Environment International 37 (2011) 960–964
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Environment International
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e n v i n t
Enhancement of AhR-mediated activity of selected pollutants and their mixtures
after interaction with dissolved organic matter
Michal Bittner a,⁎, Petra Macikova a, John P. Giesy b,c,d,e, Klara Hilscherova a
a
Research Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 126/3, CZ62500 Brno, Czech Republic
Department of Biomedical Veterinary Science and Toxicology Centre, Univ. of Saskatchewan, Canada
Zoology Department and Center for Integrative Toxicology, Michigan State University, East Lansing, USA
d
Biology and Chemistry Department, City University of Hong Kong, Kowloon, Hong Kong, China
e
School of Biological Sciences, University of Hong Kong, Hong Kong, China
b
c
a r t i c l e
i n f o
Article history:
Received 13 July 2010
Accepted 17 March 2011
Available online 13 April 2011
Keywords:
Humic substances
AhR
Mixture
Interaction
H4IIE-luc
Organic pollutants
a b s t r a c t
Dissolved organic matter (DOM) in freshwaters is present at concentrations ranging from 0.5 to 50 mg L−1,
and consists of various organic compounds, including humic substances (HS). HS exert a variety of direct and
indirect biological effects, including interaction with the aryl hydrocarbon receptor (AhR). AhR is a cytosolic
receptor that binds various hydrophobic organic compounds (HOCs) and mediates some of their toxic effects.
In vitro effects of binary mixtures of various DOM (mainly HS) with various HOCs on AhR-mediated responses
were studied by use of H4IIE-luc cells. Six out of 12 DOM activated the AhR even at environmentally relevant
concentrations (17 mg L−1). In simultaneous exposures of H4IIE-luc cells to DOM (17 mg L−1) and each of the
model compounds, 2,3,7,8-TCDD, PCB126, PCB169, benzo[a]pyrene, benzo[a]anthracene, dibenz[a,h]anthracene, fluoranthene, a mixture of persistent organic pollutants (POPs), a mixture of polycyclic aromatic
hydrocarbons (PAHs), and a mixture of all HOCs, either significant additive or facilitative effects were
observed when compared to activities of single HOCs. No significant decrease of effects due to possible
sorption of HOCs to DOM was observed, even in subsequent experiments when HOCs + DOM mixtures were
preincubated for six days before exposure to H4IIE-luc. Thus, DOM does not seem to protect organisms against
AhR-mediated toxic effects of HOCs (as usually predicted due to sorption of HOCs on DOM), but it can actually
enhance their potency for AhR-mediated effects in some situations.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
In polluted environments, especially in sediments, soils and water,
hydrophobic organic compounds (HOCs) such as polychlorinated
dioxins, biphenyls, and polycyclic aromatic hydrocarbons are
expected to occur in the presence of dissolved/natural organic matter
(D/NOM). An important mechanism of toxicity for some HOCs is
binding to the aryl hydrocarbon receptor (AhR). AhR-mediated effects
are considered to be markers of exposure to dioxin-like compounds
(Whyte et al., 2004). These compounds elicit a variety of adverse
effects in organisms, including carcinogenesis, embryotoxicity, teratogenicity, neurotoxicity, endocrine disruption and others (Poland
and Knutson, 1982; Janosek et al., 2006).
With regard to the adverse biological effects of pollutants such as
HOCs, there is a great effort to study their environmental fate which
influences their final toxicity. This includes e.g. study on possible
interactions and modes of action of HOCs in mixture with naturally
⁎ Corresponding author at: Research Centre for Toxic Compounds in the Environment,
RECETOX, Kamenice 126/3, CZ62500 Brno, Czech Republic. Tel.: + 420 549493807;
fax: + 420 549492840.
E-mail address: bittner@recetox.muni.cz (M. Bittner).
0160-4120/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2011.03.016
occurring DOM in waters. To reveal impact of DOM on HOCs' AhRmediated activity, we used in vitro bioassay based on H4IIE-luc cell
line. This bioassay is designed for detecting and estimating the relative
potency of pure compounds or complex mixtures in various matrices
to interact with the AhR. Commercially available clone of this bioassay
is registered as DR-CALUX® (Chemical-Activated Luciferase Gene
eXpression) assay (Murk et al., 1996), and analogous XDS-CALUX®
bioassay has already been approved as U.S. EPA Method 4435 for HOC
analysis (US EPA, 2008).
In the aquatic environment, approximately 75 to 99% of organic
carbon is particulate and dissolved non-living matter (the rest is
present in living organisms). Concentrations of DOM in most natural
freshwaters are in the range 0.5 to 50 mg L−1. The DOM is
predominantly comprised of humic substances (HS) that form about
40–80% of the total DOM (Steinberg et al., 2006). In sediments,
organic carbon consisting of HS can represent up to 20% of their total
dry weight (Akkanen et al., 2005). HS are natural products formed by
diagenesis of organic matter decomposition, which includes degradation and condensation processes. Generally, HS can be divided into
humic acids (HA), fulvic acids (FA) and colloidal/insoluble humins,
which differ in both their physicochemical and biological properties.
Due to the wide range of nominal molecular size and functional
M. Bittner et al. / Environment International 37 (2011) 960–964
groups, these compounds are generally functionally defined by their
solubilities in alkaline solution or spectral characteristics (Steinberg,
2003). Several processes in aquatic systems can be affected by DOM/
HS. These include acidification and water warming, light penetration
and photochemistry, microbial metabolism, ecosystem buffering,
indirect and direct biological effects (Tipping, 2002). Indirect effects
such as modulation of the availability of toxicants or nutrients via
interactions with various HS structures have been known for a long
time (Haitzer et al., 1998). Direct physiological effects such as changes
of heat shock protein expression and biotransformation enzyme
activity, or hormone-like effects in animals have been documented
more recently (Steinberg et al., 2008).
In a previous in vitro study, DOM-driven changes in AhR-mediated
effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin were described (Bittner
et al., 2009). In the present paper, in vitro effects of 12 DOM on AhRmediated effects of seven HOCs and their mixtures are investigated. A
paired experimental design was used. This consisted of exposure to
non-preincubated mixtures of HOCs and DOM, and to mixtures
preincubated for six days to simulate two situations that can occur in
the environment. At the time of accidental releases of HOCs into the
aquatic environment, organisms are exposed to fresh mixtures of
DOM and HOCs. This situation was simulated by exposure of H4IIE-luc
cells to non-preincubated HOCs + DOM mixtures. Nevertheless, HOCs
can interact with DOM during longer co-occurrence in the environment, which can result in modulation of toxicity of these mixtures.
The effect of this aging of HOCs + DOM mixtures was examined by
exposure of H4IIE-luc to HOCs + DOM mixtures that had been
preincubated for six days.
2. Materials and methods
DOM isolated from different matrices were purchased from Fluka
(now Sigma-Aldrich), Prod. No. 53680 (Prague, Czech Republic) —
Fluka HA; Sigma-Aldrich, Prod. No. H16752 (Prague, Czech Republic) —
Aldrich HA; Humintech GmbH (Düsseldorf, Germany) — HuminFeed;
and the International Humic Substances Society (IHSS, St. Paul, MN,
USA) — remaining nine DOM samples. DOMs were dissolved in
0.05 M NaOH to achieve a final concentration of 17 mg L−1. This
concentration was chosen based on previous findings where some
HA exerted AhR-mediated activity in vitro at this environmentallyrelevant concentration (Bittner et al., 2009). 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was purchased from Dr. Ehrenstorfer
(Augsburg, Germany), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) and
3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB169) were purchased from
Absolute Standards Inc. (Hamden, CT, USA), benzo[a]pyrene (B[a]P),
benzo[a]anthracene (B[a]A), dibenz[a,h]anthracene (DB[a,h]A), and
fluoranthene (Flu) were purchased from Sigma-Aldrich (Prague,
Czech Republic). Experiments were conducted using HOCs dissolved
in ethanol.
H4IIE-luc (rat hepatocarcinoma) cells stably transfected with the
luciferase gene under control of the dioxin response enhancer were
used for determination of AhR activation (Murk et al., 1996). In these
cells, when the appropriate substrates are added, binding of ligands to
the AhR results in emission of light that is proportional to the potency
of the mixture. This potency can be reported as 2,3,7,8-TCDD
equivalents (TCDD eq.). Cells were grown and maintained in DMEM
medium 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 the density of 15,000 cells/well in medium supplemented
with 1% gentamicin. After 24 h, H4IIE-luc cells were exposed in
triplicate to single HOCs or mixtures of HOCs + DOM for 24 h at 37 °C.
Single HOCs were tested at the following concentrations: 1.2 pM
2,3,7,8-TCDD, 10 pM PCB126, 1000 pM PCB169, 40 nM B[a]P, 240 nM
B[a]A, 1 nM DB[a,h]A, and 40,000 nM Flu. These concentrations were
chosen based on the fact that they are environmentally relevant and
961
they elicited significant in vitro effects of approximately 25% of
maximal AhR-mediated light emission caused by AhR saturating
concentration of 2,3,7,8-TCDD (500 pM), when compared to blanks.
The mixture of polycyclic aromatic hydrocarbons (PAHs Mix)
contained equal proportions of each tested PAH (a quarter concentration compared to the concentration of the compound if tested
alone): 10 nM B[a]P, 60 nM B[a]A, 0.25 nM DB[a,h]A, and 10,000 nM
Flu. The mixture of persistent organic pollutants (POPs Mix)
contained equal proportions of 0.4 pM 2,3,7,8-TCDD, 3.3 pM PCB126,
and 333.3 pM PCB169 (a third concentration compared to the
concentration of the compound if tested alone). The mixture of all
studied HOCs (HOCs Mix) contained equal proportions of the PAHs
Mix and POPs Mix (a half of each mixture). Each DOM was tested at a
concentration of 17 mg L−1 in a mixture with the above mentioned
HOCs and their mixtures. Cells exposed to DMEM with 0.5% EtOH and
0.5% 0.05 M NaOH were used as the appropriate vehicle controls.
Intensity of luciferase luminescence, which is proportional to
activation of AhR-mediated processes, was measured after 24 h of
exposure by use of the Promega Steady Glo Kit (Promega, Mannheim,
Germany). Three independent assays were conducted for each
compound/combination and concentration tested, except for the
mixtures with 2,3,7,8-TCDD and PCB169 where six independent
assays were conducted. More repetitions of the tests with these
exposure variants were conducted because of the greater variance
among the measurements. All samples including HOCs alone, and
each of the HOCs + DOM mixtures were added to the medium over
cells either immediately, or after preincubation. Preincubation was
conducted in culture medium supplemented with 1% gentamicin by
shaking in glass vials in the dark for the duration of six days. Such
paired experimental designs were used to simulate two situations
that can occur in real environmental mixtures as described above. The
6-day duration of preincubation was chosen on the basis of previous
findings (Bittner et al., 2009), which showed that while the results of
tests with shorter incubations were not significantly different from
non-preincubated samples, longer incubations usually resulted in
bacterial contamination of incubated samples, even when gentamicin
was used to prevent such bacterial progress.
Bioassay-derived data were examined statistically by use of
Statistica for Windows 8.0 (StatSoft, Tulsa, USA). Due to the small
sample size (mainly 3), data were not tested for normality or
homogeneity of variance. Because a one sample t-test was used to
determine if means of HOCs + DOM mixtures were significantly
different from a certain fixed value (100% response of pure HOCs) the
bias introduced by failure to meet these assumptions would likely be
minimal. Experiments were conducted in six independent repetitions
(2,3,7,8-TCDD + DOM and PCB169 + DOM) or three independent
repetitions (other HOCs + DOM mixtures), each performed in
triplicates. An F-test was used only for 2,3,7,8-TCDD + DOM, which
was done in 6 repetitions. In that case the data met the assumptions of
normality and homogeneity of variance. The level of significance used
was p = 0.05.
3. Results
Single HOCs at the tested concentrations exerted AhR-mediated effects in the range
of 12 to 38% of maximal AhR-mediated response (caused by the AhR saturating
concentration of 500 pM 2,3,7,8-TCDD). The observed up-regulations expressed as
percent of maxima for each compound were as follows: 1.2 pM TCDD — 17%, 10 pM
PCB126 — 38%, 1000 pM PCB169 — 12%, 40 nM B[a]P — 30%, 240 nM B[a]A — 38%,
1 nM DB[a,h]A — 29%, and 40,000 nM Flu — 16%. The AhR-mediated effect of the PAHs
Mix was 18%, POPs Mix 59%, and the HOCs Mix 53% of maximal 2,3,7,8-TCDD induction
(Table 1, last column). In the case of the 6-day preincubation, there were only
insignificant changes in AhR-mediated activities of HOCs compared to activities of nonpreincubated samples.
Of the 12 DOMs used in experiments (all at a concentration of 17 mg L−1), six
samples (only HA, no FA or NOM samples) elicited significant AhR-mediated activities
with and without preincubation. These levels of expression were similar in each
experimental design and the values ranged between 7 and 14% of maximal AhRmediated response caused by the AhR saturating concentration of 2,3,7,8-TCDD
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M. Bittner et al. / Environment International 37 (2011) 960–964
Table 1
Comparison of AhR-mediated activity of DOM relative to AhR-mediated activity of HOCs (%). There are shown AhR-active DOM (all HA) only, activity of other DOM was not
significantly different from the activity of vehicle control. Concentration of each DOM sample was 17 mg L−1; concentrations of HOCs are listed in the table. Values were derived from
ratio of AhR-mediated activity of tested DOM/AhR-mediated activity of tested HOCs, both expressed as percentage of 500 pM TCDD AhR-mediated activity (results in last column and
last row).
AhR-mediated activity of DOM × AhR-mediated activity of HOCs (%)
Flu 40,000 nM
PCB126 0.01 nM
PCB169 1 nM
B[a]P 40 nM
DB[a,h]A 1 nM
POPs Mix
B[a]A 240 nM
TCDD 1.2 pM
PAHs Mix
HOCs Mix
TCDD 500 pM
Leonardite HA
Fluka HA
Elliot Soil HA
Aldrich
HA
HuminFeed
Florida
Peat HA
81
34
108
43
45
22
34
76
72
25
13
81
34
108
43
45
22
34
76
72
25
13
88
37
117
47
48
24
37
82
78
26
14
56
24
75
30
31
15
24
53
50
17
9
50
21
67
27
28
14
21
47
44
15
8
44
18
58
23
24
12
18
41
39
13
7
% TCDD
500 pM⁎
16
38
12
30
29
59
38
17
18
53
100
⁎ Values in this column equal to AhR-mediated activity of HOCs relative to AhR-mediated activity of TCDD 500 pM (%).
(500 pM). The proportions of maximal expression caused by DOM themselves were as
follows: Leonardite HA — 13%, Fluka HA — 13%, Elliot Soil HA — 14%, Aldrich HA — 9%,
HuminFeed — 8%, Florida Peat HA — 7% (Table 1, last row); remaining six DOM samples
did not elicit any AhR-mediated response. More detailed information on AhR-mediated
effects of DOM has been published previously by Janosek et al. (2007). The proportions
of AhR-mediated activity of AhR-active DOM themselves compared to AhR-mediated
activity of HOCs are shown in Table 1.
Simultaneous exposure of H4IIE-luc cells to both HOCs and DOM resulted in
statistically significant modulations of the AhR-mediated activity compared to single
HOC exposure in 63 of the 120 combinations without preincubation. All of the observed
significant effects were increases of AhR-mediated activity of HOCs in the presence of
DOM. Responses of these HOCs + DOM mixtures ranged from 111 to 557% with a
median interaction that resulted in 201% compared to each HOCs' alone activity (100%;
Table 2).
In the case of HOCs + DOM mixtures that were preincubated six days prior to
exposure, statistically significant response enhancement was observed for 38 HOCs
+ DOM combinations. The degree of induction with these mixtures ranged from 127 to
834% with a median of 219% compared to activity of HOCs preincubated alone (100%).
Only one DOM type, NOM Nordic Reservoir, caused statistically significant decrease of
AhR-mediated expression when co-exposed with PAHs Mix after preincubation.
However, the change was relatively small with a decrease from 100% to 93%. Greater
decreases in the range of 74 and 76% of the POPs Mix response were observed after
preincubation of POPs Mix with several DOM (Table 2), nevertheless, these were not
statistically significant due to the assay variability.
AhR-mediated responses of single HOCs (or its mixtures) in combinations with
various DOM samples differed in dependence on time of preincubation. Nevertheless,
there was no uniform trend — both enhancement and decrease of AhR-mediated
responses of analogous HOCs + DOM mixtures with/without preincubation were
observed. To evaluate the overall influence of the preincubation, the average values of
AhR-mediated responses of single HOCs mixed with each DOM sample (i.e. average
value from each row in Table 2 was calculated) in the experiment with/without
preincubation were compared. If the difference between the average activity of nonpreincubated and preincubated samples was greater than 50% (set arbitrarily), it was
deemed to be significant. Preincubation for six days led to significantly different results
for four HOCs in mixture with all the types of DOM compared to analogous experiments
with non-preincubated samples (depicted in the last column of Table 2). Preincubated
mixtures of DOM with PCB126, B[a]P, and POPs Mix resulted in significantly less AhRmediated activity, compared to analogous non-preincubated samples. The average
Table 2
Summary of AhR-mediated activities of HOCs + DOM mixtures. Concentration of each DOM sample was 17 mg L−1; concentrations of HOCs are shown in the table. 100% in each
row represents AhR-mediated activity of corresponding HOCs at listed concentration. The table presents results for each co-exposure variant dosed to cells either immediately after
mixing or after 6-day preincubation. Values represent the mean and SD (values in brackets) of six independent experiments (2,3,7,8-TCDD + DOM and PCB169 + DOM) or three
independent experiments (other HOCs+ DOM mixtures), each performed in triplicates. Bold font denotes values significantly different (pb 0.05) from AhR-mediated activity of
corresponding HOCs alone (100%). Length of the gray bands graphically represents the values in the table (only the means). Δ indicates changes of AhR-mediated activity of single
HOCs in co-exposure with all DOM in experiments with and without preincubation (− denotes no significant change, ↗ denotes increase of activity, ↘ denotes decrease of activity).
AhR-mediated activity (% of HOCs induction)
Pre- Leonardite Fluka
incub. HA
HA
(days)
Flu 40,000 nM
PCB126 0.01 nM
PCB169 1nM
B[a]P 40nM
DB[ah]A 1nM
POPs Mix
B[a]A 240nM
TCDD 1.2 pM
PAHs Mix
HOCs Mix
0
6
0
6
0
6
0
6
0
6
0
6
0
6
0
6
0
6
0
6
557 (91)
548 (228)
442 (79)
396 (14)
477 (255)
671 (179)
305 (121)
273 (78)
319 (90)
299 (69)
339 (69)
169 (14)
232 (51)
228 (60)
176 (39)
168 (68)
149 (17)
143 (60)
147 (32)
150 (40)
383 (130)
462 (191)
499 (143)
403 (6)
469 (256)
834 (421)
256 (90)
253 (75)
231 (30)
296 (41)
256 (31)
158 (20)
203 (15)
184 (10)
174 (33)
158 (29)
114 (6)
145 (3)
121 (9)
158 (38)
Elliot Soil Aldrich
HA
HA
554 (219)
486 (120)
494 (238)
328 (4)
459 (216)
651 (261)
288 (78)
207 (25)
241 (43)
209 (46)
316 (72)
160 (29)
188 (44)
158 (21)
192 (53)
133 (39)
121 (7)
119 (17)
138 (16)
127 (18)
337 (99)
316 (104)
394 (147)
334 (62)
335 (130)
461 (212)
279 (123)
212 (51)
219 (31)
248 (26)
234 (46)
127 (1)
193 (44)
177 (30)
162 (41)
144 (34)
113 (6)
129 (37)
114 (9)
119 (12)
HuminFeed Florida
Peat HA
394 (115)
377 (136)
392 (77)
306 (44)
320 (150)
454 (104)
262 (41)
189 (10)
214 (15)
218 (28)
200 (47)
117 (29)
186 (41)
162 (15)
157 (30)
128 (32)
105 (6)
95 (9)
110 (6)
107 (3)
309 (101)
275 (102)
338 (128)
239 (13)
330 (155)
367 (128)
231 (69)
144 (26)
201 (66)
174 (29)
285 (54)
130 (30)
168 (44)
149 (25)
145 (22)
113 (25)
145 (12)
144 (33)
148 (10)
122 (22)
Waskish
Peat HA
250 (39)
312 (162)
212 (55)
230 (79)
148 (33)
307 (168)
214 (62)
224 (51)
198 (31)
220 (39)
126 (11)
97 (15)
182 (30)
179 (29)
131 (15)
124 (33)
111 (5)
108 (10)
114 (14)
107 (7)
Suwannee Nordic
Suwannee Suwannee Nordic
River NOM Reservoir River HA River FA Reservoir
FA
NOM
256 (41)
237 (90)
190 (109)
156 (61)
139 (35)
183 (73)
253 (75)
160 (48)
211 (75)
170 (42)
143 (24)
74 (27)
186 (60)
157 (35)
138 (16)
118 (26)
111 (3)
123 (57)
113 (9)
108 (12)
234 (31)
260 (99)
220 (46)
198 (16)
123 (27)
168 (77)
236 (40)
188 (71)
202 (41)
191 (22)
119 (11)
76 (35)
181 (65)
158 (45)
129 (20)
110 (29)
103 (15)
111 (44)
104 (3)
96 (11)
233 (56)
200 (15)
200 (34)
218 (59)
125 (21)
227 (131)
223 (45)
172 (41)
170 (20)
154 (42)
139 (15)
99 (25)
157 (20)
192 (46)
124 (14)
112 (20)
106 (5)
118 (29)
116 (5)
108 (13)
196 (44)
223 (119)
164 (72)
148 (49)
124 (46)
260 (113)
223 (49)
173 (46)
194 (32)
174 (33)
102 (20)
81 (23)
195 (59)
144 (19)
131 (10)
111 (29)
97 (1)
112 (23)
106 (11)
101 (1)
197 (46)
136 (19)
165 (94)
92 (8)
190 (68)
139 (89)
161 (23)
112 (16)
154 (66)
99 (9)
176 (29)
90 (14)
134 (22)
120 (9)
125 (6)
93 (17)
138 (8)
93 (2)
126 (7)
109 (11)
Δ
M. Bittner et al. / Environment International 37 (2011) 960–964
decreases for the various combinations were: 55% for PCB126 + DOM, 52% for B[a]P
+ DOM, and 88% for the POPs Mix. On the other hand, AhR-mediated response to
PCB169 + DOM mixtures that were preincubated prior to exposure was significantly
greater compared to analogous non-preincubated samples with average increase of
124%. AhR-mediated activities of other HOCs + DOM mixtures were not significantly
influenced by preincubation if compared to non-preincubated samples; the average
differences between each appropriate pair ranged from − 23 to 2%.
4. Discussion
To visually depict the differences in induction of AhR-mediated
response (expression of luciferase) by H4IIE-luc cells among treatments with combinations of DOM and HOCs, combinations of DOM
and HOCs that resulted in the greatest AhR-mediated expression are
grouped in the upper left corner of Table 2. Alternatively, mixtures
resulting in the least AhR-mediated expression are grouped in the
lower right corner (Table 2). This arrangement highlights those DOM
that most affected AhR-mediated responses of the transactivation
assay to HOCs as well as those HOCs that exerted greater AhRmediated response when mixed with DOM.
In the case of mixtures of HOCs with AhR-active HA, increases in
AhR-mediated expression were probably caused mainly by additive
effects. This can be derived from comparison of detected AhRmediated activity of HOCs mixed with AhR-active DOM (first six
columns in Table 2) with relative AhR-mediated activity of DOM
themselves (Table 1, complementary to Table 2). The mixtures of
AhR-active DOM with HOCs were consequently the most AhR-active.
Nevertheless, all the observed effects caused by DOM from relatively
great up-regulation of AhR-mediated response by HOCs to moderate
down-regulation cannot be explained by simple additive effects of
HOCs and DOM. There must be mentioned also facilitation, which
increases bioavailability of HOCs by both enhancement of its water
solubility and changes in biomembrane permeability by DOM (Doring
and Marschner, 1998; Glover and Wood, 2005). In addition, uptake of
hydrophobic organic compounds into organisms is often limited by
the diffusive transport through a thin boundary layer, and it has been
established recently that a variety of DOM at environmental levels can
enhance diffusive mass transfer in various transport scenarios (Mayer
et al., 2007). This mechanism can explain both enhancement of AhRmediated response caused by HOCs in mixture with non-AhR-active
DOM (especially FA and NOM), and more than additive effect of AhRactive DOM (HA samples in Table 1 and also in the first six columns of
Table 2) on AhR-mediated activity of Fluoranthen, PCB126 and
PCB169 (Table 2).
Another mechanism, sorption, is a well known ability of HOCs (e.g.
POPs and PAHs) to bind onto DOM structures (Haitzer et al., 1998;
Perminova et al., 2001), where the decrease of HOCs' toxicity is
usually expected. In this paper, the observed decrease of AhRmediated activity of the three groups of HOCs + DOM, i. e. PCB126
+ DOM, B[a]P + DOM, and POPs Mix + DOM in preincubated mixtures
compared to analogous non-preincubated samples could be explained
by sorption. The combination of facilitative and sorptive effects caused
by DOM has also been described for copper toxicity to Simocephalus
serrulatus (Daphnidae) in DOM-rich pond (Giesy et al., 1983). Specific
combination of these involved mechanisms, with facilitation as a most
important mechanism that has increased during 6-day preincubation,
could also explain the greater AhR-mediated expression after 6-day
preincubation (124% greater induction, in average) compared to
analogous non-preincubated PCB169 + DOM mixtures (to confirm
this quite unexpected observation, six independent experiments with
PCB169 + DOM, both with and without preincubation, were performed). Besides the above mentioned mechanisms, cross-talk
between AhR and other receptor pathways could be involved in the
effects of some compounds or mixtures. For example, recent studies
document interactive regulatory cross-talk between glucocorticoid
receptor (GR) and AhR (e.g. Dvorak et al. 2008). However, there is no
evidence of the potential interaction of DOM with GR.
963
AhR-active DOM including Fluka HA, Aldrich HA, HuminFeed and
Leonardite HA are all derived from brown coal and thus are not
relevant to effects of DOM in natural environments. However, if coalderived HA are considered to be used as so called “environmentally
friendly” sorbents for HOCs decontamination in situ (Perminova et al.,
2001), observed AhR-mediated effects including interaction with
HOCs should be taken into account as a possible side effect. Other
DOM types of environmental origin either had no effect or enhanced
the AhR-mediated potency of HOCs. To generalize effects of DOM on
HOCs activity, a range of both DOM and HOCs was used. Nevertheless,
the significance of our in vitro findings needs to be investigated in vivo
before the relevance for ecosystems can be addressed. There is one
study with fish describing similar additive effect of coal-derived HA
and crude oil (rich for PAHs) on expression of cytochrome P450 1A
(Matsuo et al., 2006) that is directly linked to AhR activation. These
findings indicate that our in vitro results could be also anticipated if
tested in vivo, but more studies with other DOM samples (not only of
coal origin) are needed for generalization.
Acknowledgments
We would like to thank to Dr. Jiri Novak for valuable assistance in
in vitro testing, and to Dr. Klara Kubosova for valuable assistance in
data analysis. This research has been supported by project GACR 525/
08/P464, INCHEMBIOL framework project MSM0021622412, and
project CETOCOEN (CZ.1.05/2.1.00/01.0001) granted by the European
Union and administered by the Ministry of Education, Youth and
Sports of the Czech Republic. Prof. Giesy was supported by the Canada
Research Chair program and at large Chair Professorship at the
Department of Biology and Chemistry and State Key Laboratory in
Marine Pollution, City University of Hong Kong.
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