Chemosphere 67 (2007) 1096–1101
www.elsevier.com/locate/chemosphere
AhR-mediated and antiestrogenic activity of humic substances
J. Janošek a, M. Bittner
b
a,*
, K. Hilscherová a, L. Bláha a, J.P. Giesy
b,c,d
, I. Holoubek
a
a
RECETOX, Masaryk University, Brno, 625 00 Brno, Czech Republic
Department Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
c
Zoology Department, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University,
E. Lansing, MI 48823, USA
d
Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
Received 19 May 2006; received in revised form 23 November 2006; accepted 24 November 2006
Available online 16 January 2007
Abstract
Humic substances (HS) were for decades regarded as inert in the ecosystems with respect to their possible toxicity. However, HS have
been recently shown to elicit various adverse effects generally attributed to xenobiotics. In our study, we used MVLN and H4IIE-luc cell
lines stably transfected with luciferase gene under control of estrogen receptor (ER) and Ah receptor (AhR; receptor connected with socalled dioxin-like toxicity) for assessment of anti/estrogenic and AhR-mediated effects of 12 commercially available humic substances. Out
of those, five humic acids were shown to induce AhR-mediated activity with relative potencies related to TCDD 2.6 · 108–7.4 · 108.
Organic extracts of HS solutions also elicited high activities what means that lipophilic molecules are responsible for a great part of effect.
However, relatively high activity remaining in extracted solution suggests also presence of polar AhR-agonists. Contribution of persistent
organic compounds to the observed effects was ruled out by H2SO4 treatment. Eight out of twelve HS elicited significant antiestrogenic
effects with IC50 ranging from 40 to 164 mg l1. The possible explanations of the antiestrogenic effect include sorption of 17-b-estradiol
(E2) on HS, changes in membrane permeability for E2 or another specific mechanism.
2006 Elsevier Ltd. All rights reserved.
Keywords: Ah receptor; AhR-mediated activity; Antiestrogenic effects; Endocrine disruption
1. Introduction
Humic substances (HS) are ubiquitous natural products
of decomposition of dead organic matter. Generally, HS
can be divided into three groups: humic acids (HA), fulvic
acids (FA) and humins. In the aquatic environment, HS
form approximately 50–70% of dissolved/natural organic
matter (D/NOM, Timofeyev et al., 2004), which is in most
natural freshwaters in the concentration range 0.5–50
mg l1 (Steinberg, 2003).
Until recently, only indirect effects of HS in the ecosystems such as changes in bioavailability of organic pollutants and/or complexation of inorganic ions, particularly
*
Corresponding author. Tel.: + 420 549491462; fax: + 420 549492840.
E-mail address: bittner@recetox.muni.cz (M. Bittner).
0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2006.11.045
heavy metals, have been reported (Steinberg, 2003). The
presumed large molecules of HS were not considered to
be able to penetrate into cells. However, recent studies
focused on HS uptake showed that at least parts of natural
as well as artificial HS can be assimilated by plant cells in
culture (Wang et al., 1999) as well as crustaceans (Gammarus pulex) or even vertebrates (Rana arvalis tadpoles; Steinberg et al., 2003). Furthermore, biological effects of HS
have been reported for algae, invertebrates as well as vertebrates (Pflugmacher et al., 2001; Steinberg et al., 2003).
Direct toxic effects of HS were reported for the freshwater snail Lymnea stagnalis. Exposure to natural concentrations of HS from the Suwannee River (0.5 mg l1) caused
death of up to 20% of individuals (Steinberg et al., 2003).
Responses of nonspecific markers of toxic effects of
HS were also observed. Elevated levels of detoxification enzymes glutathione-S-transferase and glutathione
J. Janošek et al. / Chemosphere 67 (2007) 1096–1101
peroxidase were reported in crustaceans Daphnia magna
following HS treatment at concentration range 0.5–
100 mg l1 (Wiegand et al., 2003). An increase in peroxidase activity as well as release of heat shock proteins was
also observed in several amphipod species (Timofeyev
et al., 2004) and carp (Wiegand et al., 2003). Hoss et al.
(2001) reported that natural organic matter (NOM) altered
reproduction of nematodes Caenorhabditis elegans what
was further confirmed in a study with broader spectra of
HS (Steinberg et al., 2002).
A synthetic humic substance HS1500 (a polyphenol oxidation product) was shown to elicit significant estrogenic
and anti-thyroidal effects on Xenopus laevis tadpoles (Lutz
et al., 2005). Furthermore, long-term exposure of swordtail
fish Xiphophorus helleri fry to a high concentration of
HS1500 (from 5 to 180 mg l1) lead to changes of sex ratio
in favor of females as well as to body mass increase in both
males and females (Meinelt et al., 2004), while survival of
fry has not decreased even at exposure to 500 mg l1 (Steinberg et al., 2003). However, the same very high concentration of HS1500 has significantly decreased the survival rate
of the exposed embryos of fish Danio rerio, whereas exposure to lower concentrations of 5–50 mg l1 slightly increased the survival rate of embryos relative to the control
(Steinberg et al., 2003).
In spite of these in vivo experiments, no mechanistic
in vitro studies had been conducted to determine the mode
of action of direct effects. With respect to observed estrogenic effects as well as increase in levels of detoxification
enzymes, we decided to investigate possible interactions of
a wide spectrum of HS (including HA, FA and NOM
obtained from various sources) with estrogen receptor
(ER) and aryl hydrocarbon receptor (AhR). The latter is
responsible for so-called dioxin-like mode of action resulting in a wide range of in vivo effects in vertebrates including,
e.g. enormous enhancement of activity of detoxification
enzymes.
2. Material and methods
2.1. Chemicals
HS isolated from different matrices were purchased from
various sources: HA-Fluka (Product No. 53680; Fluka,
Switzerland), HA sodium salt (Product No. H16752;
Sigma–Aldrich, Prague, Czech Republic). The following
reference HS were purchased from the International Humic
Substances Society (IHSS, Denver, USA): Suwannee River
HA (Product No. 2S101H), Suwannee River FA (1S101F)
and Suwannee River NOM (1R101N), Florida Peat HA
(1S103H) and Florida Peat FA (2S103F), Nordic Aquatic
FA (1R105F), Nordic Reservoir NOM (1R108N), Waskish
Peat HA (1R107H), Elliot Soil HA (1S102H) and Leonardite HA (1S104H). Reference compounds were purchased
from Dr. Ehrenstorfer, Augsburg, Germany (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) and Sigma–Aldrich (17-bestradiol, E2).
1097
2.2. Sample preparation
HS were dissolved in 0.05 M NaOH and final concentrations ranging from 1.9 to 150 mg l1 were studied. To evaluate the nature of active molecules, further investigations
were conducted. The alkaline solution of HS was extracted
three times with a 3:1 hexane:dichloromethane mixture.
Half of this extract was transferred into DMSO for
in vitro assays (further regarded as organic extract); the
other half was analyzed using GC–MS for known xenobiotics such as seven indicator PCB (IUPAC No. 28, 52, 101,
118, 138, 153 and 180) and 16 PAH monitored by the
United States Environmental Protection Agency (USEPA).
To determine possible contributions of other persistent
organic compounds that might have been present in standard materials, the alkaline solutions (200 ll) were transferred into excess of 96% H2SO4 (5 ml) and liquid–liquid
extraction with hexane:dichloromethane (3 · 5 ml) was
conducted. Using this method, only persistent compounds
such as PCDD, PCDF and PCB were extracted. The original alkaline solutions were examined for both AhR-mediated and anti/estrogenic activity, organic extracts as well
as extraction residues of alkali solutions and H2SO4-treated
extracts were tested for AhR-mediated activity.
2.3. In vitro assays
H4IIE-luc (rat hepatocarcinoma) and MVLN (human
breast carcinoma) cells stably transfected with luciferase
gene under control of AhR and ER, respectively, were used
for analysis of receptor activation. Both cell lines are well
established models for evaluation of AhR-mediated
(H4IIE-luc) and anti/estrogenic (MVLN) activities of pure
substances as well as environmental samples (Demirpence
et al., 1993; Machala et al., 2001; Villeneuve et al., 2002).
H4IIE-luc cells: Cells were grown and maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) containing
10% fetal calf serum (both PAA laboratories, Pasching,
Austria) at 5% CO2 and 37 C. Once the cells reached
about 70% confluence they were passaged and seeded into
a sterile 96-well plate at density 15 000 cells/well. After
24 h, the cells were exposed in triplicates to the tested samples (concentrations 1.9, 5.6, 16.7, 50 and 150 mg l1) or
reference compound (dilution series 0.1–100 pM TCDD)
for 24 h at 37 C (final vehicle concentration was 0.5%
v/v). Cells exposed to DMEM with 0.5% DMSO and
0.5% 0.05 M NaOH were used for the appropriate vehicle
controls. Intensity of luciferase luminescence was measured
using Promega Steady Glo Kit (Promega, Mannheim,
Germany) after 24 h exposure. At least three independent
assays have been conducted for each concentration tested.
MVLN cells: Cells were grown in DMEM-F12 without
phenol red (Sigma Aldrich, USA) containing 10% fetal calf
serum. For experiments, cells were grown in medium containing fetal calf serum treated with dextran-coated charcoal (strongly reduced concentrations of natural steroids).
For studies of anti/estrogenic effects of HS, cells were
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J. Janošek et al. / Chemosphere 67 (2007) 1096–1101
exposed to HS with/without simultaneous exposure to
33 pM E2 with E2 calibration series ranging from 1 to
100 pM. Other conditions were identical with those
described for H4IIE-luc cells.
Table 1
AhR-mediated and antiestrogenic activity of crude alkali solutions of HS
2.4. Calculations
HA – Fluka
HA sodium salt
Suwannee River
HA
Elliot soil HA
Florida Peat HA
Leonardite HA
Waskish Peat HA
Suwannee River
FA
Florida Peat FA
Nordic Aquatic
FA
Suwannee River
NOM
Nordic Reservoir
NOM
Results were calculated using equi-effective approach
described by Villeneuve et al. (2000) and expressed as relative potencies (REP) with respect to TCDD. REP were calculated from EC50 values. For determination of mass
balance of extracts, toxic equivalents (TEQ) expressed as
ng TEQ g-1 HA were calculated from EC25 because some
of the extracts and extraction residues did not reach 50%
effect of maximal TCDD induction.
3. Results
3.1. AhR-mediated activity
Of the 12 HS tested, five elicited a significant AhR-mediated activity in H4IIE-luc cells (Fig. 1). All of the active
samples were HA, while no FA and no NOM samples
showed any substantial activity. Relative potencies (related
to TCDD) of all the AhR-active HA were in a relatively
narrow range of 2.6–8.4 · 108. All of the active substances
elicited significant AhR-mediated effects at concentration
as little as 1.9 mg l1, except for Florida Peat HA (LOEC
16.7 mg l1; see Table 1). The organic extracts of HA
induced significant activities what confirmed presence of
non-polar AhR activators. However, the extraction residues also elicited relatively great responses (Fig. 2).
Increase in luciferase activities caused by the residues was
even greater than that of organic extracts (Table 2) except
for HA-Fluka and HA sodium salt. This result suggests a
significant contribution of polar compounds to the AhRmediated activity of HA.
Antiestrogenic activity
LOEC
(mg l1)
REP
LOEC
(mg l1)
IC50
(mg l1)
1.9
1.9
n.ind.
4.5 · 108
7.4 · 108
n.ind.
16.7
16.7
n.eff.
43 ± 3
40 ± 4
n.eff.
1.9
16.7
1.9
n.ind.
n.ind.
8.4 · 108
2.6 · 108
4.3 · 108
n.ind.
n.ind.
50
150
50
50
50
164 ± 9a
w.eff.
132 ± 8
84 ± 6
127 ± 8
n.ind.
n.ind.
n.ind.
n.ind.
50
50
112 ± 4
83 ± 7
n.ind.
n.ind.
150
w.eff.
n.ind.
n.ind.
n.eff.
n.eff.
REP – relative potencies related to TCDD; IC50 – concentration causing
50% inhibition of E2-induced luciferase activity; n.ind. – no induction;
n.eff. – no effect; w.eff. – weak effect.
a
Approximation above measured concentration range.
Extraction with H2SO4 totally suppressed the AhRmediated activity of HA. Thus it can be concluded that
the compounds responsible for the observed effects were
not persistent organic compounds such as PCDD, PCDF
or PCB. Moreover, chemical analysis of seven indicator
PCB congeners and 16 US EPA priority PAH (indication
of possible anthropogenic contamination) did not reveal
sufficient quantities of these substances that could be
responsible for the relatively great AhR-mediated activities
of HA. TEQ calculated from chemical data for PAH using
REPs published by Machala et al. (2001) was less than 1%
of bioassay-derived TEQ for the HA.
HS - Humic Substances
NOM - Natural Organic Matter
100
Luciferase activity
(% of 100 pM TCDD induction)
AhR-mediated activity
FA - Fulvic Acids
HA - Humic Acids
80
60
40
20
ka
so
Su
di
w
um
an
ne
sa
e
lt
R
iv
er
El
H
A
lio
tt
So
Fl
or
il
H
id
A
a
Pe
at
Le
H
on
A
ar
di
W
te
as
H
Su kis
A
h
w
Pe
an
at
ne
H
e
A
R
ive
Fl
r
or
FA
id
a
N
Pe
or
at
Su dic
FA
Aq
w
an
ua
ne
tic
N
e
FA
or
R
ive
di
c
r
R
N
es
O
er
M
vo
ir
N
O
M
lu
H
A
H
A
-F
TC
D
D
0
Fig. 1. AhR-mediated activity of 12 samples of HS determined in H4IIE-luc cells; cTCDD = 3.2 · 105 mg l1; cHS = 150 mg l1. Values represent the
mean ± SD of triplicate determinations.
J. Janošek et al. / Chemosphere 67 (2007) 1096–1101
1099
Luciferase activity (% of 100 pM TCDD)
140
TCDD
Leonardite HA- alkali solution
Leonardite HA- org. extract
Leonardite HA- extr. residue
Leonardite HA- H2SO4 treated
120
100
80
60
40
20
0
10-9
10-8
10-7
10-6
10-5
10-4
1
10
100
1000
Concentration (mg l-1)
Fig. 2. Dose–response curves of TCDD, Leonardite HA and its different solutions and extracts in H4IIE-luc cells. Values represent the mean ± SD of
triplicate determinations.
Table 2
Mass balance of extraction procedure
HA – Fluka
HA sodium salt
Elliot soil HA
Florida Peat
HA
Leonardite HA
Alkali solution
(ng TEQ g1 HA)
Extract (% of
alkali solution)
Extract residue
(% of alkali
solution)
205
219
197
29
70.4
67.8
37.5
w.i.
22.2
31.1
47.1
55.0
68
30.4
46.1
TEQ – toxic equivalents derived from EC25 obtained from experiments
with alkali solutions, their organic extracts and extraction residues; w.i. –
weak induction.
3.2. Interaction with ER pathway
None of the tested HS showed any estrogenic activity
both in absence and presence of ER-agonist E2. However,
Luciferase activity
(% of induction of 33 pM E2)
120
ten out of twelve tested HS caused significant decreases of
ER-dependent gene expression (Fig. 3). Unlike the AhRmediated activity, antiestrogenic effects were observed after
administration of HA, FA and NOM. Concentrations
causing a 50% decrease of E2-dependent luciferase activity
(IC50) were observed for HS in a concentration range of
40–164 mg l1 (see Table 1). The strongest suppression of
ER signaling was caused by HA-Fluka and HA sodium
salt.
4. Discussion
The results of this study demonstrates that some HS,
particularly HA, are able to activate ‘‘dioxin-like’’ signaling
pathway. However, as the phrase ‘‘dioxin-like’’ also suggests persistence, here we refer to this activity as AhR-mediated. FA were found to be inactive in these assays.
Accordingly, since HA represents only about 6–8% of the
50 mg l -1
150 mg l -1
100
80
60
40
20
H
H
um
ic
Ac
id
E2
Fl
A
uk
S
Su
od
a
w
iu
an
ne m S
al
e
R
ive t
rH
El
A
lio
t
So
Fl
or
i
lH
id
A
a
Pe
Le
at
on
H
A
ar
W
d
as
ite
H
Su kish
A
w
Pe
an
a
ne
tH
e
A
R
i
ve
Fl
rF
or
id
A
a
N
Pe
or
d
a
Su
ic
tF
Aq
w
A
an
ua
ne
t
i
c
N
e
FA
or
R
di
ive
c
rN
R
es
O
er
M
vo
ir
N
O
M
0
Fig. 3. Antiestrogenic effects of HS in MVLN cells – HS-dependent inhibition of luciferase activity elicited by 33 pM E2. Values represent the mean ± SD
of triplicate determinations.
1100
J. Janošek et al. / Chemosphere 67 (2007) 1096–1101
total NOM (Yamamoto et al., 2004), tested NOM samples
also did not elicit any AhR-mediated activity at any of the
studied concentrations.
The traditional view of HA structures suggests relatively
large molecules that are not only unlikely to bind to the
ligand-binding region of the AhR but also unlikely to enter
cells. However, recent studies have shown that HS are able
to cross plant (Nardi et al., 2002) and mammalian cell
membranes and interact with some receptors (Beer et al.,
2000). Moreover, recent studies of HS structure suggest it
consists of aggregates of relatively low-molecular weight
(<2 kDa) organic compounds and metal ions bound
together by non-covalent interactions (Simpson et al.,
2002). This leads to a hypothesis that the AhR-active compounds may be rather small molecules released from this
complex. As for structural dissimilarity of HA with ‘‘classical’’ high affinity AhR ligands such as PCDD, PCDF
and PCB, a variety of AhR activators structurally very
different from the ‘‘classical’’ ligands have been identified recently (Denison and Heath-Pagliuso, 1998). These
‘‘non-classical’’ ligands, such as oxidized carotenoids,
indoles and heterocyclic amines, are usually very weak activators when compared to the halogenated aromatic hydrocarbons (HAH). HA also seem to belong to this group of
‘‘non-classical’’ AhR ligands.
The adverse biological effects of HAH attributed to the
AhR activation are usually thought to be due to their high
affinity to AhR and low susceptibility to biodegradation.
Many of these effects can be observed only after several
days to weeks of exposure what suggests that their cause
lies in long-term continuous activation of AhR-signaling
pathway (Denison and Heath-Pagliuso, 1998). Thus, continuous exposure to low-affinity ligands may result in
effects similar to those caused by HAH, as was shown in
brown bullhead Ameiurus nebulosus (Grady et al., 1992).
On the other hand, a single activation of AhR-signaling
pathway by the non-persistent ligands may also not only
cause no harm but even produces some beneficial effects
(Jeuken et al., 2003). Thus, AhR-mediated activity of HA
may be of importance namely for vertebrates (i.e. animals
with ligand-binding domain of AhR) living in aquatic systems with great concentrations of HS.
Concentrations of DOM in surface water are in range of a
few mg l1, in eutrophic lakes and marshes up to tens of
mg l1 and can reach more than 100 mg l1 in raised peat
bogs (Steinberg, 2003) or pore water (Zsolnay, 1996). As
mentioned above, HA form only a minor fraction of DOM
(6–8%). Since LOEC was 1.9 mg l1 for all AhR-active HA
except for peat HA (16.7 mg l1), the AhR-mediated activity
of HA may be of environmental concern particularly for animals living in eutrophic aquatic systems, peat bogs or for soil
organisms exposed to pore water. Furthermore, ecotoxicological significance of AhR activity of HA can be also
emphasized by the fact that LOEC values of all AhR-active
HA are much lower than, e.g. above mentioned HS1500
concentration significantly reducing the survival rate of
the exposed embryos of fish Danio rerio,which was
500 mg l1, while survival of Xiphophorus helleri fry has
not been influenced even at such high concentration of
HS1500 (Steinberg et al., 2003).
It is also important to emphasize that maximal response
obtained with AhR-active HS samples, even at very high
concentrations (150 mg l1), reached only about half of
maximal induction caused by standard agonist TCDD.
Thus, AhR-active HA samples can be considered ‘‘partial
agonists’’ because they are unable to elicit a full response.
According to pharmacologic receptor principles, partial
agonists could act as antagonists against potent full agonists.
It is possible that AhR-active HS (weak agonists) could actually antagonize the toxicity of potent AhR agonists such as
TCDD, potentially reducing its toxicity in the analogous
way such as, e.g. weak AhR-agonists a-naphthoflavone
(Santostefano et al., 1993) or 4 0 -iodoflavone (Lu et al.,
1996). From this point of view, HS might help to protect
water organisms from toxicity of full potent AhR agonists.
Extraction of the alkali solution with organic solvents
resulted in an interesting outcome – the AhR-mediated
activity was distributed between both organic extract and
extraction residue. The mass balance was quite good for
the more active compounds (Table 2). However, in the least
active sample (Florida Peat HA) activity of the organic
extract did not reach the 25% of induction caused by 100
pM TCDD. Thus, its activity could not be calculated. Anyway, induction of AhR-mediated effects by both organic
and water solutions demonstrate presence of both lipophilic and hydrophilic AhR activators.
Antiestrogenic effects of HS are not restricted only to
HA; FA as well as NOM were also shown to negatively
affect E2 induced luciferase activity. However, this finding
is not consistent with the recent results of studies on swordtail fish Xiphophorus helleri (Meinelt et al., 2004) and African clawed frog Xenopus laevis (Lutz et al., 2005), where
feminization of tadpoles was observed after exposure to a
synthetic HS1500. However, these studies were conducted
with a synthetic humic substance (an oxidation product
of caffeic acid) and whole organisms, which may cause
the differences in results. Other effects on reproduction
have been observed in nematodes (Steinberg et al., 2004)
where natural peat FA strongly inhibited number of offsprings of exposed individuals. A recent in vitro study with
oocytes of frog Rana temporaria (Zenkevics et al., 2005)
showed that HS reduced sensitivity of oocytes to gonadotropic hormone and thus delayed their maturation. These
authors attribute this effect to blocking membrane transport of gonadotropic hormone. Changes of membrane permeability for E2 may also be the reason of antiestrogenic
effects observed in our study. Another cause may be the
simple sorption of relatively hydrophobic molecule of E2
to carbon-rich HS in test medium. This hypothesis is
supported by a recent study on influence of NOM on
partitioning of E2 between water phase and an artificial
membrane (Yamamoto et al., 2004). The researchers have
observed 30% decrease of membrane/water partition
coefficients of E2 at concentrations as low as 3 mg l1 of
J. Janošek et al. / Chemosphere 67 (2007) 1096–1101
HA (Sigma) and 20% decrease for 7.7 mg l1 of Suwannee
River FA, respectively. That corresponds with our finding
that HA (Sigma) elicits much higher antiestrogenic activity.
Unfortunately, Yamamoto et al. (2004) did not study
greater concentrations of HS, and therefore we cannot
directly compare their results with ours. At such low concentrations we have not observed greater effect than 10%,
if any. On the other hand, sorption is a relatively nonspecific process what would suggest all samples to be active.
However, four samples (two samples of NOM, one HA and
one FA) elicited no or only weak antiestrogenic activity
even at high concentrations. Question of some more
specific mechanism of action thus still remains unsettled.
5. Conclusions
The results of this study demonstrate interactions of HS
with AhR signaling pathway. Some HA but no FA were
able to elicit significant AhR-mediated effects with relative
potencies in range of 2.6–8.4 · 108. Thus, although being
natural compounds, HS may cause some adverse effects on
organisms that are continuously exposed to high concentrations of HS, such as soil organisms or organisms from
peat bogs. In vitro antiestrogenic effects have also been
detected that may be caused by simple sorption of E2 on
HS, by reduced membrane permeability for E2 caused by
parts of HS molecules, or by another more specific mechanism. However, determination of the mode of action
requires further studies.
Acknowledgements
We highly acknowledge financial support from research
grants of Czech Ministry of Education (INCHEMBIOL
MSM 0021622412) and Grant Agency of the Czech Republic (525/05/P160). We would like to thank Dr. Jana
Klanova for the analysis of HS for PCB and PAH content,
and we also thank kind reviewers for their valuable
comments.
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