Identification of 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H- benzotriazole (PBTA-4) as a potent mutagen in river water in Kyoto and Aichi prefectures, Japan

Article abstract of DOI:10.1016/S1383-5718(01)00147-4

We have previously isolated five mutagens in blue rayon-adsorbed substances from water at a site below sewage plants in the Nishitakase River, in Kyoto, Japan, and identified two of them as 2-phenylbenzotriazole derivatives, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7- bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetylamino)-4-[(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7- bromo-4-chloro-2H-benzotriazole (PBTA-2). In the present study, we collected adsorbed materials on blue cotton (3kg×9 times) at the same location, and isolated a sufficient amount (97μg) of one of the remaining three mutagens other than PBTA-1 and PBTA-2, for structural analysis, by multiple column chromatography. The structure of mutagen, accounting for 12% of the total mutagenicity of the blue rayon-adsorbed substances, was determined to be a PBTA-1 analogue, 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H- benzotriazole (PBTA-4). PBTA-4 is a potent mutagen, inducing 190,000 and 7,800,000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. In addition to the water of the Nishitakase River, PBTA-4 was detected in water samples from two rivers that flow through other regions where textile-dyeing industries have been developed. Like other PBTA analogues, PBTA-4 might also be produced from azo dyes during industrial processes in dyeing factories and treatment at sewage plants.

Full text of DOI:10.1016/S1383-5718(01)00147-4

Mutation Research 492 (2001) 73–80
Identification of 2-[2-(acetylamino)-4-amino-
5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole
(PBTA-4) as a potent mutagen in river water in Kyoto and Aichi
prefectures, Japan
Haruo Nukayaa, Tatsushi Shiozawab, Atsuko Tadac, Yoshiyasu Teraob, Takeshi Ohed,
Tetsushi Watanabee, Masaharu Asanomaf, Hiroyuki Sawanishig, Takao Katsuharah,
Takashi Sugimurac, Keiji Wakabayashic,∗
a
School of Pharmaceutical Science, University of Shizuoka, 52-1, Yada, Shizuoka 422-8526, Japan
b
Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1, Yada, Shizuoka 422-8526, Japan
c
Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
d
Department of Food and Nutrition Science, Kyoto Women’s University,
Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan
Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
e
f
Nagoya City Public Health Research Institute, 1-11 Hagiyama-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8615, Japan
g
Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan
h
Central Research Laboratories, TSUMURA & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1155, Japan
Received 25 December 2000; received in revised form 7 February 2001; accepted 7 February 2001
Abstract
We have previously isolated five mutagens in blue rayon-adsorbed substances from water at a site below sewage plants in
the Nishitakase River, in Kyoto, Japan, and identified two of them as 2-phenylbenzotriazole derivatives, 2-[2-(acetylamino)-4-
[bis(2-methoxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-[2-(acetyl-
amino)-4-[(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2). In the
present study, we collected adsorbed materials on blue cotton (3 kg × 9 times) at the same location, and isolated a sufficient
amount (97 ␮g) of one of the remaining three mutagens other than PBTA-1 and PBTA-2, for structural analysis, by multiple
column chromatography. The structure of mutagen, accounting for 12% of the total mutagenicity of the blue rayon-adsorbed
substances, was determined to be a PBTA-1 analogue, 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-amino-7-bromo-4-
chloro-2H-benzotriazole (PBTA-4). PBTA-4 is a potent mutagen, inducing 190,000 and 7,800,000 revertants of Salmonella
typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix. In addition to the water of the
Nishitakase River, PBTA-4 was detected in water samples from two rivers that flow through other regions where textile-dyeing
∗Corresponding author. Tel.: +81-3-3542-2511/ext. 4500;
fax: +81-3-3543-9305.
E-mail address: kwakabay@gan2.ncc.go.jp (K. Wakabayashi).
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00147-4

74
H. Nukaya et al. / Mutation Research 492 (2001) 73–80
industries have been developed. Like other PBTA analogues, PBTA-4 might also be produced from azo dyes during industrial
processes in dyeing factories and treatment at sewage plants. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Mutagen; PBTA-4; Azo dye; Blue cotton; River water
1. Introduction
be suggested, as for the PBTA-1 and PBTA-2
cases.
Genotoxic compounds from industrial and do-
mestic effluents are often discharged into surface
waters such as rivers [1]. Water of the Yodo River,
which serves as the main supply of drinking water
for people living in the Osaka area, Japan, has been
shown to be markedly mutagenic to Salmonella ty-
phimurium TA98 and YG1024 with S9 mix [2–5].
We previously investigated blue rayon-adsorbed sub-
stances from the Nishitakase River, a tributary of
the Yodo River, to clarify their chemical structures,
and isolated five mutagens, compounds I–V. Among
them, compounds I and II have been identified as
2-[2- (acetylamino)-4-[bis(2-methoxyethyl)amino]-5-
methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-ben-
zotriazole (PBTA-1) [6] and 2-[2-(acetylamino)-4-
[(2-cyanoethyl)ethylamino]-5-methoxyphenyl]-5-am-
As described above, we previously determined
the structures of two out of five mutagens, com-
pounds I–V, which were isolated from the Nishitakase
River in Kyoto. In the present study, among the
three mutagens whose structures remained unclear,
compound IV, which accounted for 12% of the to-
tal mutagenicity of blue rayon-adsorbed substances
[6], could be collected in sufficient quantities for its
structural analysis. As a result, it was determined to
be 2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-
amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4),
by spectral analyses and chemical synthesis. The mu-
tagenicity of PBTA-4 was comparable with those of
the three previously identified PBTA-type mutagens.
The distribution of PBTA-4 in river waters other than
the Nishitakase River and the possible formation route
are also described.
ino-7-bromo-4-chloro-2H-benzotriazole
(PBTA-2)
[7], respectively. PBTA-1 and PBTA-2 accounted for
21 and 17% of the total mutagenicity of the blue
rayon-adsorbed substances, respectively. Based on
synthesis studies, both PBTA compounds would be
expected to have been formed from corresponding
dinitrophenylazo dyes, used as industrial materials
[7,8]. Dinitrophenylazo dyes might be converted to
the non-chlorinated derivatives of PBTA compounds
(non-ClPBTAs) in which a chlorine atom of PBTA
compound is replaced with a hydrogen atom, by
treatment with reducing reagents such as sodium
hydrosulfite, employed in textile-dyeing processes.
PBTA compounds could then be formed from the
non-ClPBTAs by chlorination reagents such as sodium
hypochlorite during treatment of industrial effluent at
sewage plants, and discharged into rivers. This hy-
pothesis is supported by results of quantification of
PBTA-1 and PBTA-2 in the Yodo River system [9]. In
addition, we have isolated another PBTA-type muta-
gen, 2-[2-(acetylamino)-4-[(2-hydroxyethyl)amino]-
5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-
benzotriazole (PBTA-3), from the river water sam-
ple of the Nikko River in Aichi prefecture, Japan
[10]. A similar route for PBTA-3 formation could
2. Materials and methods
2.1. Isolation of compound IV from river water
Compound IV was originally isolated from the
adsorbate on 24 g of blue rayon, which was hung in
the river for 24 h at a site downstream of two sewage
plants along the Nishitakase River, by HPLC as pre-
viously reported [6]. Using this original sample as a
standard marker, a large amount of compound IV suf-
ficient for structural determination was purified from
river water samples, collected with 27 kg (3 kg × 9
times) of blue cotton from March 1995–June 1996
[6]. The materials adsorbed to each 3 kg of blue
cotton, prepared as described previously [11], were
extracted by stirring in a methanol/ammonia water
solution, and the extract was then evaporated to dry-
ness. The residue (2.6 g) was dissolved in 20 ml of
methanol, filtered through a glass filter and applied to
a Sephadex LH-20 column (50 mm × 877 mm, Phar-
macia, Uppsala, Sweden). The materials were eluted

H. Nukaya et al. / Mutation Research 492 (2001) 73–80
75
with methanol and the fractions at elution volumes of
2.3. Synthesis of 5-amino-2-[(2-bromo-4,6-
2710–3220 ml were found to contain compound IV.
These fractions were combined and evaporated. The
residue (61 mg) was dissolved in 1.0 ml of methanol,
and then applied again to a Sephadex LH-20 column
(12 mm×1322 mm) with methanol as a mobile phase.
The fractions that contained compound IV were
eluted at elution volumes of 215–221 ml, and then
combined and evaporated. The residue was dissolved
in 250 ␮l of methanol and finally purified by HPLC
on a semi-preparative YMC-pack ODS-AM 324 col-
umn (5 ␮m particle size, 10 mm ×300 mm, YMC Co.
Ltd., Kyoto). The mobile phase of 75% methanol was
pumped in isocratically at a flow rate of 2 ml/min, and
compound IV was found in peak fractions with reten-
tion times of 16–18 min. The above processes were
repeated nine times to give 97 ␮g of compound IV.
The presence of a peak corresponding to authentic
compound IV was confirmed by HPLC on an analyt-
ical YMC-Pack ODS-A 303 column (5 ␮m particle
size, 4.6 mm×250 mm; YMC Co. Ltd.) with a mobile
phase of 35% acetonitrile in 25 mM phosphate buffer
(pH 2.0) at a flow rate of 1 ml/min.
dinitrophenyl)azo]-5-amino-4-methoxyacetanilide
The azo coupling reaction of 2-bromo-4,6-dinitro-
benzenediazonium sulfate, which was prepared from
2-bromo-4,6-dinitroaniline (2.6 g, 10 mmol), sodium
nitrite (800 mg, 11 mmol) and concentrated sulfu-
ric acid (10 ml), with 3-amino-4-methoxyacetanilide
(1.8 g, 10 mmol) was carried out in a manner similar
to that described previously [8] to yield a green pow-
der (4.08 g, 90%), mp: 240◦C (decomp.). FABMS
m/z: 453 (M++1, 79Br), 455 (M++1, 79Br). 1H-NMR
(DMSO-d6) δ: 2.17 (3H, s, NCOCH3), 3.40 (2H,
br, NH2), 3.84 (3H, s, OCH3), 7.12 (1H, s, ArH),
7.80 (1H, s, ArH), 7.90 (1H, br, CONH), 8.66 (1H,
d, J = 2.3 Hz, ArH), 8.68 (1H, d, J = 2.3 Hz,
ArH).
2.4. Synthesis of 2-(2-acetylamino-4-amino-5-
methoxyphenyl)-6-amino-4-bromo-2H-benzotriazole
(non-ClPBTA-4)
Iron powder (3 g, 53 mmol) and magnesium
chloride·6H2O (6.4 g, 31 mmol) were added to a solu-
tion of 5-amino-2-[(2-bromo-4,6-dinitrophenyl)azo]-
5-amino-4-methoxyacetanilide (2.3 g, 5 mmol) in
200 ml of a mixed solvent (tetrahydrofuran/methanol/
water = 2/2/1). The mixture was stirred at 80◦C
for 1 h. Insoluble materials were filtered off and
washed with tetrahydrofuran. The filtrate was con-
centrated to 1/3 of its original volume under reduced
pressure and extracted with dichloromethane. The or-
ganic solution was washed with brine and dried over
magnesium sulfate. Removal of the solvent under
reduced pressure gave a dark-colored oil, which was
subjected to column chromatography on silica gel
(eluent: 2% methanol in chloroform) and Sephadex
LH-20 (eluent: methanol) to yield a yellow pow-
der (150 mg, 7.7%), mp > 240◦C. FABMS m/z:
391 (M++1, 79Br), 393 (M++1, 81Br). 1H-NMR
(CDCl3) δ: 2.25 (3H, s, NCOCH3), 3.95 (3H, s,
OCH3), 3.96 (2H, br, NH2), 4.13 (2H, br, NH2), 6.88
(1H, d, J = 2.0 Hz, ArH), 7.14 (1H, d, J = 2.0 Hz,
ArH), 7.69 (1H, s, ArH), 8.04 (1H, s, ArH), 11.11
(1H, br, CONH). 13C-NMR (DMSO-d6) δ: 30.25,
55.65, 92.34, 105.85, 107.72, 109.10, 119.54, 123.23,
124.97, 137.61, 138.78, 142.47, 145.29, 148.56,
167.64.
All HPLC procedures were carried out at ambient
temperature, and the eluates were monitored for ab-
sorbance at 260 nm using a Shimazu SPD-10AV spec-
trometric detector (Kyoto).
2.2. Spectral measurement
UV absorption spectra were measured with a Tosoh
PD-8020 photodiode array detector and a Beckman
1
DU 640 spectrophotometer. H-NMR and 13C-NMR
spectra were taken of solutions in chloroform-d and
DMSO-d6 with a JEOL JNM-GSX270 (1H: 270 MHz,
13C: 67.5 MHz) or a JNM-GSX500 (1H: 500 MHz,
13C: 125 MHz) Fourier transform spectrometer. The
following abbreviations are used: s: singlet, d: dou-
blet, t: triplet, q: quartet, br: broad. Chemical shifts are
shown in ppm using tetramethylsilane as an internal
standard. High-resolution mass spectra were measured
using a JEOL JMS-DX300 or a JEOL JMS-AX505w
mass spectrometer, equipped with a direct inlet sys-
tem. Fast atom bombardment mass spectra (FAB-MS,
matrix: m-nitrobenzyl alcohol) were measured with
a JEOL JMS-SX103 mass spectrometer. All melting
points were obtained on a Yazawa micro melting point
apparatus (5Y-1) and are given as uncorrected values.

76
H. Nukaya et al. / Mutation Research 492 (2001) 73–80
2.5. Synthesis of PBTA-4
All HPLC procedures were carried out at ambient
temperature, and the eluates were monitored for ab-
sorbance at 260 nm.
An aqueous solution of sodium hypochloride (1 ml,
available chlorine minimum of 1%) was added drop-
wise to a stirred solution of non-ClPBTA-4 (40 mg,
0.1 mmol) in dichloromethane (100 ml) at room tem-
perature. After stirring for 1 h, the organic layer
was separated, washed with brine, and dried over
magnesium sulfate. The solvent was removed un-
der reduced pressure to yield a pale yellow solid,
which was subjected to column chromatography
on Sephadex LH-20 (eluent: methanol) to yield a
yellow powder (35 mg, 82%), mp > 230◦C. UV
max (MeOH): 219, 264, 389 nm. FABMS m/z: 425
(M++1, 79Br, 35Cl) 427 (M++1, 81Br, 35Cl & 79Br,
37Cl) 429 (M++1, 81Br, 37Cl). 1H-NMR (CDCl3)
δ: 2.26 (3H, s, COCH3), 3.97 (3H, s, COCH3), 4.16
(2H, brs, NH2), 4.34 (2H, brs, NH2), 7.21 (1H, s,
ArH), 7.74 (1H, s, ArH), 8.06 (1H, s, ArH), 11.13
(1H, brs, NHCO). 13C-NMR (DMSO-d6) δ: 30.26,
55.63, 96.15, 106.00, 107.92, 119.78, 122.59, 124.74,
127.78, 137.45, 139.14, 142.54, 144.02, 151.35,
167.66.
2.7. Mutagenesis assay
All test samples were dissolved in 100 ␮l of 50%
dimethyl sulfoxide and examined for mutagenicity by
the preincubation method [12] using S. typhimurium
TA98, TA100, YG1024 and YG1029 with or without
S9 mix. S. typhimurium YG1024 and YG1029, pro-
duced by introducing plasmids containing the acetyl-
transferase gene from TA1538 into TA98 and TA100,
respectively [13], were kindly provided by Dr. T.
Nohmi, National Institute of Health Sciences, Tokyo.
The S9 mix contained 5 ␮l of S9 (25 mg protein/ml)
(Kikkoman Co. Ltd., Noda, Japan), which was pre-
pared from the livers of male Sprague–Dawley rats
treated with phenobarbital and 5,6-benzoflavone, in a
total volume of 500 ␮l. Mutagenic activities of the test
samples were calculated from the linear portions of the
dose-response curves, obtained with four or five doses,
with duplicate plates, in two independent experiments.
2.6. Detection of the mutagen in water samples
from rivers other than the Nishitakase River
3. Results and discussion
3.1. Isolation and structural analysis of compound IV
Samples were collected from the Nikko River in
Aichi in September 1997 and from the Uji River in
Kyoto in February 1998. At each sampling site, 50 g of
blue cotton was hung for 24 h in the river and then col-
lected and treated by the method described above. Blue
cotton extracts were separated by Sephadex LH-20
column chromatography (12 mm × 1322 mm) with
methanol as a mobile phase. The fractions at elution
volumes of 215–221 ml were evaporated to dryness
and the residue was dissolved in methanol and finally
purified by HPLC on a semi-preparative YMC-pack
ODS-AM 324 column. The mobile phase of 75%
methanol was pumped in isocratically at a flow rate of
2 ml/min.
The presence of a peak corresponding to stan-
dard PBTA-4 was confirmed by HPLC on an an-
alytical YMC-Pack ODS-A 303 column (5 ␮m
particle size, 4.6 mm × 250 mm; YMC Co. Ltd.)
with a mobile phase of 35% acetonitrile in 25 mM
phosphate buffer (pH 2.0) at a flow rate of
1 ml/ min.
From material adsorbed to 27 kg of blue cotton,
mutagenic compounds were separated by Sephadex
LH-20 column chromatography twice and finally
compound IV (97 ␮g) was isolated as a single UV
absorption peak by HPLC on semi-preparative ODS
columns, as shown in Fig. 1. The UV absorption
spectrum of compound IV was found to be similar
to those of PBTA-1, PBTA-2 and PBTA-3 [6,7,10],
suggesting possession of a 2-phenylbenzotriazole
skeleton (Fig. 2). Analysis of compound IV, using
high-resolution mass spectrometry, indicated its
molecular formula to be C15H14BrClN6O2 (m/z,
424.0050, calcd. 424.0051). Compared with the struc-
ture of PBTA-1, compound IV has the same number
of nitrogen atoms, but six less carbon atoms, 12 less
protons and two less oxygen atoms.
The 1H-NMR spectrum of compound IV in
chloroform-d indicated the presence of 14 protons
in the molecule, and most of the chemical shifts

H. Nukaya et al. / Mutation Research 492 (2001) 73–80
77
Fig. 2. UV absorption spectrum of the mutagen on the first
YMC-Pack ODS-AM 324 column with a photodiode array detec-
tor. The material was eluted with 75% methanol.
Fig. 1. Purification of mutagenic compound IV by HPLC. Mu-
tagenic fractions from the second Sephadex LH-20 column with
elution volumes of 215–221 ml were purified on a YMC-Pack
ODS-AM 324 column. A single UV absorption peak showing mu-
tagenicity was detected at a retention time of 17 min. The UV ab-
sorbance and mutagenicity are shown by the upper line and lower
bar, respectively.
due to a primary amino and an amido groups were
observed as broad singlets at 4.33 (2H) and 11.12
(1H) ppm, respectively. Moreover, a broad singlet
of an exchangeable proton was observed at 4.15
(2H) ppm, indicating the presence of a primary amino
group instead of the –N(CH2CH2OCH3)2 in PBTA-1.
Based on the results of the spectral analyses described
above, the chemical structure of the mutagen was
deduced to be PBTA-4 (Fig. 3).
were similar to those of PBTA-1, as shown in Fig. 3.
Three aromatic protons were observed at 7.22, 7.74
and 8.06 ppm as singlets, and two singlets at 2.26
(3H) and 3.97 (3H) ppm were assigned to acetyl and
methoxy groups, respectively. Exchangeable protons
To confirm the structure of compound IV, PBTA-4
was synthesized from an azo dye, 5-amino-2-
Fig. 3. Chemical structures of PBTA-1 and PBTA-4. 1H-NMR chemical shifts in chloroform-d are presented as ppm; s: singlet, t: triplet,
q: quartet, br: broad.

78
H. Nukaya et al. / Mutation Research 492 (2001) 73–80
Scheme 1. Synthesis of PBTA-4.
[(2-bromo-4,6-dinitrophenyl)azo]-5-amino-4-metho-
xyacetanilide, according to the method used for
PBTA-1 [6], as shown in Scheme 1.
collected as blue cotton-adsorbed materials from the
Nikko River and the Uji River in Aichi and Kyoto
prefectures, respectively. These rivers flow through
regions where dyeing industries have been developed.
The blue cotton extracts were separated by Sephadex
LH-20 column chromatography, and fractions cor-
responding to authentic PBTA-4 were then further
purified by HPLC on a YMC-Pack ODS-AM 324 col-
umn. A single UV absorption peak was observed at a
retention time of 17 min. The retention time and the
UV spectrum of each peak fraction were identical to
those of PBTA-4. Moreover, the retention time of the
peak fraction from each river sample on a YMC-Pack
ODS-A 303 column was 26 min, which is the same
as that of authentic PBTA-4. Thus, PBTA-4 was de-
tected in the water samples from the Nikko and Uji
Rivers, at concentrations of 21 and 33 ng/g of blue
cotton, respectively. Under the same separation con-
ditions, the level of PBTA-4 in the Nishitakase River
was estimated to be 32 ng/g of blue cotton.
Iron powder reduction of 5-amino-2-[(2-bromo-
4,6-dinitrophenyl)azo]-5-amino-4-methoxyacetanilide
yielded a 2-phenylbenzotriazole skeleton product,
non-ClPBTA-4, at a yield of 7.7%. As reported previ-
ously [8], synthesis of non-ClPBTA-4 was also carried
out by the reduction of 5-amino-2-[(2-bromo-4,6-dini-
trophenyl)azo]-5-amino-4-methoxyacetanilide with
sodium hydrosulfite, with a yield down to 1.5%. The
monochlorinated product, PBTA-4, was produced
from the non-chlorinated derivative (non-ClPBTA-4)
by treatment with sodium hypochlorite, at a yield
of 82%. The UV, MS and 1H-NMR spectra of the
synthesized compound, PBTA-4, were the same as
those of compound IV, isolated from water samples
from the Nishitakase River. The 13C-NMR spectrum
was also consistent with the corresponding structure.
Thus, we concluded the new PBTA-type mutagen
isolated from the Nishitakase River to be PBTA-4.
3.3. Mutagenicities of PBTA-4, non-ClPBTA-4, and
the azo dye
3.2. Detection of PBTA-4 in water of rivers other
than the Nishitakase River
In the presence of S9 mix, synthesized PBTA-4,
non-ClPBTA-4, and the azo dye, 5-amino-2-[(2-
bromo-4,6-dinitrophenyl)azo]-5-amino-4-methoxyac-
To investigate the distribution of PBTA-4 in rivers
other than the Nishitakase River, water samples were

H. Nukaya et al. / Mutation Research 492 (2001) 73–80
79
Table 1
and the chlorinating reagent sodium hypochlo-
rite is used as a disinfecting agent in sewage
plants. The azo dye used for PBTA-4 synthe-
sis, 5-amino-2-[(2-bromo-4,6 -dinitrophenyl)azo]-5-
amino-4-methoxyacetanilide, is listed as an
industrial material [14], like other azo dyes, which
are possible precursors of PBTA-1 and PBTA-2 [7,8].
Therefore, PBTA-4 could be formed from the azo
dye 5-amino-2-[(2-bromo-4,6-dinitrophenyl)azo]-5-
amino-4-methoxyacetanilide during industrial activ-
ity and disinfecting processes at sewage plants, and
then discharged into rivers. In addition, the possibility
that 5-amino-2-[(2-bromo-4,6-dinitrophenyl)azo]-5-
amino-4-methoxyacetanilide is formed by the deh-
ydroxyethylation of azo dyes, such as 2-[(2-bromo-
4,6-dinitrophenyl)azo]-4-methoxy-5-[bis(2-acetoxye-
thyl)amino]acetanilide (Color Index Name, Disperse
Blue 79:1), cannot be ruled out, because similar de-
hydroxyethylation has been reported to occur for
ethanolamines on treatment with hypochlorous acid
or chlorine dioxide [15]. Since dinitrophenylazo dyes
such as Disperse Blue 79:1 are very popular in dyeing
factories throughout the world, it is plausible that the
formation of PBTA-4 from azo dyes results in pollu-
tion not only of river water in Japan, but also world-
wide, wherever there are textile-related industries.
In order to estimate the influence of PBTA com-
pounds on the aquatic ecosystem and human health,
quantification of the levels of these mutagens in the
water of various rivers and determination of their bio-
logical activities, including carcinogenicity in rodents,
are needed. Furthermore, the development of new
methods that prevent the formation and promote the
degradation of PBTA compounds is very important.
Mutagenicities of azo dye, non-ClPBTA-4 and PBTA-4 in four
Salmonella strains with S9 mix
Compound
Revertantsa/␮g
TA98 TA100
600
YG1024
YG1029
Azo dyeb
Non-ClPBTA-4
PBTA-4
19
35
2700
370000
7800000
750
1400
15000
950
190000
310
a Numbers of revertants are averages obtained from two inde-
pendent experiments.
b Azo dye: 5-amino-2-[(2-bromo-4,6-dinitrophenyl)azo]-5-amino-
4-methoxyacetanilide.
etanilide were all mutagenic in four strains (TA98,
TA100, YG1024 and YG1029) of S. typhimurium, as
shown in Table 1. PBTA-4 induced 190,000 revertants
for S. typhimurium TA98, 310 for TA100,7,800,000
for YG1024 and 15,000 for YG1029 per micro-
gram in the presence of S9 mix. Compared to TA98
and TA100, YG1024 and YG1029, which origi-
nate from TA98 and TA100, respectively, with a
high O-acetyltransferase activity, were more sensi-
tive to PBTA-4. Therefore, acetyltransferase seems
to be required for the mutagenicity of PBTA-4. In
addition, PBTA-4 showed a higher mutagenicity in
S. typhimurium TA98 and YG1024, detectors of
frame-shift mutations, than in S. typhimurium TA100
and YG1029, detectors of base-pair-change muta-
tions. The mutagenic potency was found to be about
twice as high as those of PBTA-1, PBTA-2 and
PBTA-3 [7,10]. Non-Cl PBTA-4 and the azo dye were
also mutagenic in the four strains in the presence of
S9 mix, again especially high in the strains detecting
frame-shift mutations. The mutagenic activities of
PBTA-4 in TA98 and YG1024 were 200 and 21 times
higher than those of non-ClPBTA-4, and 320 and 900
times higher than those of the azo dye, respectively. In
the absence of S9 mix, none of the three compounds
were mutagenic in any of the strains, at up to 10 ␮g.
We have successfully synthesized PBTA-4 from the
azo compound, 5-amino-2-[(2-bromo-4,6-dinitrophe-
nyl)azo]-5-amino-4-methoxyacetanilide, through non-
ClPBTA-4. In the present study, synthesis of
non-ClPBTA-4 was carried out with the reducing
reagent sodium hydrosulfite, as well as iron pow-
der, and sodium hypochlorite was used for the
chlorination of non-ClPBTA-4. Sodium hydrosulfite
is used for discharge printing in dyeing factories,
Acknowledgements
This study was supported by Grants-in-Aid for Can-
cer Research from the Ministry of Health and Welfare
of Japan and funds under a contract with the Environ-
ment Agency of Japan.
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