Environment International 33 (2007) 812 – 816
www.elsevier.com/locate/envint
Changes of AhR-mediated activity of humic substances after irradiation
M. Bittner a,⁎, K. Hilscherová a , J.P. Giesy b,c,d
a
b
RECETOX, Masaryk University, Kamenice 126/3, 62500 Brno, Czech Republic
Department of 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, United States
d
Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
Received 30 November 2006; accepted 22 March 2007
Available online 30 April 2007
Abstract
Humic substances (HS) and natural organic matter (NOM) are natural organic compounds ubiquitous in the environment. However, some
studies indicate that both HS and NOM can act as xenobiotics, e.g. induce hormone-like effects in fish, amphibians and invertebrates. Molecules
of these substances contain a number of aromatic rings and conjugated double bonds — the so called chromophores. Irradiation of dissolved HS
and NOM can lead to a series of photochemical reactions which can act on these substances itself, or on other substances present in aquatic
environment along with HS and NOM such as e.g. xenobiotics. In our previous study, we have found significant interactions of five humic acids
(HA) with cytosolic aryl hydrocarbon receptor (AhR) in an in vitro bioassay based on H4IIE-luc cells. In the present study, we have studied the
changes in AhR-mediated activities both of HS and NOM after irradiation that simulated natural solar light. Nine different HS and two NOM
samples were irradiated in Pyrex tubes with a medium-pressure mercury lamp for a duration of 0 to 52 h (which corresponds to 0–52 d natural
solar radiation). Original concentrations of the samples were 50 mg L− 1, and the greatest concentration of HS and NOM photoproducts
subsequently tested in the bioassay was 17 mg L− 1, which is an environmentally relevant concentration. After irradiation the absorbances of all the
samples were less than the original materials. The AhR-mediated activity of the HA-Fluka and HA Sodium Salt were partially decreased by
irradiation. The activities of other HS and NOM, that were either AhR-active or -inactive were not changed by irradiation. The results of the study
demonstrate that AhR-mediated activities of two active HA is caused by both photo-stable and photo-labile AhR activators, while the other three
active HA contain only photo-stable AhR activators. Potential mechanisms of the observed irradiation-induced changes in AhR-mediated
activities are discussed.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Humic acids; Ah receptor; H4IIE-luc; Absorbance; NOM
1. Introduction
Humic acids (HA) and fulvic acids (FA) are important
fractions of the group of organic compounds called humic
substances (HS) that are ubiquitous natural products of
decomposition of dead organic matter. In the aquatic environment, HS form approximately 50–70% of dissolved/natural
organic matter (D/NOM, Timofeyev et al., 2004) found in most
natural freshwaters at concentrations of 0.5 to 50 mg L− 1, but
can occur at concentrations as great as 100 mg L− 1 in raised
⁎ Corresponding author. Tel.: +420 549491462; fax: +420 549492840.
E-mail address: bittner@recetox.muni.cz (M. Bittner).
0160-4120/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2007.03.011
peat bogs (Steinberg, 2003), where the HA fraction forms about
6–8% of the total NOM (Yamamoto et al., 2004).
Although both HS and NOM have been regarded to be
without any direct biological activity, recently it has been found
that these substances alone can act as xenobiotic chemicals. One
of the rather non-specific mechanisms of action is NOMdependent modulation of heat shock proteins 70 in carp and
Daphnia magna, and induction of biotransformation enzymes
glutathione-S-transferases, oxidative stress enzymes peroxidase
and glutathione peroxidase in amphipods (Wiegand et al.,
2003). A more specific mechanism of action is both HS and
NOM-dependent modulation of photosynthetic oxygen release
in alga Scenedesmus armatus, water moss Vesicularia dubyana
M. Bittner et al. / Environment International 33 (2007) 812–816
and hornwort Ceratophyllum demersum (Pflugmacher et al.,
2006; Steinberg et al., 2003). Additionally, it has been reported
that synthetic HA (HS1500) affect physiological condition and
slightly the sex ratio of the swordtail fish (Xiphophorus helleri,
Meinelt et al., 2001). Furthermore, hormone-like effect of HS
on the nematode Caenorhabditis elegans (Hoss et al., 2001;
Steinberg et al., 2002), and increased mortality of amphipods
and biochemical changes have been found (Timofeyev et al.,
2004).
Dissolved HS are yellow colored and exert relatively strong
absorption in the UV region of solar radiation (Hessen and
Faerovig, 2001; Rasmussen et al., 1989) correlated to the
presence of delocalized π-electron systems, which are available
from aromatic rings or conjugated double bonds (Steinberg,
2003). Photo-transformation of HS in surface waters is a natural
biogeochemical process that leads to changes of their biological,
chemical, and physical properties (Amador et al., 1991;
Frimmel, 1994, 1998; Polewski et al., 2005). Experiments
with irradiation of NOM samples performed by Frimmel (1998)
have shown that there is a general bleaching effect (i.e. decrease
of absorbance) in irradiated samples accompanied with a
change in the molecular size distribution of NOM. From an
ecotoxicological point of view, photo-degradation of HS can
lead to either beneficial or harmful effects on water organism.
The beneficial effect has been shown for bacterial population
since photo-degradation of HS leads to the release of more
easily bioavailable low-molecular organic molecules that can
stimulate bacterial production (Bertilsson and Tranvik, 1998;
Wetzel et al., 1995). On the other hand, irradiation of HS
induces production of reactive oxygen molecules (ROS,
Frimmel, 1994) that can restrict viability of aquatic organisms,
such as bacteria (Scully et al., 2003), invertebrates such as the
water flea (Daphnia magna, Frimmel, 1998) and algae Selenastrum capricornutum (Gjessing and Kallovist, 1991). However, ecotoxicological studies on the effects of irradiated HS on
aquatic organisms are still rare.
In our previous in vitro studies (Bittner et al., 2006; Janosek
et al., 2007), we have described a specific mechanism of HS
action, such as induction of biotransformation enzymes via
activation of intracellular aryl hydrocarbon receptor (AhR) by
HA. The activation of AhR by HA was also confirmed by in vivo
experiments using the Amazonian fish tambaqui (Colossoma
macropomum, Matsuo et al., 2006). In that study, induction of
Cytochrome P450 1A (the biomarker of exposure to pollutants)
by HA-Fluka was shown by use of Western blot analysis, catalytic assay, and immunohistochemistry.
The traditional view of HS structures suggests relatively
large molecules that are not only unlikely to bind with 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 suggest that the HS structure consists of
aggregates of relatively low-molecular weight (b 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
813
released from this complex. As mentioned above, irradiation of
HS leads to decrease of their mean molecular weight. Thus, we
hypothesized that irradiation of both HS and NOM could
enhance the amount of smaller molecules that can easily
penetrate the cell membrane and subsequently interact with
intracellular AhR. In our present study, we have evaluated the
changes in ability of irradiated HS and NOM to activate the
AhR. First of all, we irradiated all eleven samples under
laboratory conditions. After that, subsequent changes in both
biological and absorption properties of irradiated HS and NOM
samples were studied.
2. Materials and methods
2.1. Materials
Humic substances (HS) and natural organic matter (NOM) isolated from
different matrices were purchased from various sources: humic acid (HA-Fluka;
product No. 53680) from Fluka, Switzerland, humic acid sodium salt (HA
Sodium Salt; Product No. H16752) from Sigma Aldrich, USA. The following
reference substances were purchased from IHSS, USA: Suwannee River HA
(Product No. 2S101H), Suwannee River FA (1S101F) and Suwannee River
Natural Organic Matter (NOM; 1R101N), Florida Peat HA (1S103H), Nordic
Aquatic FA (1R105F), Nordic Reservoir NOM (1R108N), Waskish Peat HA
(1R107H), Elliot Soil HA (1S102H) and Leonardite HA (1S104H). Reference
compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was purchased from
Dr. Ehrenstorfer, Germany. HS and NOM used for irradiation were first
dissolved in 0.05 M NaOH to obtain stock concentration 2 g L− 1, and after that
these solutions were diluted by distilled water to obtain a final concentration
50 mg L− 1 used for irradiation experiments.
2.2. Cell culture and exposure
The H4IIE-luc cells are stably transfected with a DRE-driven firefly
luciferase reporter gene construct; the transcriptional activation of which occurs
in an AhR-dependent manner (Sanderson et al., 1996). Recombinant rat
hepatoma H4IIE-luc cells were grown and maintained in Dulbecco's Modified
Eagle's Medium (DMEM) containing 10% fetal calf serum (PAA laboratories,
Austria) at 5% CO2 and 37 °C. Cells were grown to about 70% confluence in a
sterile 96-well plate for 24 h, and subsequently incubated with reference TCDD
(dissolved in DMSO) and tested samples (with addition of corresponding
amount of DMSO) for up to 24 h at 37 °C. Concentration of irradiated HS tested
was 17 mg L− 1, that is concentration relevant for environmental conditions. All
the samples were tested at resulting pH 8.3. Cells exposed to DMEM with 0.5%
DMSO were used for the appropriate vehicle controls.
2.3. Luciferase reporter gene assay
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 luminometer Ascent (Thermo Electron Corp., USA). Final values are
expressed as a percentage of maximal TCDD induction (after subtraction of the
solvent control activity).
2.4. Irradiation and measurement of absorbance
Both HS and NOM samples were irradiated in 13 × 180 mm Pyrex
(eliminates wavelengths below 300 nm) tubes, sealed with septa using a watercooled 125W medium-pressure mercury lamp (Teslamp, Czech Republic).
Initial volume of the air above samples constituted about a quarter of the total
volume of tubes. The concentration of all irradiated samples in aqueous solution
was 50 mg L− 1 with pH in the range of 9.9–10.2. Sub-samples (2 mL) of the
irradiated samples were taken at the following times [h]: 0, 0.5, 1, 3, 6, 12, 20, 37
814
M. Bittner et al. / Environment International 33 (2007) 812–816
Table 1
Summary of relative potencies (REP), LOEC values, decay equations of both absorbance and AhR-mediated activities, and calculated t1/2 of degradation of all
irradiated samples
Compound
AhR-mediated activity
Changes after irradiation
Absorbance
LOEC [mg L− 1]
REP
HA-Fluka
HA Sodium Salt
Elliot Soil HA
Florida Peat HA
Leonardite HA
Suwannee River HA
Waskish Peat HA
Suwannee River FA
Nordic Aquatic FA
Suwannee River NOM
Nordic Reservoir NOM
−8
4.5 × 10
7.4 × 10− 8
8.4 × 10− 8
2.6 × 10− 8
4.3 × 10− 8
–
–
–
–
–
–
1.9
1.9
1.9
16.7
1.9
–
–
–
–
–
–
Decay equation
(− 0.083x)
y = 0.48e
+ 0.19
y = 0.50e(− 0.075x) + 0.21
y = 0.53e(− 0.076x) + 0.57
y = 0.64e(− 0.095x) + 0.23
y = 0.73e(− 0.071x) + 0.36
y = 0.33e(− 0.076x) + 0.28
y = 0.41e(− 0.012x) + 0.27
y = 0.19e(− 0.015x) + 0.15
y = 0.27e(− 0.014x) + 0.17
y = 0.17e(− 0.021x) + 0.28
y = 0.13e(− 0.019x) + 0.12
AhR-mediated activity
t1/2 [h]
8.4
9.2
9.1
7.3
9.8
9.1
5.7
4.8
5.1
3.3
3.6
Decay equation
(− 0.20x)
y = 16.25e
+ 6.72
y = 6.52e(− 0.18x) + 5.67
–
–
–
–
–
–
–
–
–
t1/2 [h]
3.5
3.9
–
–
–
–
–
–
–
–
–
REP and LOEC values are related to activity of positive control — TCDD. t1/2 values were calculated from exponential decay equations described in Materials and
methods.
and 52, where approximately one hour of lab-irradiation represents one day of
direct natural solar irradiation. All these irradiated samples were used for both
bioassay and spectro-photometric measurement. Absorbance of irradiated
samples was measured in plastic cuvettes using UV–VIS spectrophotometer
Cary 50 Bio (Varian, USA) at a range of wavelengths 250–650 nm.
2.5. Statistical analysis and calculations
Bioassay-derived data were examined statistically using Statistica for
Windows 6.0 (StatSoft, USA) at level of significance of P = 0.05. In figures,
means ± SD of triplicate determinations are shown. The same software was used
for calculation of one phase exponential decay equations for both absorbance
and bioassay data. Half-life values (t1/2) of both absorbance and AhR-mediated
activity decreases were obtained from the calculated one phase exponential
decay equations, and mean rate of decrease from the highest value (time 0) of
absorbance/AhR-mediated activity to plateau value (not to zero). Plateau values
of one phase exponential decay equations are the intercepts of the equations in
Table 1.
3. Results
Based on our previous findings (Janosek et al., 2007), only five HA
out of twelve samples (ten HS and two NOM) tested elicited a
significant AhR-mediated activity in H4IIE-luc cells. Relative
potencies (REP) and LOEC values of these five HA are summarized
in Table 1. The AhR-active samples were HA-Fluka, HA Sodium Salt,
Elliot Soil HA, Florida Peat HA and Leonardite HA, while some other
HA, FA and NOM samples did not show any substantial AhR-mediated
activity.
The absorbance of all eleven irradiated HS and NOM decreased
significantly as a function of duration of irradiation. The decrease in
absorbance of all the samples can be described using one phase
exponential decay functions (Table 1). The subsequently calculated t1/2
values of photo-degradation of all irradiated samples are also given.
Although decreases in absorbance were observed across the wavelengths spectra (as an example are depicted irradiated HA-Fluka
samples, Fig. 1), the most pronounced decrease of absorbance was
observed by spectrophotometric measurement at approximately
λ = 350 nm. Thus, calculations (Table 1) and comparison of absorbance
of irradiated samples (Fig. 2) were performed at λ350.
Of the eleven samples, irradiations of only two, HA-Fluka and HA
Sodium Salt, resulted in a statistically significant decrease of their
AhR-mediated activities (Fig. 3). The AhR-mediated activities of other
three of the five AhR-active HA (Leonardite HA, Elliot Soil HA and
Florida Peat HA), was decreased only slightly by irradiation (Fig. 4).
Because 1.0 h of the simulated irradiation represents approximately
1.0 d of direct solar irradiation under natural conditions, this small
decrease could be regarded as non-significant. The AhR-mediated
activities of the other four HS and two NOM (without any substantial
AhR-mediated activity) have not changed after irradiation.
The intrinsic AhR-mediated activity of HA-Fluka as well as the
decrease of its AhR-mediated activity after irradiation was greater
compared to those of HA Sodium Salt (Fig. 3). After 6 h irradiation, the
AhR-mediated activity of HA-Fluka decreased to 58% of original
activity of non-irradiated sample, while HA Sodium Salt decreased to
74% of original activity after the same time. Nevertheless, even after
52 h of laboratory irradiation, the photo-induced decay of HA-Fluka
and HA Sodium Salt AhR-mediated activities did not reach zero. The
AhR-mediated activity of both HA samples reached significant plateau
values — 6.7% for HA-Fluka and 5.7% for HA Sodium Salt (i.e. % of
maximal standard TCDD induction), which corresponds to 29% of
original activity of HA-Fluka sample, resp. 46% of original activity of
HA Sodium Salt sample. The rate of decrease of the AhR-mediated
activity of both HA samples exhibited t1/2 values of 3.5 h and 3.9 h, for
the HA-Fluka and sodium salt, respectively. This t1/2 values are
Fig. 1. Absorption spectra of HA-Fluka samples irradiated for nine different
times: 0, 0.5, 1, 3, 6, 12, 20, 32 and 52 h. All HA-Fluka samples were irradiated
at a concentration of 50 mg L− 1. Distilled water was used as a blank.
M. Bittner et al. / Environment International 33 (2007) 812–816
Fig. 2. Decrease of absorbance of irradiated AhR-active HA samples measured
at λ = 350 nm. Four other HS samples and two NOM samples exerted analogous
exponential decay of absorbance (not shown). Exponential decay equations and
t1/2 of absorbing fraction for all irradiated samples are described in Table 1.
Samples were irradiated at a concentration of 50 mg L− 1.
equivalent to approximately 3.5 and 3.9 d under natural solar
irradiation.
4. Discussion
The fate and properties of NOM and HS as naturally
occurring compounds are significantly affected by the environmental conditions common in the water environment, including
solar irradiation. Since both HS and NOM also exert significant
biological activity, we have evaluated impact of irradiation on
both absorption properties and AhR-mediated activity.
The results of the studies which are reported here demonstrate
that all eleven examined samples (nine HS and two NOM)
elicited significant photo-bleaching effect, as measured by
absorbance, when exposed to simulated irradiation. These
results are similar to those reported by other researchers
(Frimmel, 1998; Grzybowski, 2000). Decoloration of irradiated
solutions is probably caused by degradation of charge transfer
complexes of HS that are responsible for absorption of radiation
with λ N 350 nm (Del Vecchio and Blough, 2004). This
phenomenon is accompanied by decrease of the mean molecular
size of light absorbing organic matter with release of molecules
with lower mean molecular size (Bertilsson and Tranvik, 1998;
Opsahl and Benner, 1998). Based on this fact, we developed the
hypothesis that smaller molecules produced by photo-degradation of HS samples could be more able to activate AhR than the
original HS samples. This hypothesis can be rejected, since
either no change or in some cases even a decrease in AhRmediated activities after irradiation were observed.
Based on the results of our study, we now suppose that
decrease of AhR-mediated activity of both HA-Fluka and HA
Sodium Salt after simulated irradiation could be related to
indirect photolysis of AhR-active parts of HA by reactive
oxygen molecules (ROS). Our previous results (Janosek et al.,
2007) suggest the AhR-activating agents of HS are both
hydrophilic and lipophilic non-persistent molecules, such as
polycyclic aromatic hydrocarbons (PAHs, Grady et al., 1992;
Safe, 1990) and their various, often more hydrophilic
derivatives e.g. aza-PAHs (Sovadinova et al., 2006).
It is well known that ROS are generated in surface water after
photo-chemical excitation of HS (Aguer et al., 1999; Frimmel,
815
Fig. 3. Decrease of AhR-mediated activities of irradiated HA-Fluka and HA
Sodium Salt. The results are related to maximal standard (100 pM TCDD)
induction. Values represent the mean ± SD of triplicate determinations.
Exponential decay equations and t1/2 of AhR-active fraction are described in
Table 1. Concentration of tested HA samples was 17 mg L− 1.
1994; Hoigne et al., 1989), and are able to oxidize surrounding
dissolved organic molecules. HS-induced ROS production has
been shown to oxidize numerous chemicals including DDT
(Kulovaara et al., 1995) or pesticides Irgarol, fenuron (AmineKhodja et al., 2006), carboxin, oxycarboxin, and vinclozolin
(Hustert and Moza, 1997; Hustert et al., 1999). The photolysis
of these compounds can change their properties including
toxicity, in some cases it can even significantly increase their
toxic potential, such as in the case of vinclozolin. These
observations together with our results indicate that AhR-active
agents within HS could be degraded by ROS leading to
products with different toxicological properties.
Either a decrease or no significant changes of AhR-mediated
activity of both HS and NOM samples after irradiation were
observed in our studies. Nevertheless, the photo-induced
decrease of AhR-mediated activity of HA-Fluka and HA
Sodium Salt also show photo-induced degradation of AhRactive fraction or compartments of HA is not complete, but only
to a certain plateau of the AhR-mediated activity. Then the
activity did not decrease further, which was similar that
observed for the other three AhR active HA samples. These
findings suggest a combination of photo-stable and photo-labile
AhR activators in both HA samples with decrease of their AhRmediated activity (HA-Fluka and HA Sodium Salt, Fig. 3), and
presence of only photo-stable AhR-activators in the other three
AhR-active HA samples without any significant decrease in
Fig. 4. AhR-mediated activities of irradiated Leonardite HA, Elliot Soil HA and
Florida Peat HA. The results are related to maximal standard (100 pM TCDD)
induction. Values represent the mean ± SD of triplicate determinations.
Concentration of tested HA samples was 17 mg L− 1.
816
M. Bittner et al. / Environment International 33 (2007) 812–816
AhR-mediated activity (Elliot Soil HA, Leonardite HA and
Florida Peat HA, Fig. 4).
Overall, the results of our studies suggest that potency of HA
samples to act through this specific mechanism of action –
activation of AhR – either stays the same or decreases after
irradiation. Thus, in some cases, irradiation of dissolved HA can
be beneficial in the sense of decrease of AhR active substances
based stress to aquatic vertebrates. However, generalization of
this ecotoxicological conclusion is strongly limited to fact that
each HS and NOM is of different origin and thus can elicit
rather specific properties. It is also important to keep in mind
that properties of especially HS samples can be partially
affected by the isolation procedure of these substances.
Acknowledgement
We highly acknowledge scientific support of Dr. Jaroslav
Janosek, Dr. Ludek Blaha and Prof. Ivan Holoubek. This work
was supported by grant GACR 525/05/P160 and Ministry of
Education Grant (Project “INCHEMBIOL” VZ0021622412 of
RECETOX, Masaryk University).
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