Activation of Ah Receptor by Pure Humic Acids
Michal Bittner,1 Jaroslav Janošek,1 Klára Hilscherová,1 John Giesy,2,3
Ivan Holoubek,1 Luděk Bláha1
1
Masaryk University in Brno, RECETOX, Kamenice 126/3, 625 00 Brno, Czech Republic
2
National Food Safety and Toxicology Center, Center for Integrative Toxicology,
Department of Zoology, Michigan State University, East Lansing, Michigan 48824-1222, USA
3
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue,
Kowloon, Hong Kong SAR, China
Received 17 June 2005; accepted 21 March 2006
ABSTRACT: Humic substances (HS) are ubiquitous in the environment. However, some studies indicate
that HS could induce direct adverse effects on human health and hormone-like effects in fish, amphibians,
and invertebrates. In this study we investigated a possible biochemical mechanism of HS toxicity via activation of the aryl hydrocarbon receptor (AhR). AhR mediates the toxic and biological effects of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but a number of structurally
diverse compounds has also been found to activate AhR. Alkali solutions of humic acids (HA) were prepared, and subsequently, lipophilic compounds (including parts of HA) were extracted by liquid–liquid
extraction with hexane/dichloromethane. Organic extract of HA was further treated with sulfuric acid to
study the role of possible trace persistent contaminants. In vitro dioxin-like activities of obtained extract
and HA solutions have been evaluated using H4IIE.luc cells by determining the ethoxyresorufin-O-deethylase (EROD) activity and induction of AhR-dependent reporter luciferase. Traces of nonpersistent residues
in HA with known AhR activity were identified and quantified by GC-MS. Our results show that an alkali
solution as well as organic extract of HA were active in both EROD and luciferase assays, while H2SO4treated extract activity was negligible. Only nonsignificant levels of AhR-inducing contaminants (PAHs and
PCBs) were found in the HA samples. Our results indicate that HA or their fragments can elicit significant
inductions of AhR-mediated effects in vitro. To our best knowledge, this study is the first in providing direct
evidence of dioxin-like effects of HA. Further efforts should focus on detailed characterization of potential
toxic effects of various HSs. # 2006 Wiley Periodicals, Inc. Environ Toxicol 21: 338–342, 2006.
Keywords: aryl hydrocarbon receptor; H4IIE.luc; luciferase; EROD; humic substances
INTRODUCTION
Humic acids (HA) and fulvic acids are certain fractions of
the group of organic compounds called humic substances
(HS). HS are ubiquitous organic compounds that occur in
soils and aquatic ecosystems. In aquatic ecosystems, they
Correspondence to: J. Janošek; e-mail: janosek@recetox.muni.cz
Supported by: The Ministry of Education, Czech Republic (No. FRVS
2510/05).
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/tox.20185
C
2006 Wiley Periodicals, Inc.
338
are the main components of dissolved organic matter that is
found in most natural freshwaters in concentrations 0.5–
50 mg L1 (Steinberg, 2003). Until recently, HS were generally considered to be inert and only the sorption of metals
and organic contaminants on HS was considered to be important (McCarthy, 1987; McGeer et al., 2002; Mezin and
Hale, 2004). However, some recent works have shown that
HS can act as xenobiotic chemicals. Meinelt et al. (2001)
found that synthetic HA (HS1500) affects physiological
condition and slightly also the sex ratio of the fish Xiphophorus helleri. Furthermore, the hormone-like effect of HS
on nematode Caenorhabditis elegans (Hoss et al., 2001;
Ah RECEPTOR ACTIVATION BY PURE HUMIC ACIDS
Steinberg et al., 2002), and increase in amphipod mortality
and change of biochemical parameters (Timofeyev et al.,
2004) were shown. Recent studies conducted in Taiwan
have suggested that HS can even be the causative agents
of endemic human diseases such as Blackfoot disease
(Cheng et al., 1999; Gau et al., 2001; Hseu et al., 2002;
Huang et al., 2003) or Kashin-Beck disease (Liang et al.,
1999).
So far, two types of mechanisms of HS action have been
studied. Nonspecific mechanisms of action were examined
with Daphnia magna, three other amphipod species, and
common carp Cyprinus carpio (Wiegand et al., 2003).
Dose–response relationships between HS and/or natural organic matter (NOM) exposure and modulation of heat
shock proteins 70 (Hsp 70) were found in carp. Additionally, HS induced Hsp 70 as well as the biotransformation
enzymes glutathione S-transferases, oxidative-stress enzymes peroxidase and glutathione-peroxidase in amphipods
and D. magna (Wiegand et al., 2003). A more specific
mechanism of action is modulation of photosynthetic oxygen release induced by HS and NOM in alga Scenedesmus
armatus, water moss Vesicularia dubyana, and hornwort
Ceratophyllum demersum (Pflugmacher et al., 1999; Steinberg et al., 2003).
Another specific mechanism of direct action of HS, the
activation of cytosolic aryl hydrocarbon receptor (AhR),
was suggested by Pflugmacher (unpublished results, from
Steinberg, 2003). The hypothesis of binding interaction
between HS and AhR was derived from indirect evidence
that Hsp 70 might be induced after activation of the AhR.
However, we are not aware of any research evidence of this
fact. The aim of this study was to examine whether the HA
is able to act as an activator of the AhR. For this purpose,
we used two different endpoints (reporter luciferase and
ethoxyresorufin-O-deethylase (EROD) assays) for determination of the ability of HA to elicit AhR-mediated response
in vitro in H4IIE.luc cell line. The advantage of the EROD
assay is a natural AhR-mediated expression of measured
protein activity, while the expression of luciferase, being
foreign to the cell, is probably not affected by posttranscriptional and -translational events which influence CYP1A1
expression measured as EROD (Sanderson et al., 1996).
339
10 g L1, was extracted with 5 mL of hexane/dichloromethane, 3:1 v/v). This LLE was repeated three times and all
organic extracts were pooled and concentrated under a
stream of nitrogen to a volume of 1 mL (‘‘HA-extract’’).
One half of the concentrated organic extract was transferred
into 1 mL dimethyl sulfoxide (DMSO) and prepared for
bioassay analysis; another half was analyzed for the presence of 16 US EPA priority PAHs using GC-MS (HewlettPackard, USA) and 7 indicator PCBs (IUPAC No. 28, 52,
101, 118, 138, 153, and 180) using GC-ECD (HewlettPackard, USA). One-milliliter aliquot of remaining alkali
HA solution after extraction (‘‘alkali HA remaining after
LLE’’) was also stored for subsequent bioassay analysis.
An extraction of only persistent organic compounds (e.g.,
PCDDs, PCBs, and PCDFs) from alkali HA was conducted
in a manner similar to that described earlier, but with the
addition of 5 mL of H2SO4 to the extraction mixture with
only 0.25 mL of HA (i.e., HA þ H2SO4 þ hexane/DCM)
and incubation of the mixture in water bath. The scheme
of preparation for all of the tested samples is shown in
Figure 1. Appropriate extraction control samples were prepared to preclude any possible contamination with AhR
activators from the used solvents or equipment.
Cell Culture and Exposure
The H4IIE.luc cells are stably transfected with DRE-driven
firefly luciferase reporter gene construct; its transcriptional
activation occurs in an AhR-dependent manner (Sanderson
et al., 1996). Recombinant rat hepatoma H4IIE.luc cells
were grown and maintained in DMEM medium containing
10% fetal calf serum (PPA laboratories, Austria) at 5%
CO2 and 378C. Cells were grown to about 70% confluence
in a sterile 96-well plate for 24 h, and subsequently incubated with reference tetrachlorodibenzo-p-dioxin (TCDD)
and tested samples for up to 24 h at 378C (final volume of
vehicle was 0.5% v/v). Cells exposed to DMEM with 0.5%
DMSO or 0.5% 0.05 M NaOH were used for the appropriate vehicle controls.
MATERIALS AND METHODS
Materials
Humic acids (HA) was purchased from Fluka, Switzerland
(prod. no. 53680). HA dissolved in 0.05 M NaOH (‘‘alkali
HA’’) was used for testing. Extraction of lipophilic organic
compounds from alkali HA solution (fragments of HA,
but also possible AhR-active organic contaminants like
PCDDs, PCDFs, PCBs, PAHs, etc.) was carried out by
liquid–liquid extraction (LLE; 3 mL of alkali HA solution,
Fig. 1. Preparation scheme of tested solutions as described in Materials and Methods.
Environmental Toxicology DOI 10.1002/tox
340 BITTNER ET AL.
PAHs were calculated using the equation chem-TEQ ¼
S [PAHi REPi], using the REP values for individual
PAHs suggested by Machala et al. (2001).
RESULTS AND DISCUSSION
Fig. 2. Luciferase activity induced by TCDD, alkali HA, alkali HA remaining after LLE, HA-extract, and H2SO4-treated
HA extract. The results are related to max. luciferase induction of TCDD. Values represent the mean 6 SD of triplicate
determinations.
Luciferase Reporter Gene Assay
and EROD Activity
After incubation with test substances, cells were washed
twice with phosphate-buffered saline, and luminescence
was measured with Steady-Glo Kit (Promega, USA)
according to manufacturer’s instructions, using an automated microplate fluoro-/luminometer GENios (TECAN,
Switzerland). Final values are expressed as percentage of
maximal TCDD induction (after subtraction of the solvent
control activity). EROD activity was measured by a procedure slightly modified from methods described by Jung
et al. (2001). We measured fluorescence at 550 nm excitation and 612 nm emission. Fluorescence was measured
using a microplate fluoro-/luminometer GENios.
The three greatest tested concentrations of HA (5, 15, and
50 mg L1) significantly increased luciferase reporter gene
activity in H4IIE.luc cells (Fig. 2) in a dose-dependent
manner. Activity was related to reference TCDD and resulting REP of HA determined by luciferase induction assay
equaled 6 108. HA extract elicited practically an identical luciferase response as the original alkali HA (Fig. 2).
This indicates that almost all of the AhR activators present
in HA were extracted into the organic phase. Induction of
luciferase activity at maximal tested concentration of alkali
HA remaining after LLE was about 3.5-fold less than the
activity induced by the original alkali HA, but still significantly greater than control sample (Fig. 2). However,
dioxin-like activity of alkali HA and HA extract were practically identical. Accordingly, we assume that removal of
extracted parts of HA has broken the balance in HA solution and thus other AhR-activating compounds could be
released from the complex HA. It was not possible to determine the REP of alkali HA remaining after LLE because
even the greatest tested concentration (50 mg L1) did not
achieve 50% maximal standard induction. Data obtained
from EROD assay (Fig. 3) corresponded very well with the
results from luciferase assay.
The structure of humic substances (HS) studied using
multidimensional NMR is presented as a macromolecular
aggregate composed from a mixture of relatively low-molecular weight (<2kDa) organic compounds holding together through a complex combination of hydrophobic,
Statistical Analysis
Data were examined statistically using Statistica for Windows 6.0 (StatSoft, USA) and a level of significance P ¼
0.05. In figures, mean 6 SD of triplicate determinations are
shown. Simple log-linear regression models were calculated for linear portions of the dose–response curves of
standard TCDD and tested HA samples. Relative luciferase-induction potency of HA (expressed as relative potency, REP) was calculated using the equi-effective
approach (Jones and Anderson, 1999; Villeneuve et al.,
2002). Concentrations of the HA inducing 50% of the
TCDD-max response (CEQ-50) were compared with the
EC50 of the reference TCDD, and the REPHA ¼ CEQ-50/
EC50 was derived for the HA. For risk characterization of
AhR-active compounds concept of toxic equivalents (TEQ,
Van der Berg et al., 1998) was used. Bioassay-determined
TEQs of HA (bioassay-TEQHA) were calculated using the
equation bioassay-TEQHA ¼ [HAFluka] REPHA. Chemical analyses-derived TEQs (chem-TEQ) of determined
Environmental Toxicology DOI 10.1002/tox
Fig. 3. EROD activity induced by TCDD, alkali HA, HA
extract, and alkali HA remaining after LLE related to max.
EROD induction by TCDD. Values represent the mean 6 SD
of triplicate determinations.
Ah RECEPTOR ACTIVATION BY PURE HUMIC ACIDS
charge-transfer and hydrogen bond interactions, and metal
bridging (Simpson et al., 2002). This finding, in conjunction with our results, implies that activation of AhR in
H4IIE.luc cells could be caused mainly by lipophilic parts
of macromolecular ‘‘humic aggregate’’ that were released
from HA macromolecule.
Possible false-positive results caused by the contamination of HA with classical AhR activators (such as PCDDs,
PCDFs, PCBs, and PAHs) were investigated by both
H2SO4-treatment of HA extract as well as chemical analyses. No significant luciferase activity of HA extract after
H2SO4 treatment ruled out possible contribution of persistent AhR activators to observed luciferase activity of alkali
HA and HA extract. The bioassay data were confirmed by
chemical analyses. None of the 7 indicator PCB congeners
was detected (<LOD), and only trace concentrations of
some PAHs known to activate AhR were determined (concentrations in ng g1): Fluoranthen-80, Pyren-376, Indeno
(123cd)pyren-160. Chem-TEQ calculated from chemical
data for detected PAHs equaled 4.8 102 ng TCDD g1,
what accounted for only about 0.1% of total observed AhRmediated activity of HAFluka (bioassay-TEQHA 60 ng
TCDD g1).
The ability of alkali HA and HA extracts to induce AhR
observed in our study is, at the first sight, surprising
because HA (or their fragments) has apparently different
molecular structure than the prototypical and most potent
AhR agonists such as PCDDs, PCDFs, PCBs, or PAHs
(Safe, 1990; Simpson et al., 2002). However, several studies have revealed AhR activators with very diverse structures (Denison and Heath-Pagliuso, 1998; Denison and
Nagy, 2003; Jeuken et al., 2003), and consequently AhR
might be considered as a relatively nonspecific receptor.
In conclusion, the results of our study have shown that
both alkali HA and its organic extract cause significant
inductions of AhR-mediated responses in vitro (as shown
by both reporter luciferase inductions and EROD bioassays). Possible false-positives due to the presence of AhRactive contaminants were excluded by chemical analyses.
In vitro dioxin-like effects of HA were observed at environmentally relevant concentrations and the determined
REPHA was 6 108. Therefore, our study indicates that
HS (or their constituents) might act as AhR-inducing substances in vivo, and can thus significantly affect various
toxicological processes. However, HS are not uniform compounds; their structure and properties differ with regard to
the place of origin. Our further experiments focus on
detailed characterization of AhR modulations by a wider
range of available HS.
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