Isolation and identification of xenobiotic aryl hydrocarbon receptor ligands in dyeing wastewater

Article abstract of DOI:10.1021/es061500g

Dyeing wastewater collected in Kyoto city, Japan, was investigated for the occurrence of aryl hydrocarbon receptor (AhR) ligands by using an AhR-responsive reporter gene assay. Concentrated extracts of wastewater samples elicited a dose-dependent increase in AhR ligand activity, and several hydrophobic HPLC fractions of the extracts were highly effective in inducing AhR ligand activity. Three potential AhR ligands were isolated from these fractions and identified to be Disperse Red 92, Disperse Yellow 64, and 3′-hydroxybenzo[b] quinophthalone by using HPLC and LC-MS/MS. Disperse Red 92, which has also been detected in the treated effluent from a sewage plant receiving the wastewater, is an anthraquinone disperse dye showing weak AhR binding affinity in the assay. Disperse Yellow 64 and 3′-hydroxybenzo[b]quinophthalone are quinoline disperse dyes capable of activating the AhR at nanomolar concentrations. In particular, Disperse Yellow 64 is a highly potent AhR ligand that was 3 times more effective in inducing AhR ligand activity than β-naphthoflavone in the assay. Quinoline disperse dyes are suggested to be a new class of xenobiotic AhR ligands which pose a danger to aquatic biota and human health.

Full text of DOI:10.1021/es061500g

Environ. Sci. Technol. 2007, 41, 652-657
Among the toxic dyes that were banned, sudan dyes or
Isolation and Identification of
Xenobiotic Aryl Hydrocarbon
Receptor Ligands in Dyeing
Wastewater
methyl yellow have been shown to interact with the aryl
hydrocarbon receptor (AhR) (11, 12), a ligand-activated
transcription factor that mediates many biological and toxic
effects of environmental contaminants such as dioxins or
polycyclic aromatic hydrocarbons. The ligand-AhR complex
enters the nucleus after ligand binding and dimerizes with
the AhR nuclear translocator protein. The heterodimer
subsequently interacts with the xenobiotic responsive ele-
ment and activates the transcription of downstream genes,
including those encoding xenobiotic-metabolizing enzymes.
The AhR is also involved in many physiological functions,
such as cell cycle regulation, apoptosis, development, and
reproduction (13-15). Activation of the AhR by xenobiotic
ligands may lead to metabolical detoxification by metaboliz-
ing enzymes or altered gene expression that relates to various
toxic responses, including teratogenicity, immunotoxicity,
or carcinogenicity. Thus, binding of a xenobiotic ligand to
the AhR is suggested to be an indication of possible toxicity.
Environmental AhR ligands have been detected in in-
dustrial effluent such as pulp and paper mill wastewater or
produced water from offshore oil and gas production (16,
17). As for dyeing industries, dioxazine or phthalocyanine
dyes have been found to be contaminated with polychlo-
rodibenzodioxins (PCDDs) and polychlorodibenzofurans
(PCDFs) due to the use of chlorinated aromatic solvents or
chloranil containing high levels of PCDD/PCDFs during
manufacturing processes (18, 19). In addition, several
synthetic azo dyes or anthraquinone dyes have also been
reported to activate the AhR (11, 12, 20). Since it is presumable
that dyeing wastewater contains chemicals capable of binding
to the AhR, investigating the occurrence of potential AhR
ligands in dyeing wastewater is necessary to provide basic
information for hazard reduction.
In this study, untreated dyeing wastewater was collected
in the vicinity of a sewage plant that treats the wastewater
together with domestic wastewater. Xenobiotic AhR ligands
and mutagens have been detected in the treated effluent of
the sewage plant and are suggested to be dyes or dye
derivatives (9, 10, 20). A bioassay-directed analysis, which
consists of a yeast-based reporter gene assay for measuring
AhR ligand activity, high-performance liquid chromatography
(HPLC) for separating target compounds, and HPLC-tandem
mass spectrometry (LC-MS/MS) for identifying ligand
candidates, was used to investigate potential AhR ligands in
the wastewater. The results revealed that several commercial
dyes currently used in Japan elicit AhR ligand activity, and
quinoline disperse dye is suggested to be a new class of
xenobiotic AhR ligands.
P E I - H S I N C H O U , † S A B U R O M A T S U I , †
K E N T A R O M I S A K I , ‡ A N D
T O M O N A R I M A T S U D A * , †
Department of Technology and Ecology, Graduate School of
Global Environmental Studies, Kyoto University, Sakyo-Ku
Yoshida-Honmachi, Kyoto 606-8501, Japan, and Department
of Urban and Environmental Engineering, Graduate School of
Engineering, Kyoto University, Nishikyo-Ku
Kyotodaigaku-Katsura, Kyoto 615-8540, Japan
Dyeing wastewater collected in Kyoto city, Japan, was
investigated for the occurrence of aryl hydrocarbon receptor
(AhR) ligands by using an AhR-responsive reporter gene
assay. Concentrated extracts of wastewater samples elicited
a dose-dependent increase in AhR ligand activity, and
several hydrophobic HPLC fractions of the extracts were
highly effective in inducing AhR ligand activity. Three potential
AhR ligands were isolated from these fractions and
identified to be Disperse Red 92, Disperse Yellow 64, and 3′-
hydroxybenzo[b]quinophthalone by using HPLC and LC-
MS/MS. Disperse Red 92, which has also been detected in
the treated effluent from a sewage plant receiving the
wastewater, is an anthraquinone disperse dye showing
weak AhR binding affinity in the assay. Disperse Yellow 64
and 3′-hydroxybenzo[b]quinophthalone are quinoline
disperse dyes capable of activating the AhR at nanomolar
concentrations. In particular, Disperse Yellow 64 is a
highly potent AhR ligand that was 3 times more effective
in inducing AhR ligand activity than â-naphthoflavone in the
assay. Quinoline disperse dyes are suggested to be a
new class of xenobiotic AhR ligands which pose a danger
to aquatic biota and human health.
Introduction
Dyeing wastewater contains many hazardous chemicals and
has been shown to elicit genotoxicity and mutagenicity (1,
2). Although certain carcinogenic dyes have been prohibited,
toxicological data for dyes or dye derivatives are still
insufficient, and at least 14 dye products in use in European
countries were reported to be mutagenic in 2004 (3-6).
Additionally, while many dyes are not readily biodegradable,
ozonation or chlorination have also been shown to increase
the toxicity of dyeing wastewater, indicating the formation
of more toxic intermediates during the treatment processes
(7-10). Thus, discharging non-treated or inappropriately
treated dyeing effluent poses a threat to the receiving water
body.
Materials and Methods
Reagents. Disperse Red 92 [Sumikaron Brilliant Red S-BLF;
C.I. 60752] was kindly provided by Sumitomo Chemicals
(Osaka, Japan). 2,3-Naphthalenedicarboxylic anhydride was
obtained from Tokyo Chemical Industries (Tokyo, Japan).
3-Methylcholanthrene, 3-hydroxy-2-methyl-4-quinolinecar-
boxylic acid, and Disperse Yellow 64 [Latyl Yellow 3GB; C.I.
47023] were purchased from Aldrich (WI). Commercially
obtained dyes were purified using HPLC to remove impurities
before use. Other chemicals were of analytical grade and
were purchased from Wako (Osaka, Japan).
Synthesis of 3′-Hydroxybenzo[b]quinophthalone. 3′-
Hydroxybenzo[b]quinophthalone (1H-Benz[f]indene-1,3-
(2H)-dione, 2-(3-hydroxy-2(1H)-quinolinylidene), CAS 341-
34-7) was synthesized according to the method described in
ref 21. In brief, equimolar amounts of 2,3-naphthalenedi-
* Corresponding author phone: +81-753-5171; fax: +81-753-3335;
e-mail: matsuda@eden.env.kyoto-u.ac.jp.
† Department of Technology and Ecology, Graduate School of
Global Environmental Studies.
‡ Department of Urban and Environmental Engineering, Graduate
School of Engineering.
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652 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 2, 2007
10.1021/es061500g CCC: $37.00
2007 American Chemical Society
Published on Web 12/14/2006

carboxylic anhydride and 3-hydroxy-2-methyl-4-quinoline-
carboxylic acid were dissolved in nitrobenzene, stirred, and
heated overnight under reflux. Nitrobenzene was removed
by distillation under reduced pressure, and the target
compound was purified repeatedly by using silica gel column
chromatography (eluent: hexane/chloroform) followed by
recrystallization from methanol. Further purification was
performed by HPLC.
Sampling and Extraction. Dyeing wastewater was col-
lected from an open channel containing effluent of dyeing
factories in the southern area of Kyoto city, Japan, in July
2003, March 2004, and October 2004. Samples collected in
July 2003 and March 2004 (500 mL) were filtered through
0.45 µm glass fiber filters and passed through Sep-pak Plus
C18 environmental cartridges (Waters, MA) at a flow rate of
10 mL/min. Each cartridge was washed with 10 mL of water
and then eluted with 10 mL of methanol. The eluents were
combined, evaporated to dryness in a centrifugal vacuum
concentrator (Tomy, Tokyo, Japan), and redissolved in a small
amount of dimethyl sulfoxide (DMSO).
Dyeing wastewater was taken again in October 2004 (1500
mL) to obtain sufficient amounts of potential AhR ligands.
The wastewater sample was filtered and passed through Sep-
Pak Vac 35 cc C18 cartridges (Waters, MA) using a flow rate
of 10 mL/min. The cartridges were washed with water and
then eluted with 60 mL of 50% methanol in water, 60 mL of
75% methanol in water, and 120 mL of methanol. Three
extracts were collected separately, evaporated to dryness,
and redissolved in DMSO. Dilution series of all concentrated
extracts were prepared in DMSO.
HPLC Fractionation, Quantification, and Ligand Isola-
tion. An aliquot of each concentrated extract was injected
into a Shim-pack FC-ODS column (particle size 5 µm, 150
× 4.6 mm; Shimadzu, Kyoto, Japan) for HPLC fractionation
and quantification. The column was eluted in a linear gradient
of 10% methanol in water to 100% methanol within 20 min
followed by holding at 100% methanol for a further 20 min
at a flow rate of 1 mL/min. Fractions were collected every
minute for 36 min by using a fraction collector (FRC-10A,
Shimadzu, Kyoto, Japan) or manually. Each fraction was
evaporated to dryness and then redissolved in DMSO. The
HPLC system was equipped with a SPD-M10Avp diode array
detector (Shimadzu, Kyoto, Japan), and concentrations of
AhR ligand candidates in the extract of wastewater collected
in March 2004 were estimated by monitoring the absorption
of eluate at 254, 443, and 456 nm.
The methanol extract of wastewater sample taken in Oct
2004 was also fractionated by a Wakosil-II 5C18HG column
(particle size 4.2-4.7 µm, 20 × 50 mm; Wako, Osaka, Japan)
eluted using a flow rate of 2.5 mL/min with gradient elution,
which started at 75% methanol in water and increased to
100% methanol within 5 min followed by holding at 100%
methanol for another 25 min. AhR ligand candidates in the
fractions collected from retention times of 9-10 min and
18-21 min were further isolated and purified for several times
using the Shim-pack FC-ODS column and the Wakosil-II
5C18HG column.
AhR-Dependent Reporter Gene Assay. AhR ligand activity
was measured by a reporter gene assay using the recombinant
yeast YCM3 strain (22), which contains the human AhR and
AhR nuclear translocator (Arnt) expression construct and a
pTXRE5-Z (LacZ) reporter plasmid responding to the ligand-
induced AhR/Arnt complex. DMSO that showed no activity
in the assay was use as a vehicle for dissolving and diluting
test samples. The bioassay was basically carried out as
described in ref 23. In brief, the yeast was grown in a synthetic
glucose medium (2% glucose, lacking tryptophan) at 30 °C
for 18 h. One µL of a test sample was mixed with 200 µL of
synthetic galactose medium (2% galactose, lacking tryp-
tophan) and 5 µL of the saturated culture in a 96-well
microplate, and subsequently incubated at 30 °C for another
18 h. After the incubation, cell density was determined by
reading the absorbance at 595 nm (A595) using a microplate
reader (Bio-Rad Laboratories, CA), and 10 µL of the cell
suspension was transferred into a new microplate and mixed
thoroughly with 140 µL of Z-buffer (22) and 50 µL of
o-nitrophenyl-â-D-galactopyranoside solution (4 mg/mL,
made in Z-buffer). The absorbance at 405 nm (A405) was
measured after incubating at 37 °C for 60 min, and the
â-galactosidase activity (reported as LacZ units) was calcu-
lated by the following formula: LacZ units ) 1000 × A405
/
(A595 × 0.01 (mL of cell suspension added) × 60 (minutes of
reaction time)). Each sample was tested in triplicate, and
data are shown as mean values ( standard deviation.
â-Naphthoflavone (â-NF) was used as the positive control.
â-NF equivalent concentration was calculated using the
dose-response curve of â-NF.
LC/ESI-MS/MS. Potential AhR ligands isolated from the
dyeing wastewater were analyzed by an electrospray ioniza-
tion tandem mass spectrometry (ESI-MS/MS). Experiments
were carried out using a Micromass Quattro Ultima Pt mass
spectrometer (Waters, MA) equipped with a Shim-pack FC-
ODS column and a SPD-10Avp UV-vis detector (Shimadzu,
Kyoto, Japan). The column was eluted in a linear gradient of
10% methanol in water to 100% methanol within 20 min
followed by holding at 100% methanol for another 40 min
at a flow rate of 0.4 mL/min. Data acquisition was performed
in the positive ion mode. The experimental conditions were
set as follows: capillary voltage 3.5 kV, cone voltage 35 V, ion
source temperature 130 °C, desolvation temperature 380 °C,
desolvation gas flow rate 700 L/hr. Argon was used as the
collision gas, and the collision energy was set at 25 eV for
acquiring MS/MS spectra.
Results
Detection of AhR Ligand Activity. Concentrated extracts of
dyeing wastewater samples collected in July 2003 (Jul-03
sample) and March 2004 (Mar-04 sample) showed a dose-
dependent increase in AhR ligand activity measured by using
the yeast-based reporter gene assay (Figure 1A). Significant
increases in the activity were detected when the concentration
factor of both extracts was as low as 0.004 (p < 0.05, t-test),
indicating that even the diluted extracts equivalent to the
concentrations of 250-fold diluted dyeing wastewater were
able to induce AhR signaling in the assay. Figure 1B shows
the AhR ligand activity of HPLC fractions of the two extracts.
High AhR ligand activity was induced by the 23rd to 28th and
33rd to 34th fractions of the Jul-03 sample, and the 23rd to
28th, 31st, and 34th to 35th fractions of the Mar-04 sample.
Activation of the AhR was not detected in the polar fractions
collected from 0 to 20 min of both extracts.
Though the composition of wastewater samples varied
between months, several peaks showing similar UV spectra
and HPLC retention times were detected in both extracts.
Compounds considered to be potential AhR ligands were
chosen for further isolation and identification due to the
following reasons. A red compound (R1) observed in the
25th fraction drew attention because its retention time and
UV spectrum were similar to those of an AhR ligand candidate
detected in the treated sewage effluent in our previous studies
(20). In addition, a yellow compound (Y1) was chosen because
it was detected in the 33rd to 34th fractions of the Jul-03
sample and the 34th to 35th fractions of the Mar-04 sample
that elicited high AhR ligand activity. A second yellow
compound (Y2) observed in the 31st fraction of the Mar-04
sample was also selected because it was the major compound
in the fraction eliciting high AhR ligand activity. Figure 1C
shows the HPLC chromatogram at UV absorbance 254 nm
of the Mar-04 sample.
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FIGURE 2. (A) AhR ligand activity of DMSO (solvent blank) and
three extracts of the Oct-04 sample, E1 (50% methanol in water),
E2 (75% methanol in water), and E3 (100% methanol) (final
concentration factor 0.6). (B) HPLC chromatogram (UV absorbance
at 254 nm versus retention time) of E3. R1 and Y1 are AhR ligand
candidates detected in fractions eliciting AhR ligand activity. (C)
AhR ligand activity of the HPLC fractions of E3 (final concentration
factor 4.9)
12.5 nM), suggesting that the loss in AhR ligand activity during
the fractionation step was very low.
E3 was further fractionated to obtain R1 and Y1, and the
two compounds were isolated from the fractions collected
in retention times of 9-10 min and 18-21 min, respectively.
In addition, Y2 was separated from the concentrated extract
of the Mar-04 sample. These AhR ligand candidates were
purified using HPLC and were then analyzed by LC-MS/MS
and the yeast-based reporter gene assay.
Identification of Xenobiotic AhR Ligands. A base peak
at m/z 497 was observed in the full-scan MS spectrum of R1,
and the MS/MS spectrum of the base peak ion showed a
major fragment ion at m/z 330 (Figure 3A). The mass of R1
was suggested to be approximately 496, which corresponded
to that of a commercial anthraquinone disperse dye, Disperse
Red 92 (DR 92; C25H24N2O7S, molecular weight 496.53; Figure
3B). The UV-visible spectrum of R1 (λmax: 254, 519, and 553
nm in methanol; Figure 3A) was also similar to that of DR
92 (24). Thus, DR 92 was obtained commercially and analyzed
by HPLC and LC-MS/MS. R1 was identified to be DR 92 due
to the identical HPLC retention time, UV-visible spectrum,
and MS/MS spectrum.
The mass of Y1 was suggested to be approximately 339
because a base peak at m/z 340 was observed in the full-scan
mass spectrum. In addition, fragment ions generated from
the base peak were detected at m/z 323 and 155 (Figure 4A).
3′-Hydroxybenzo[b]quinophthalone (3′-HB[b]QP; C22H12NO3,
molecular weight 339.34; Figure 4B), a quinoline disperse
dye which was studied as a potential substitute for the toxic
dye cadmium yellow in Japan during the 1970s, was suggested
to be the candidate of Y1 due to its similar molecular weight
FIGURE 1. (A) AhR ligand activity of the serial dilution of the Jul-03
sample and Mar-04 sample. The asterisk indicates the lowest
concentration factor eliciting a significant increase in AhR ligand
activity (0.004 for both samples; p < 0.05, t-test). (B) AhR ligand
activity of the HPLC fractions of the Jul-03 sample and Mar-04
sample (final concentration factor 1.2). (C) HPLC chromatogram (UV
absorbance at 254 nm versus retention time) of the Mar-04 sample.
Peaks indicated as R1, Y1, and Y2 are three AhR ligand candidates
observed in fractions eliciting AhR ligand activity.
Isolation of Potential AhR Ligands. The wastewater
sample taken in October 2004 (Oct-04 sample) was used to
obtain sufficient amounts of potential AhR ligands for
chemical identification. Since the polar HPLC fractions of
the Jul-03 sample and Mar-04 sample were incapable of
inducing AhR ligand activity, hydrophobic compounds were
separated from hydrophilic ones by eluting each cartridge
with three eluents as described in the Materials and Methods
section. Figure 2A shows the AhR ligand activity of the three
corresponding extracts, referred to as E1 (50% methanol in
water), E2 (75% methanol in water), and E3 (100% methanol).
E3 elicited the highest activity and R1 and Y1 were observed
in its HPLC chromatogram (Figure 2B). Figure 2C shows the
AhR ligand activity of the HPLC fractions of E3. The â-NF
equivalent concentration of AhR ligand activity elicited by
each HPLC fraction (final concentration factor: 4.9) was
calculated and summed up to be 12.9 nM, which was close
to that of the 4.9-fold concentrated extract (estimated to be
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654 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 2, 2007

FIGURE 3. (A) MS/MS spectrum of the [M + H]+ ion 497 of R1 and
the UV-visible spectrum of DR 92 in methanol (λmax: 254, 519, 553
nm). (B) Chemical structure of DR 92.
FIGURE 5. (A) MS spectrum and the UV spectrum in methanol (λmax
:
443, 422, 285 nm) of Y2. (B) MS/MS spectrum of the [M + H]+ ion
368 of Y2. (C) Chemical structure of DY 64.
FIGURE 4. (A) MS/MS spectrum of the [M + H]+ ion 340 of Y1 and
the UV-visible spectrum of 3′-HB[b]QP in methanol (λmax: 456, 432,
276, 286, 368 nm). (B) Chemical structure of 3′-HB[b]QP.
and UV-visible spectrum (21). 3′-HB[b]QP was synthesized
and then subjected to HPLC and LC-MS/MS analysis. Y1
was confirmed to be 3′-HB[b]QP because the HPLC retention
time, UV-visible spectrum (λmax: 456, 432, 276, 286, and 368
nm in methanol; Figure 4A), and MS/MS spectrum of 3′-
HB[b]QP were identical to those of Y1.
Y2 exhibited a characteristic isotope pattern for bromine
in its MS spectrum (peaks at m/z 368 and 370 corresponding
to 79Br and 81Br isotopes, respectively), and λmax at 443, 422,
and 285 nm were observed in its UV-visible spectrum (Figure
5A). The MS/MS spectrum of the base peak atm/z368 showed
major fragment ions at m/z 350, 289, and 245 (Figure 5B).
A monobrominated quinoline disperse dye, Disperse Yellow
64 (DY 64; C18H10BrNO3, molecular weight 368.18; Figure 5C),
was considered to be the candidate of Y2. Commercially
obtained DY 64 was purified and then analyzed by HPLC
and LC-MS/MS. Y2 was confirmed to be DY 64 because
identical data were obtained in the analyses (Figure 5A and
B).
FIGURE 6. Dose-response curves of AhR ligand activity elicited
by DR 92, 3′-HB[b]QP, DY 64, 3-MC, and â-NF. The asterisk indicates
the lowest concentration eliciting a significant increase in AhR
ligand activity (DY 64 0.5 nM; 3′-HBQP 2.7 nM; DR 92 971 nM; p <
0.01, t-test)
AhR Ligand Activity and Concentrations of Potential
Ligands. Figure 6 shows the dose-response curves of AhR
ligand activity of the three potential AhR ligands isolated
from the dyeing wastewater (DR 92, 3′-HB[b]QP, DY 64) and
two typical AhR ligands (3-methylcholanthrene (3-MC) and
â-NF). DY 64 is the most potent AhR ligand identified in the
dyeing wastewater and was capable of inducing AhR signaling
in the assay at the concentration of 0.5 nM (p < 0.01). The
effective concentration of DY 64 that induced lacZ units
equivalent to 50% of the maximal response (EC50) was
approximately 3 nM. 3′-HB[b]QP induced significant increase
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in AhR ligand activity at the concentration of 2.7 nM (p <
0.01) and was as potent as 3-MC when the concentration
was lower than 100 nM. However, it was incapable of inducing
maximal activity in the higher concentration range. DR 92
showed weak AhR binding affinity and elicited significant
increase in AhR ligand activity when the concentration was
higher than 971 nM (p<0.01). It also failed to induce maximal
activity.
The concentrations of DY 64, 3′-HB[b]QP, and DR 92 in
the Mar-04 sample were estimated to be 34.5, 8.7, and 639
nM by using HPLC analysis. The â-NF equivalent concentra-
tion was calculated to be 201, 2.6, and less than 1 nM,
respectively. DY 64 and 3′-HB[b]QP accounted for ap-
proximately 22% and 3% of the AhR ligand activity elicited
by the wastewater extract, suggesting that other unidentified
AhR ligands may contribute to 75% of the activity.
90% of DY 64 has been eliminated by a good settling activated
sludge from a wastewater treatment plant (33), the major
mechanism of removal was suggested to be the adsorption
of dyes to fungal cells or sludge instead of biodegradation
(32, 33). The presence of these potential AhR ligands in the
treated sewage effluent or in the activated sludge is presum-
able, and actually DR 92 has been detected in the treated
sewage effluent in our previous study (20), and DY 64 has
also been found in the drying sludge of a municipal waste
treatment plant receiving textile wastewater (34). Chemical
treatment such as chlorination has also been shown to be
incapable of treating certain disperse dyes appropriately (35).
In addition, 3′-HB[b]QP has been reported to be highly
resistant to photodegradation (21, 36), and no chemical
degradation was detected in the electrochemical treatment
of DY 64 using aluminum electrodes (37).
The quinoline disperse dyes are suggested to have
significant potential to adsorb to sediment rather than being
degraded in the environment due to their hydrophobic
property. The water/organic carbon partition coefficient (Koc)
of DY 64 has been reported to be logKoc )5.9, which suggested
that DY 64 and the more hydrophobic dye 3′-HB[b]QP are
very likely to partition into sediments (38). The hypothesis
is further supported by the detection of DY 64 in the sediment
of a creek below a municipal waste treatment plant receiving
textile effluent (34). However, the bioconcentration factor
(BCF) in fish for DY 64 has been reported to be low (log BCF
0.70) due to its low lipid solubility (39). It has also been
reported that disperse dyes did not bioaccumulate in fish
because of their low bioavailability in water and the ag-
gregation tendency that hampered the transport across
membranes (40).
Degradation of quinoline disperse dyes in anoxic sediment
has also been indicated to be very low (34, 41). The mean
half-lives of azo or anthraquinone disperse dyes to be
degraded in sediment soaked in lake water have been
reported to range from 2.6 h to 15 days. However, a much
longer half-life has been observed for the degradation of DY
64, which required at least 240 days (34). Another structurally
related quinoline dye, Solvent Yellow 33, has been shown to
be stable in the sediment as well, with a half-life of 82-200
days (41). Thus, DY 64 and 3′-HB[b]QP are suggested be
refractory and persistent in the environment. Their occur-
rence in the water environment, especially for the highly
potent AhR ligand DY 64, may pose a danger to the aquatic
biota and human health since their toxicological data are
still insufficient. Further studies concerning their environ-
mental behavior and toxicological data are necessary.
Discussion
Quinoline Disperse Dyes as Novel AhR Ligands. Quinoline
disperse dyes are suggested to be a new class of xenobiotic
AhR ligands because two quinoline disperse dyes, DY 64 and
3′-HB[b]QP, were identified as potent ligands capable of
activating the AhR at nanomolar concentrations in the yeast-
based reporter gene assay (Figure 6). 3′-HB[b]QP showed
AhR ligand activity comparable to that of 3-MC in the low
concentration range (from approximately 3-100 nM), and
DY 64 elicited higher AhR ligand activity than â-NF and 3-MC
at all concentrations. The EC50 of DY 64, â-NF, and 3-MC was
approximately 3, 10, and 100 nM, respectively, indicating
that DY 64 was 3 and 33 times more effective than â-NF and
3-MC, respectively, in the assay.
The yeast bioassay is suggested to be a useful screening
tool since the yeast HSP90 homolog and other cofactors in
the yeast have been reported to interact with the human
AhR properly to generate a receptor competent in signal
transduction (25). However, it is important to note that the
results of this bioassay are not always correspondent with
those of other experimental systems using mammalian cells
or in vivo assays. The discrepancy may be explained by the
species difference and the metabolic degradability of different
AhR ligands. For instance, indiruin, a potent AhR ligand
isolated from human urine, has been shown to be 50 times
more potent than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
in the yeast bioassay (23) and capable of inducing CYP1A1
and CYP1A2 mRNAs in human hepatoma cell line HepG2 at
concentrations 10 times lower than that of TCDD (26).
Nevertheless, it has also been reported to be less potent than
TCDD in other studies using experimental animals or
mammalian cell lines (27). In contrast to TCDD, indirubin
is easily metabolized by the induced enzymes such as CYP1A1
(26), which may be the main reason for the difference in
toxicity elicited by these two ligands.
The potent AhR ligand DY 64 isolated from the wastewater
has been reported to be genotoxic using a SOS chromotest
in the absence of S9 liver homogenate (28). In addition, it
has also been detected as a sensitizing agent causing allergic
contact dermatitis (ACD) in humans (29, 30). It is possible
that the activation of AhR is involved in the ACD responses
caused by DY 64, because AhR-mediated transcriptional
activation has been suggested to relate to inflammatory
disorders caused by PAH exposure using transgenic mice
expressing a constitutively active form of mouse AhR (31).
Possible Occurrence of Xenobiotic AhR Ligands in the
Environment. The dyeing wastewater investigated in this
study is treated by biological treatment and post-chlorination
in a sewage treatment plant, but the identified AhR ligands
are considered to be poorly biologically or chemically
degraded following conventional treatment. Although DR
92 have been reported to be efficiently removed by using the
fungus Cunninghamella polymorpha in 72 h (32) and over
Acknowledgments
This work was supported in part by the Grant-in-Aid for
Scientific research from the Ministry of Health, Labour and
Welfare, Japan, and the Grant-in-Aid for Scientific research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan, 16201012. We address special thanks to
Dr. Charles A. Miller III of the Department of Environmental
Health Sciences and Center for Bioenvironmental Research,
Tulane University, for kindly providing the YCM3 strain. We
would also like to thank Dr. Robert A. Kanaly for his help in
revising this manuscript.
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Received for review June 24, 2006. Revised manuscript re-
ceived October 23, 2006. Accepted October 30, 2006.
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