995
y = 1.159 x
r2 = 0.974
y = 1.0 x
r2 = 1.0
a
Calculated based on triplicate measures for each sample.
Same sample measured on different ELISA plates; total number of plates = 11.
c
Liner regression between nominal and measured spike concentrations. Spike concentrations were as follows: P, 0, 250, 500, 1000, 5000 pg/ml; T, 0, 100, 250, 500,
and 3000 pg/ml; E2, 0, 100, 1000 pg/ml.
d
Detection limit of the assay represents the lowest detectable amount of hormone per milliliter standard.
e
S-LOQ: Sample limit of quantification. Lowest detectable concentration of each hormone in medium after 48 h and at a cell density of 200,000 cells per milliliter
medium within the tested linear range of dilutions.
b
Hormone measurements. Frozen medium was thawed on ice, the hormones
were extracted twice with diethyl ether (5 ml) in glass tubes, and phase
separation was achieved by centrifugation at 2000× g for 10 min. Solvent was
evaporated under a stream of nitrogen, and the residue was dissolved in ELISA
assay buffer and was either immediately measured or frozen at − 80 °C for later
analysis. Hormones in culture medium were measured by competitive ELISA
using the manufacturer's recommendations (Cayman Chemical Company, Ann
Arbor, MI; Progesterone [Cat # 582601], Testosterone [Cat # 582701], 17βEstradiol [Cat # 582251]). However, since there is a 61% cross-reactivity of the
progesterone antibody with pregnenolone, progesterone concentrations are
expressed as total progesterone + pregnenolone (P). Extracts of culture medium
were diluted 1:2, 1:5, and 1:10 for estradiol, and 1:50, 1:100, and 1:150 for
testosterone and P prior to use in the ELISAs.
Statistical analyses. Statistical analyses of hormone data were conducted
using SYSTAT 11 (SYSTAT Software Inc., Point Richmond, CA). All data are
expressed as means and standard deviations. Student's t test was used to
evaluate differences in hormone production between high and low confluency
cells and to examine differences between cells cultured in regular and charcoalstripped (DCC) medium. Analysis of variance (ANOVA) was conducted to test
for differences among all treatments. Differences between controls and cells
exposed to model compounds were evaluated using Dunnett's test applied to
relative changes in hormone production. Relative changes were calculated as
follows (Eqs. (1) and (2)):
If SC > treatment :
SC
1 1
Treatment
Results
Hormone measurement
All ELISA systems used in this study to measure hormones
were validated in terms of sensitivity, accuracy, precision,
linearity, and parallelism to the standard curves (Table 1). To
evaluate the need for extraction, hormone concentrations in
ether-extracted medium were compared to non-extracted
medium. While extraction did not have a significant effect on
T concentrations, the reported concentrations of both P and E2
concentrations were approximately 3-fold greater in the nonextracted medium. The apparent concentrations of P and E2 in
the extracted samples were 8.6 ± 1.2 ng/ml and 0.34 ± 0.01 ng/
ml, respectively, while in the non-extracted samples concentrations were 25 ± 7 ng/ml and 0.97 ± 0.22 ng/ml, respectively. This
difference was not due to poor extraction efficiency since
recovery, as monitored by the addition of a tracer amount of 3Hlabelled T to each sample before extraction, was similar
between non-extracted and extracted samples (results not
shown).
ð1Þ
Basal hormone production
Treatment
1 1
If Treatment > SC :
SC
ð2Þ
Differences with p < 0.05 were considered significant.
In regular supplemented medium, concentrations of T and E2
in wells seeded to 70% confluence were approximately double
those measured in wells seeded at 30% confluence while
Table 2
Concentrations of progesterone, testosterone and estradiol in the medium of H295R cells treated with either DMSO or with prochloraz (PRO) for 48 h a
PRO (1 μM)
0.1% DMSO
p values (t test: Nu vs. DCC) b
30%
P (ng/ml)
T (ng/ml)
E2 (ng/ml)
a
70%
30% Nu
30% DCC
70% Nu
70% DCC
30% Nu
30% DCC
70% Nu
70% DCC
Control
PRO
Control
PRO
8.0 ± 2.0
2.3 ± 0.4
0.41 ± 0.05
6.6 ± 0.8
1.7 ± 0.3
0.14 ± 0.01
4.1 ± 0.4
5.0 ± 0.1
0.91 ± 0.06
5.9 ± 0.9
6.1 ± 1.1
0.25 ± 0.02
16 ± 3
0.2 ± 0.1
0.04 ± 0.01
16 ± 0
0.17 ± 0.01
0.02 ± 0.01
13 ± 1
0.35 ± 0.05
0.04 ± 0.01
15 ± 1
0.32 ± 0.00
0.03 ± 0.00
0.249
0.073
0.000
0.838
0.410
0.048
0.018
0.132
0.000
0.223
0.654
0.475
P = progesterone/pregnenolone; T = testosterone; E2 = 17B-estradiol. Concentrations are in units of ng/ml medium and are given as mean and standard deviations.
H295R cells were seeded at 30% confluency or 70% confluency. “Nu” indicates regular cell culture medium with Nu serum while DCC indicates medium that has
been stripped prior to seeding of cells.
b
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
concentrations of P were unaffected (Table 2). When comparing
regular and DCC medium, E2 concentrations were greater in
regular medium in both 30% (t test: p < 0.001) and 70% (t test:
p < 0.001) confluent wells when compared to cells grown in
DCC medium. In contrast, P concentrations in the 70%
confluent wells (t test: p < 0.05) were greater in DCC medium
than in regular medium while P did not differ from the 30%
confluent wells. In cells treated with prochloraz (PRO) E2
concentrations were significantly less in regular medium (t test;
p < 0.05) than in DCC medium.
In the time course experiment, all three steroids showed
increases in concentration overtime with maximum P and E2
concentrations being measured at 48 h whereas T increased
throughout the experiment (Fig. 1). Consequently, all further
experiments were conducted with regular medium using cells
seeded at approximately 30% confluency (200,000 cells per ml)
for 48 h since these conditions were deemed optimal for
monitoring both positive and negative changes in hormone
production.
In the CTRs, concentrations of all three steroids showed
considerable variability among experiments (n = 5) conducted
on different days and with different batches of cells. P
concentrations from the different experiments ranged from
3.0 ± 0.6 (mean of all plates from one experiment ± SEM) to
9.2 ± 0.7 ng/ml in culture medium with an overall average of
7.0 ± 1.2 ng/ml. Mean concentrations of T ranged from 0.6 ± 0.2
to 2.3 ± 0.1 ng/ml medium (overall mean 1.6 ± 0.4 ng/ml). E2
concentrations ranged from 0.21 ± 0.05 to 0.81 ± 0.24 ng/ml
(overall mean of 0.51 ± 0.13 ng/ml). The mean coefficients of
variation (CV) for the duplicates within a plate (n = 15 plates)
were 9 ± 2 (mean ± SEM), 17 ± 3, and 8 ± 2% for P, T, and E2,
respectively.
While not consistently observed, DMSO appeared to have
some effect on hormone production. On average, hormone
production in cells treated with 0.1% DMSO was slightly less
than in the CTRs. For DMSO-treated cells, concentrations of P
were approximately 80% (±12%) (mean ± SEM; n = 5) of those
measured in the CTR while T and E2 concentrations were 82%
117
Fig. 2. Effect of prochloraz on hormone release by H295R cells. Cells were
treated for 48 h with the indicated concentrations of prochloraz. Hormone data
are expressed as relative changes compared to solvent controls (SC = 0). Values
represent the mean ± SD. Significant differences for progesterone (*), testosterone (+), and estradiol (@) are reported relative to the solvent control. Multiple
symbols indicate different significant levels: 1 symbol = p < 0.05; 3 symbols =
p < 0.001.
(± 10%) and 80% (± 15%) of CTR concentrations, respectively.
Therefore, in tests with model chemicals, hormone production
was compared to the SCs rather than to CTRs. To account for
inter-plate variability in hormone production, concentrations for
each test chemical were expressed relative to the average SC
response of each plate.
Effects of model chemicals
Cytotoxicity/Cell viability
Cytotoxicity was observed in cells treated with PRO at
concentrations greater than 3 μM (results not shown). Therefore, the prochloraz concentrations evaluated in this study
ranged from 0.001 to 1 μM. None of the concentrations tested
for any of the other chemicals affected cell viability. A summary
of the cell viability results is given in the Supplementary
materials.
Prochloraz (PRO)
Exposure to PRO resulted in significant changes in
concentrations of P, T, and E2 (Fig. 2). P concentrations
increased in a dose-dependent manner with concentrations
being significantly greater than the SCs at PRO concentrations greater than 0.01 μM. Testosterone and E2 decreased in
a dose-dependent manner at PRO concentrations greater than
0.03 and 0.003 μM, respectively. Maximal changes in
hormone concentrations were observed at the greatest noncytotoxic dose of PRO (1 μM). At this dose, P concentrations
were 5-fold greater than the SC while E2 and T concentrations were 5.5- and 3-fold less, respectively, than their
respective SCs.
Fig. 1. Time-dependent basal secretion of testosterone, progesterone and
estradiol by H295R cells under standard culture conditions. H295R cells were
cultured in medium supplemented with NuSserum. Hormone concentrations
(pg/ml) in medium are given as means ± SD.
Ketoconazole (KET)
Ketoconazole significantly inhibited E2 and T production and
induced P production by H295R cells (Fig. 3). Concentrations of
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M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
KET greater than 0.03 μM significantly reduced testosterone
production and the effect became more pronounced with
increasing concentrations. Similarly, mean E2 production
showed a steady decline with increasing KET concentration,
and the effect was significant at concentrations greater than
0.3 μM. Progesterone concentrations in medium increased
progressively, and were significantly greater than the SC at a
KET dose of 1 μM (p < 0.01). At KET concentrations greater
than 1 μM, P production started to decrease again. The effect of
KET on T production occurred at lesser concentrations than for
E2, causing a 50% reduction compared to the solvent controls at
approximately 2 μM. The least change was observed for P
(≈2.5-fold).
Fadrozole (FAD)
A significant, dose-dependent reduction (up to 4.7-fold) in
E2 production by H295R cells was observed for FAD
concentrations greater than 0.1 μM (Fig. 4). However, no
comparable response pattern was observed for T and P with
the exception that at the greatest dose, 100 μM, both mean T
(p < 0.001) and P (p < 0.05) concentrations were significantly
reduced relative to the solvent controls. Mean T production
was suggestive of a dose-dependent decrease (maximum
reduction ≈ 12-fold) at FAD concentrations greater than 1 μM,
but due to the relatively great variability in the hormone
concentrations, only the greatest dose was statistically
significant when compared to the solvent control. Although
statistically significant, the reduction of P production at
100 μM FAD was not great and was approximately 0.5-fold
that of the control levels.
Fig. 4. Effect of fadrozole treatment on testosterone (left y axis) and
progesterone and estradiol (right y axis) release by H295R cells. Cells were
treated for 48 h with the indicated concentrations of fadrozole. Hormone data are
expressed as relative changes compared to solvent controls (SC = 0). Values
represent the mean ± SD. Significant differences for progesterone (*), testosterone (+), and estradiol (@) are reported relative to the solvent control. Multiple
symbols indicate different significant levels: 1 symbol = p < 0.05; 2 symbols =
p < 0.01; 3 symbols = p < 0.001.
greater than 1 μM with a maximum response being observed
(3-fold greater) at 10 μM. Concentrations of E2 were
significantly less relative to the SCs at AMG concentrations
of 10 μM or greater with a more than 4-fold reduction at the
greatest dose tested (30 μM). Concentrations of P in the
medium decreased in a manner similar to that observed for E2
except that the only statistically significant reduction from SCs
was observed at the greatest dose, 30 μM.
Aminogluthetimide (AMG)
Treatment with AMG resulted in a dose-dependent change
in hormone concentrations (Fig. 5). T concentrations were
significantly greater than the SCs at AMG concentrations
Forskolin (FOR)
FOR exposure increased all hormone concentrations (Fig.
6). The greatest increase relative to the SCs (7-fold) was
observed for E2 at 30 μM. E2 concentrations were
Fig. 3. Effect of ketoconazole treatment on hormone release by H295R cells.
Cells were treated for 48 h with the indicated concentrations of ketoconazole.
Hormone data are expressed as relative changes compared to solvent controls
(SC = 0). Values represent the mean ± SD. Significant differences for progesterone (*), testosterone (+) and estradiol (@) are reported relative to the solvent
control. Multiple symbols indicate different significant levels: 1 symbol =
p < 0.05; 2 symbols = p < 0.01; 3 symbols = p < 0.001.
Fig. 5. Effect of aminoglutethimide treatment on hormone release by H295R
cells. Cells were treated for 48 h with the indicated concentrations of
aminoglutethimide. Hormone data are expressed as relative changes compared
to solvent controls (SC = 0). Values represent the mean ± SD. Significant
differences for progesterone (*), testosterone (+) and estradiol (@) relative to
the solvent control. Multiple symbols indicate different significant levels: 3
symbols = p < 0.001.
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
Fig. 6. Effect of forskolin treatment on hormone release by H295R cells. Cells
were treated for 48 h with the indicated concentrations of forskolin. Hormone
data are expressed as relative changes compared to solvent controls (SC = 0).
Values represent the mean ± SD. Significant differences for progesterone (*),
testosterone (+), and estradiol (@) relative to the solvent control. Multiple
symbols indicate different significant levels: 1 symbol = p < 0.05; 2 symbols =
p < 0.01; 3 symbols = p < 0.001.
significantly greater than the SCs at all doses greater than
0.1 μM. P and T concentrations increased in a dose-dependent
manner but the relative increases were less than those
observed for E2. At FOR concentrations greater than
0.1 μM, the concentrations of P and T were statistically
greater than the SCs. The maximum concentration of both P
and T (3-fold above SCs) was observed in cells treated with
30 μM FOR.
Vinclozolin (VIN)
Cell viability appeared to be slightly reduced upon
exposure to the three greatest VIN concentrations tested
(100, 300, 1000 μM), resulting in approximately 70 to 73%
viable cells in these treatments. However, since the reduction
in cell viability was not dose-dependent, all data except the
1000 μM exposure were used for hormone analyses. Exposure
of H295R cells to VIN resulted in a maximum increase in E2
concentration that was less than 1.5-fold, while the maximum
decreases for T and P were 2- and 3-fold, respectively (Fig. 7).
Decreases in P and T concentrations were characterized by a
relatively high variation among the different experiments
conducted. Changes in the relative P, T, and E2 concentrations
were statistically significant in cells exposed to 30 and
100 μM vinclozolin.
119
hormone ELISA systems utilized in this study cross-react
with steroid metabolites and conjugates such as sulfates and
glucuronates. Such compounds are likely to be produced by
the cells, and are removed during the extraction process.
However, the hormone metabolizing capabilities of H295R
cells have not yet been described, and therefore, the exact
cause for the observed differences remains unclear. Additional
experiments are needed that describe steroid metabolism by
these cells. It is also important to note that since the antibody
used to measure P has a cross-reactivity to pregnenolone
(approximately 61%), it is likely that the P concentrations
measured in this study were primarily pregnenolone since it
has been reported that H295 cells produce approximately 75fold more pregenolone than progesterone (Gazdar et al.,
1990). Absolute P concentrations measured in our study
(mean = 7.0 ng/2 × 105 cells/48 h) were comparable to those of
pregnenolone previously described (Gazdar et al., 1990)
(15.6 ng/2 × 105 cells/48 h) after applying a correction factor
(0.61) for cross-reactivity. Since the goal of this study was to
develop a test system to assess the effects of chemicals on E2
and T production, it can be assumed that the P assay as used
in our study was sufficient to evaluate the status of the
precursor pool involved in the synthesis of androgens and
estrogens. As a result, changes in the concentrations of P
would be diagnostic of up-stream events even if the assay
was not measuring only progesterone.
To date, most studies with H295R cells have measured
hormone products of the mineralcorticoid (aldosterone) and
glucocorticoid (cortisol) pathways as well as certain androgens
such as androstenedione (Ohno et al., 2002; Rainey et al.,
1993; Rainey et al., 1994; Thomson et al., 2003; Li et al.,
2004; Li and Wang, 2005). The major androgen production
pathways are thought to include formation of 17α-hydroxyprogesterone and subsequent synthesis of androstenedione
Discussion
General test performance
The ELISA determination of P, T, and E2 concentrations
was accurate and precise. However, it was necessary to
extract hormones from the medium prior to determination of
P and E2 because the antibodies used in these ELISAs appear
to cross-react with unknown compounds in the non-extracted
medium. In fact, some of the antibodies used with the
Fig. 7. Effect of vinclozolin treatment on release of hormones by H295R cells.
Cells were treated for 48 h with the indicated concentrations of vinclozolin.
Hormone data are expressed as relative changes compared to solvent controls
(SC = 0). Values represent the mean ± SD. Significant differences for progesterone (*), testosterone (+), and estradiol (@) relative to the solvent control.
Multiple symbols indicate different significant levels: 1 symbol = p < 0.05; 2
symbols = p < 0.01; 3 symbols = p < 0.001.
120
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
(Gazdar et al., 1990). Although Gazdar et al. (1990)
hypothesized that cells secrete small amounts of T and E2,
they only quantified the production of androstenedione.
However, other studies have reported T production by
H295R cells (Danesi et al., 1996). In fact, concentrations
measured for androstenedione (0.11 ng/2 × 105 cells/48 h)
(Gazdar et al., 1990) are less than the T concentrations
measured in our study (1.6 ng/2 × 105 cells/48 h). To our
knowledge, there have been no reports quantifying estrogen
production in H295R cells even though aromatase activity has
previously been measured (Sanderson et al., 2002). While the
production of E2 by H295R cells was demonstrated in our
study, E2 concentrations were approximately 20- and 6-fold
less than those of P and T, respectively.
Hormone production of H295R cells was variable among
different batches/passages of cells. This variability may be due
to several factors; first, it has been shown in various stably
transfected cell lines that physiological and biochemical
fluctuations can be linked to the cell cycle (McFerran et al.,
2001; Barker et al., 2004). It has also been reported that with
increasing age (cell passage), cells may gradually lose their
capacity to maintain specific functions such as albumin
secretion, drug metabolism capabilities, and the functionality
of the urea cycle (Guillouzo, 1998; Priesner et al., 2004).
Finally, differences in cell density may have also contributed
to the variability in hormone production. To account for some
of this variability, hormone concentrations in the treated wells
were normalized to those measured in relevant SCs. Repeated
exposure experiments with model chemicals showed that
relative changes in hormone production were more reproducible than absolute hormone concentrations among experiments and were a reliable way to express effects of
compounds on steroid hormone production. Overall, based
on the data for the changes of hormone production as a
function of cell passage, it is recommended to use cells up to
passage 10. Past this passage, basal production of E2 declines
to the extent that detecting decreases of this steroid can be
limited. Furthermore, due to the fluctuations and changes in
cell-specific functions discussed above changes or shifts in
hormone homeostasis cannot be excluded with increasing age
of the cells. Further studies are needed to better elucidate the
changes in hormone production patterns and steroid homeostasis as a function of cell age.
Cell culture medium had some effect on hormone
production by H295R cells with the greatest effect observed
for E2. The use of dextran-coated charcoal-stripped (DCC)
medium reduced E2 concentrations relative to those of cells
grown in non-stripped medium. Previous studies have
demonstrated that changes in medium components can affect
production of hormones by H295R cells; culture of cells in
serum-free medium resulted in a reduction of C21 steroids
when compared to cells grown in medium containing 2%
serum (Gazdar et al., 1990). The basis for these differences
has not yet been investigated but may involve the presence of
cholesterol and trace levels of other steroid hormones in the
supplemented medium that serve as precursors in the
production of androgens and estrogens. The fact that the
differences observed for T and P at the lower cell confluency
tested (30%) were no longer apparent when these hormones
were measured in cells exposed at 70% confluence is likely
due to a more rapid depletion of these precursors under these
conditions. In addition, components within the serum may also
alter the aldosterone and/or cortisol biosynthesis pathways
such that the androgen/estrogen pathways are enhanced.
However, the significance of these factors in affecting the
production of the steroids monitored under the culture
conditions developed in this study needs to be evaluated
prior to the final validation of this bioassay. It is important to
note that since prochloraz, an inhibitor of estrogen synthesis,
reduced E2 concentrations in both DCC and regular
supplemented medium to near the sample specific limit of
quantification (S-LOQ), it is unlikely that the differences in
basal hormone production were due to contamination of
regular medium with hormones.
Exposure to model compounds
Agonists of specific steroidogenic pathways can alter
hormone profiles in the culture of H295R cells in a manner
that is consistent with their mode of action. For instance,
angiotensin II or K+ selectively promotes the synthesis of
aldosterone in H295 cells while adrenocorticotrophic hormone
(ACTH) and other cAMP pathway inducers are associated with
production of cortisol, androgens and estrogens (Rainey et al.,
1993; Bird et al., 1993; Pezzi et al., 1996). In our study,
exposure of H295R cells to the six model chemicals resulted in
different hormone concentration patterns consistent with their
modes of action.
Imidazol fungicides, including PRO and KET, have been
found to be potent inhibitors of aromatase among the many
vertebrate species including humans and fish (Mason et al.,
1987; Ankley et al., 2005) as well as being capable of affecting
other P450-dependent enzymes (Laignelet et al., 1989).
Significant reductions of plasma androgen (T and 11-ketotestosterone) concentrations in male fathead minnows (Pimephales
promelas) and a significant decrease in plasma E2 titers in
female fish exposed to 0.3 μM PRO were observed in a study
conducted by Ankley et al. (2005). Exposure to PRO resulted in
significant decreases in testicular and plasma T concentrations
and a significant increase in testicular P concentrations in a
study with fetal rats (Vinggaard et al., 2005). A possible
explanation for this could be a suppression of 17α-hydroxylase
activities. However, this study was not designed to address this
question, and therefore future studies will be necessary to
investigate the specific effects of PRO on steroidogenic enzyme
activities. Regardless, the fact that the effect on E2 production
was more potent than that on P and T indicates that aromatase
was likely more sensitive to the inhibitory effects of PRO than
the upstream target enzymes. While the findings in the above
studies were similar to those observed here with H295R cells,
effects on T and E2 occurred at three- to ten-fold lesser PRO
concentrations compared to the in vivo studies.
The pattern of responses in hormone concentration
observed in H295R cells exposed to KET was similar to that
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
observed in cells exposed to FAD where T and E2
concentrations were reduced in treated cells when compared
to controls. In general these response patterns agree with those
observed in other studies that evaluated the effects of these
compounds on steroidogenesis in several mammalian and fish
species. In a 15-day adult male rat study, concentrations of T
as well as E2 were significantly reduced from control levels in
rats exposed to FAD (O'Connor et al., 2002). However, in a
study of male pubertal rats, T concentrations were not different
from control levels in rats exposed to 6 mg/kg/d FAD (Marty
et al., 2001). The lack of response in the pubertal assay is not
surprising in that pre-pubertal animals have been shown to be
less responsive than adult animals when exposed to potential
endocrine active compounds. Finally, exposure of fathead
minnows to FAD resulted in a decrease of brain aromatase in
both male and female fish that was also accompanied by an
increase in T in male fish (Ankley et al., 2002). While no
effects were noted on E2 concentration in exposed male
minnows, E2 concentrations in female minnows were
decreased in a concentration-dependent manner.
The patterns of response for T and E2 observed with
H295R cells exposed to KET differed somewhat from those
observed in studies conducted with adult male rats. In these
studies, there was a statistically significant decrease in T while
E2 concentrations were significantly increased compared to
controls (O'Connor et al., 2002). In a rat testis explant assay,
exposure of up to 100 μM KET resulted in decreased T
concentrations while E2 concentrations were unaffected
(Powlin et al., 1998). In flounder (Platichthys flesus), exposure
of ovarian slices to KET resulted in statistically significant
decreases in T and E2 concentrations in a manner that was
similar to that observed for the H295R assay (Monteiro et al.,
2000). Most likely, the differential results observed for T and
E2 in these tests were due to the fact that KET has a greater
effect on P450-17,20 desmolase than on aromatase and thus,
would be expected to decrease T concentrations while E2
concentrations would decrease to a lesser extent or be
unaffected (Weber et al., 1991). In the current study, the
effect of KET on T in the H295R assay was greater than that
on E2. This result is consistent with the greater effect of KET
on C17,20-lyase (desmolase) activity of CYP17 as compared
to that associated with CYP19 and its associated aromatase
activity.
Like FAD, AMG is an aromatase inhibitor used in breast
cancer treatment to downregulate endogenous estrogen
production. AMG can also downregulate other steroidogenic
pathways including the synthesis of cortisol and aldosterone
(Fassnacht et al., 1998). While AMG consistently decreased
E2 synthesis regardless of species and test system, progestin
and androgen production in in vivo and in vitro systems can
be affected differently. In rat and fish (P. flesus) ovarian
cultures, AMG increased the concentration of T and
androstendione, respectively (Berman and Laskey, 1993;
Monteiro et al., 2000). AMG exposure also resulted in
decreased production of P in rat ovarian cell cultures and
H295R cells (Johansson et al., 2002; Berman and Laskey,
1993). The decrease in P synthesis is most likely due to
121
suppression of CYP11A activity while decreases in estrogen
production and subsequent accumulation of androgens may be
due to inhibition of aromatase (Johansson et al., 2002). In
contrast, in vivo studies of rats and humans have demonstrated
that AMG exposure may decrease androgen production
(Tsuitsui, 1992; Lundgren et al., 1996). These conflicting
results may be due to the reproductive status of the test
organism. Significant differences between effects of AMG
during diestrus, proestrus, and estrus stages of cycling rats
have been reported (Berman and Laskey, 1993).
Forskolin is a diterpene that can mimic the effects of ACTH
by stimulating adenylcyclase and increasing cAMP levels in
adrenal cells (Seamon et al., 1981). In our study, exposure of
H295R cells to FOR resulted in a general increase in the
production of P, T, and E2 that was consistent with effects
observed in other studies (Johansson et al., 2002; Rainey et al.,
1993; Cobb et al., 1996). However, while the production of all
three hormones increased, the greatest relative increase was
observed for E2 where FOR exposure resulted in approximately
a 7-fold increase in E2 concentrations as compared to a 2–3fold increase in T and P concentrations. This observation is also
consistent with another study that demonstrated the upregulation of aromatase in H295R cells by FOR (Watanabe and
Nakajin, 2004).
There are only a few reports on the effects of VIN on P, T,
or E2 production in animals. In rats, exposure to 400 mg VIN/
kg for 9 days (Yu et al., 2004) and 100 mg VIN/kg for 3–24 h
(Kubota et al., 2003) resulted in greater serum T concentrations compared to controls. However, in studies with
amphibians (Xenopus laevis) (van Wyk et al., 2003), reptiles
(Alligator mississippiensis) (Crain et al., 1997), and birds
(Coturnix coturnix japonica) (Niemann et al., 2004), VIN had
no effect on sex steroid concentrations. Similarly, exposure of
H295R cells to VIN resulted in only minor (< 2 to 3-fold)
changes in P, T, and E2 concentrations. However, unlike
observations in rats, exposure of H295R cells to VIN resulted
in decreased T and P concentrations while E2 levels slightly
increased at the greatest VIN concentrations. The basis for
these differences has not yet been investigated but may be due
to inherent differences that exist between in vivo and in vitro
systems where multiple tissues can influence the serum steroid
concentrations. It has been reported that the VIN metabolites
M1 and M2 are potent androgen receptor antagonists while the
parent compound VIN binds only weakly to the receptor
(Kelce et al., 1994). Thus, the antiandrogenic action of VIN is
unlikely to be mediated directly via effects on steroidogenic
pathways but rather may be a function of feedback mechanisms mediated by the inhibition of receptor-mediated processes by VIN metabolites. It is unclear whether H295R cells
have the capacity to metabolize VIN to M1 and M2.
Furthermore, the extent to which androgen receptors are
expressed in H295 cells is not known. Therefore, it remains
uncertain whether the trends observed for P, T and E2 in this
study can be attributed to the above processes, especially in
light of the fact that steroidogenesis in H295R cells can be
affected by alterations in cAMP-mediated pathways on which
the effect of VIN is unknown.
122
M. Hecker et al. / Toxicology and Applied Pharmacology 217 (2006) 114–124
The results from this study support the use of the H295R
cell line as a screening tool to evaluate the effects of chemicals
on steroidogenesis. The bioassay is sensitive, reproducible,
and effects on steroid hormone production appear to be
consistent with most results observed in other models
including in vivo models with various mammalian species.
In addition, the bioassay provides a convenient and costeffective tool to evaluate the effects of chemicals on several
steroidogenic pathways that include the synthesis of mineralcorticoids, glucocorticolds, and androgens and estrogens.
Thus, while the focus of this study was on the synthesis of
androgens and estrogens, the H295R assay system can be used
as a more comprehensive test system to examine the mode of
action of chemicals on molecular and biochemical aspects of
steroidogenesis.
While this study emphasized the suitability of the H295R
steroidogenesis assay as a possible TIER I screening tool for
chemical effects on sex steroid production, uncertainties
remain with regard to our understanding of the specific
mechanism(s) of action and predicting effects at other levels of
organization or biological systems (e.g. different tissues or
species). In order for this cell line to become a predictive tool
for in vivo effects on hormone production, there is a need for
additional research characterizing the autoregulatory processes
of steroidogenic pathways and the presence and/or functions
of hormone receptors and metabolic capacities of the H295R
cells. Currently, studies are underway to elucidate some of
these aspects, such as metabolism of sex steroids and
expression of hormone receptors. Furthermore, comparative
studies are being conducted that compare effects of the model
chemicals described in this paper with those on different
tissues and whole organisms, including fish and amphibians,
to evaluate the relevance of the H295R steroidogenesis system
at higher levels of organization.
Acknowledgments
Funding for this project was provided by USEPA, ORD
Service Center/NHEERL, Contract Number: GS-10F-0041L.
We acknowledge many helpful discussions and manuscript
review by Dr. Gary Timm, Dr. Ralph Cooper, Dr. Jerome
Goldman, and Dr. Robert Kavlock, Endocrinology Branch,
NHEERL, US EPA Research Triangle Park, North Carolina.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.taap.2006.07.007.
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