Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan

Article abstract of DOI:10.1016/S1383-5718(01)00273-X

We previously determined the chemical structures of four 2-phenylbenzotriazole mutagens (PBTA-1, -2, -3 and -4) in blue rayon-adsorbed material from the Nishitakase River in Kyoto prefecture and the Nikko River in Aichi prefecture in Japan. On the basis o

Full text of DOI:10.1016/S1383-5718(01)00273-X

Mutation Research 498 (2001) 107–115
Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5
and PBTA-6, and their detection in river water from Japan
Tetsushi Watanabea,∗, Haruo Nukayab, Yoshiyasu Teraoc, Yoshifumi Takahashia,
Atsuko Tadad, Takeji Takamurad, Hiroyuki Sawanishie, Takeshi Ohef,
Teruhisa Hirayamaa, Takashi Sugimurad, Keiji Wakabayashid
a
Department of Public Health, Kyoto Pharmaceutical University, 5 Nakauchicho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
b
School of Pharmaceutical Science, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
c
d
Cancer Prevention Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
e
Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan
f
Department of Food and Nutrition Science, Kyoto Women’s University, Kitahiyoshi-cho,
Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan
Received 18 May 2001; received in revised form 10 July 2001; accepted 10 July 2001
Abstract
We previously determined the chemical structures of four 2-phenylbenzotriazole mutagens (PBTA-1, -2, -3 and -4) in
blue rayon-adsorbed material from the Nishitakase River in Kyoto prefecture and the Nikko River in Aichi prefecture
in Japan. On the basis of a synthesis study, these four PBTA derivatives were deduced to have originated from corre-
sponding dinitrophenylazo dyes by reduction and chlorination. 2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-[bis(2-acetoxyethyl)
amino]-4-methoxyacetanilide (Color Index Name, Disperse Blue 79:1; CAS Registry Number, 75497-74-4) is a very common
dinitrophenylazo dye used in textile dyeing factories. In the present study, we synthesized 2-[4-[bis(2-acetoxyethyl)amino]-2-
(acetylamino)-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5) from Disperse Blue 79:1 by reduc-
tion with sodium hydrosulfite and subsequent chlorination with sodium hypochlorite. On hydrolysis of PBTA-5 with alkali,
2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-
6)wasobtained. BothPBTA-5and-6werepotentmutagens, inducing723,000revertantsand485,000revertantspermicrogram
of Salmonella typhimurium YG1024, respectively, in the presence of S9 mix. To clarify whether PBTA-5 and -6 exist in the
environment, water samples were collected from five rivers flowing through regions where textile dyeing industries are de-
veloped. PBTA-6 was detected at levels of 3–134 ng/g blue rayon in all water samples that were examined. On the other hand,
the amount of PBTA-5 in the samples was less than the detection limit. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: PBTA-5; PBTA-6; Disperse Blue 79:1; River water
1. Introduction
Numerous studies have revealed that genotoxic
substances can enter surface waters from a wide
range of industrial and municipal sources [1–7]. For
example, 33 effluents, which were collected from 27
∗ Corresponding author. Tel.: +81-75-595-4650;
fax: +81-75-595-4769.
E-mail address: watanabe@mb.kyoto-phu.ac.jp (T. Watanabe).
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00273-X

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T. Watanabe et al. / Mutation Research 498 (2001) 107–115
industries, were evaluated using the Salmonella muta-
tion assay, and it was clarified that several industries
such as dye manufacturers and organic manufacturers
emitted genotoxic effluents [1]. In the reviews of stud-
ies on the genotoxicity of industrial effluents, Houk
[3] documented that the genotoxic potency of indus-
trial wastes can vary over 10 orders of magnitude in
the Salmonella assay, from virtually non-detectable to
highly potent, and that the untreated effluents of sim-
ilar industrial processes produced effluents of similar
genotoxicity. However, it was reported that domestic
wastewaters also constitute a great genotoxic hazard
to aquatic systems and their associated biota [7]. To
clarify the sources of genotoxic substances and to
minimize the contaminants efficiently, the identifica-
tion of genotoxins in surface water is of importance.
Recently, we isolated five major mutagens from
water collected at a site below sewage plants of the
Nishitakase River in Kyoto, Japan [8]. Among these
five mutagens, three compounds were identified
as 2-phenylbenzotriazole derivatives, i.e. 2-[2-(acetyl-
amino)-4-[bis(2-methoxyethyl)amino]-5-methoxy-
phenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole
(PBTA-1) [8], 2-[2-(acetylamino)-4-[N-(2-cyano-
ethyl)ethylamino]-5-methoxyphenyl]-5-amino-7-bro-
mo-4-chloro-2H-benzotriazole (PBTA-2) [9] and
2-[2-(acetylamino)-4-amino-5-methoxyphenyl]-5-
amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4)
[10]. In addition, 2-[2-(acetylamino)-4-[(2-hydroxy-
ethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-
chloro-2H-benzotriazole (PBTA-3), was also isolated
from samples taken at a site below a sewage plant
of the Nikko River in Aichi prefecture, Japan [11].
The chemical structures of PBTA-1, -2, -3 and -4
are shown in Fig. 1. At the sewage plants of these
rivers, abundant effluents from textile dyeing facto-
ries are treated, as well as domestic wastewater. In
synthesis studies, these four PBTA derivatives were
suggested to be formed from corresponding dinitro-
phenylazo dyes via reduction by sodium hydrosulfite
and subsequent chlorination with sodium hypochlo-
rite [9–12]. 2-[(2-Bromo-4,6-dinitrophenyl)azo]-5-
Fig. 1. Chemical structures of PBTA-1, -2, -3, -4, -5 and -6. 1H NMR chemical shifts in CDCl3 are presented as ppm, s: singlet, t: triplet,
br: broad.

T. Watanabe et al. / Mutation Research 498 (2001) 107–115
109
[bis(2-methoxyethyl)amino]-4-methoxyacetoanilide
[12], 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-[N-(2-
Hitachi U-2001 spectrophotometer. 1H and 13C NMR
spectra were obtained with a JEOL JMN-GSX270
(1H, 270 MHz; 13C, 67.5 MHz) Fourier transform
spectrometer, using tetramethylsilane as an internal
standard. Electron impact mass spectrometry (EI-MS)
and high-resolution mass spectrometry (HRMS) were
conducted with a JEOL JMS-GC mate mass spec-
trometer. Fast atom bombardment mass spectrometry
(FAB-MS) (matrix: m-nitrobenzyl alcohol) was per-
formed using a JEOL JMS-SX102 mass spectrometer.
All melting points were obtained on a Yazawa micro-
melting point apparatus and are given as uncorrected
values.
cyanoethyl)ethylamino]-4-methoxyacetoanilide [9]
and 2-[(2-bromo-4,6-dinitrophenyl)azo]-5-amino-4-
methoxyacetanilide [10] were precursors of PBTA-1,
-2 and -4, respectively, and are listed as compounds
used for textile industrial purposes. 2-[(2-Bromo-
4,6-dinitrophenyl)azo]-5-[bis(2-acetoxyethyl)amino]-
4-methoxyacetanilide (Color Index Name, Disperse
Blue 79:1; CAS Registry Number, 75497-74-4)
is very commonly used in dyeing factories and
also has the 2-[(2-bromo-4,6-dinitrophenyl)azo]-4-
methoxyacetanilide moiety in the structure, similar to
azo compounds which are precursors of PBTA-1, -2
and -4. Although 2-[(2-bromo-4,6-dinitrophenyl)azo]-
4-methoxy-5-[(2-hydroxyethyl)amino]acetanilide,
which was a precursor of PBTA-3, is not listed as an
industrial material, it is possible that this azo com-
pound is formed by hydrolysis of ester bond and
subsequent dehydroxyethylation of Disperse Blue
79:1 [11]. Thus, it is postulated that these PBTA-type
mutagens occurred in dyeing factories and sewage
plants, and were discharged into the rivers [9–13].
We report here the synthesis of 2-[4-[bis(2-acetoxy-
ethyl)amino] -2 -(acetylamino) -5 -methoxyphenyl] -5-
amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5
) and 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)-
amino]-5-methoxy-phenyl]-5-amino-7-bromo-4-
chloro-2H-benzotriazole (PBTA-6) from Disperse
Blue 79:1 and the mutagenicity of these PBTA deriva-
tives. Moreover, the attempts to detect PBTA-5 and
-6 from several river waters are also described.
2.3. Synthesis of 2-[2-(acetylamino)-4-
[bis(2-acetoxyethyl)amino]-5-methoxyphenyl]-6-
amino-4-bromo-2H-benzotriazole (non-ClPBTA-5)
Sodium hydrosulfite (2 g) was added by portions
to a vigorously stirred mixture of Disperse Blue
79:1, which was synthesized by the method
reported previously [14] (620 mg, 1 mmol) in 100 ml
of methanol and 50 ml of water at room tempera-
ture. After the mixture had been stirred at 40–45◦C
for 2 h, insoluble materials were filtered off, and
washed with methanol. The filtrate was concentrated
to one-third of its original volume under reduced
pressure, and extracted with dichloromethane. The
organic solution was washed with brine and dried
over magnesium sulfate, concentrated under reduced
pressure, and subjected to column chromatography
on silica gel (eluent: 20% acetonitrile in chloro-
form) and Sephadex LH-20 (eluent: methanol) to
afford a yellow powder (25 mg, 4.5%); mp 85–86◦C;
FAB-MS m/z: 562, 564 (M+); UVmax (methanol)
nm: 266, 389; 1H NMR (CDCl3) δ: 2.03 (6H, s,
2 × –OCOCH3), 2.23 (3H, s, –COCH3), 3.58 (4H, t,
J = 5.4 Hz, –CH2–), 3.94 (3H, s, –OCH3), 4.03 (2H,
br, –NH2), 4.24 (4H, t, J = 5.4 Hz, –CH2–), 6.86
(1H, d, J = 1.9 Hz, Ar–H), 7.19 (1H, d, J = 1.9 Hz,
Ar–H), 7.77 (1H, s, Ar–H), 8.35 (1H, s, Ar–H), 11.10
(1H, s, –NHCO–); 13C NMR (CDCl3) δ: 170.95
(2C), 168.48, 148.24, 146.58, 144.79, 139.54, 138.95,
124.78, 123.74, 121.89, 113.34, 110.59, 106.41,
95.22, 62.39 (2C), 56.04, 51.29 (2C), 25.42, 20.89
(2C).
2. Materials and methods
2.1. Chemicals
Blue rayon was obtained from Funakoshi Co.
Ltd. (Tokyo, Japan). HPLC-grade acetonitrile and
methanol were purchased from Wako Pure Chemical
Industries Co. Ltd. (Osaka, Japan). All other chemi-
cals were of analytical grade.
2.2. Spectral measurement
UV absorption spectra were measured using a Shi-
madzu SPD-M10AV photodiode array detector and a

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T. Watanabe et al. / Mutation Research 498 (2001) 107–115
2.4. Synthesis of 2-[4-[bis(2-acetoxyethyl)-
amino]-2-(acetylamino)-5-methoxyphenyl]-5-amino-
7-bromo-4-chloro-2H-benzotriazole (PBTA-5)
(4H, t, J = 5.4 Hz, –CH2–), 3.93 (3H, s, –OCH3),
4.32 (2H, br, –NH2), 7.18 (1H, s, Ar–H), 7.79 (1H, s,
Ar–H), 8.43 (1H, s, Ar–H), 11.01 (1H, s, –NHCO–);
13C NMR (CDCl3) δ: 168.65, 148.87, 144.83, 143.77,
141.20, 138.46, 125.12, 123.59, 116.00, 108.85 (2C),
96.56, 95.95, 59.46 (2C), 56.56, 55.17 (2C), 24.06.
A diluted sodium hypochlorite solution (2.5 ml,
available chlorine, minimum 1%) was added dropwise
to an ice-cooled solution of non-ClPBTA-5 (274 mg,
0.48 mmol) in a mixture of dichloromethane (15 ml)
and acetic acid (300 ␮l, 0.50 mmol) with stirring. The
mixture was stirred for 1 h in an ice bath and for an
additional hour at room temperature. The reaction
mixture was diluted with water and extracted with
dichloromethane (20 ml) three times. The organic
layer was washed with brine (5 ml) three times, dried
over magnesium sulfate, concentrated under reduced
pressure, and subjected to column chromatography on
silica gel (eluent: 10% acetonitrile in chloroform) and
Sephadex LH-20 (eluent: methanol) to afford a yel-
low powder (140 mg, 43.7%); mp 142–143◦C; HRMS
m/z: calcd. for C23H26BrClN6O6, 596.0785; found,
596.0777 (M+); UVmax (methanol) nm: 265, 389; 1H
NMR (CDCl3) δ: 2.04 (6H, s, 2 × –OCOCH3), 2.27
(3H, s, –COCH3), 3.59 (4H, t, J = 5.4 Hz, –CH2–),
3.96 (3H, s, –OCH3), 4.25 (4H, t, J = 5.4 Hz,
–CH2–), 4.41 (2H, br, –NH2), 7.19 (1H, s, Ar–H),
7.81 (1H, s, Ar–H), 8.35 (1H, s, Ar–H), 11.12 (1H, br,
–NHCO–); 13C NMR (CDCl3) δ: 170.93 (2C), 168.51,
148.17, 142.59, 142.47, 139.91, 138.50, 124.99,
122.95, 121.50, 113.11, 109.04, 106.40, 99.57, 62.38
(2C), 56.14, 51.35 (2C), 25.48, 20.94 (2C).
2.6. Mutagenicity assay
All test samples were dissolved in 100 ␮l of 50%
dimethyl sulfoxide and mutagenicity was assayed by
the preincubation method [15] in the presence or ab-
sence of the S9 mix. Salmonella typhimurium strains
TA98 [16], TA100 [16], YG1024 [17], and YG1029
[17] were utilized. The S9 mix contained 10 ␮l of S9
in a total volume of 500 ␮l. S9 was prepared from
the livers of male Sprague–Dawley rats which were
treated with phenobarbital and ␤-naphthoflavone. Mu-
tagenic activities of samples were calculated from lin-
ear portions of the dose–response curves, which were
obtained with four or five doses and duplicate plates,
in two independent experiments.
2.7. Detection of PBTA-5 and PBTA-6
from river waters
Sample collection was carried out at a site down-
stream from the textile dyeing factories along the To-
bei River in Ishikawa prefecture in April 1998 and the
Asuwa River in Fukui prefecture in March 1998 and at
sites below municipal sewage plants treating effluents
from textile dyeing factories on the Nishitakase, Kat-
sura and Uji Rivers in Kyoto prefecture in February
1999. At each sampling site, 15 g of blue rayon (5 g
per bag) was hung in the river for 24 h. Then the blue
rayon was washed with distilled water several times.
The materials adsorbed to the blue rayon were ex-
tracted by the method described previously [18] with
minor modification. The adsorbed substances were ex-
tracted by shaking in 80 ml of methanol, instead of
methanol/ammonia water (50:1, v/v), per gram of blue
rayon for 30 min twice. The extracts were combined
and evaporated to dryness. The residue was dissolved
in 1 ml of 75% methanol. One-half of the sample so-
lution was applied to a semi-preparative YMC-Pack
ODS-AM 324 column (5 ␮m particle size, 10 mm ×
300 mm, YMC Co. Ltd., Kyoto, Japan) for HPLC,
and then eluted with the following gradient system:
2.5. Synthesis of 2-[2-(acetylamino)-4-[bis-
(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-
7-bromo-4-chloro-2H-benzotriazole (PBTA-6)
A sodium hydroxide solution (1 mol/l, 0.5 ml) was
added to a solution of PBTA-5 (140 mg, 0.27 mmol)
in methanol (37 ml) and stirred for 0.5 h. The mix-
ture was concentrated under reduced pressure, diluted
with water, and extracted with dichloromethane. The
organic layer was washed with brine and then dried
over magnesium sulfate. Removal of the solvent un-
der reduced pressure gave a yellow powder (100 mg,
71.3%); mp 200–203◦C (decomp.); HRMS m/z: calcd.
for C19H22BrClN6O4, 512.0574; found, 512.0576
(M+); UVmax (methanol) nm: 230, 266, 390; 1H
NMR (CDCl3) δ: 2.20 (3H, s, –COCH3), 2.88 (2H,
br, 2 × –OH), 3.31 (4H, t, J = 5.4 Hz, –CH2–), 3.61

T. Watanabe et al. / Mutation Research 498 (2001) 107–115
111
0–25 min, 75% methanol, 25–40 min, linear gradient
of 75–80% methanol, 40–50 min, linear gradient of
80–90% methanol, 50–90 min, 90% methanol at a flow
rate of 1.6 ml/min. This process was repeated twice.
Fractions corresponding to PBTA-5, with retention
times of 29–31 min on the YMC-Pack ODS-AM 324
column, were combined and evaporated to dryness.
The residue was dissolved in 0.5 ml of 45% acetoni-
trile, and were injected into a CAPCELL PACK C18
UG80 column (5 ␮m particle size, 4.6 mm × 250 mm,
Shiseido Co. Ltd., Tokyo, Japan) with a mobile
phase of 45% acetonitrile in 25 mM Tris–HCl (pH
6.5) at a flow rate of 1 ml/min. The fractions corre-
sponding to PBTA-5 were eluted at retention times
of 21–23 min. These fractions were evaporated to
dryness. The residue was dissolved in 0.5 ml of
45% acetonitrile, and separated by HPLC using a
YMC-Pack ODS-A303 column (5 ␮m particle size,
4.6 mm × 250 mm, YMC Co. Ltd., Kyoto, Japan).
The materials were eluted with 45% acetonitrile in
25 mM H3PO4/NaH2PO4 (pH 2.0) at a flow rate of
1.0 ml/min. The peak due to authentic PBTA-5 was
detected at 28 min.
eluted at retention times of 13–15 min. These fractions
were evaporated to dryness. The residue was dissolved
in 0.4 ml of 25% acetonitrile and applied to the YMC-
Pack ODS-A303 column with a mobile phase of 25%
acetonitrile in 25 mM H3PO4/NaH2PO4 (pH 2.0) at a
flow rate of 1.0 ml/min. The peak due to PBTA-6 was
detected at 18.5 min.
All HPLC procedures were carried out at ambient
temperature, and the UV absorption spectra of eluates
were monitored for structural confirmation with the
Shimadzu SPD-M10AV photodiode array detector.
3. Results and discussion
3.1. Synthesis of PBTA-5 and PBTA-6
As shown in Scheme 1, Disperse Blue 79:1 was
reduced and chlorinated by a method similar to
that for synthesizing PBTA-1 from 2-[(2-bromo-4,6-
dinitrophenyl)azo]-5-[bis(2-methoxyethyl)amino]-
4-methoxyacetanilide [12]. Reduction of Disperse
Blue 79:1 with sodium hydrosulfite gave a 2-
phenylbenzotriazole skeleton product, i.e. non-
ClPBTA-5. The UV absorption pattern of non-
ClPBTA-5 was similar to those of PBTA derivatives
[8–12]. PBTA-5 was subsequently produced from
non-ClPBTA-5 with sodium hypochlorite. The HRMS
spectrum of PBTA-5 indicated a molecular formula
of C23H26BrClN6O6. In the 1H NMR spectrum of
PBTA-5, aromatic protons and several other protons
Fractions corresponding to PBTA-6, with a reten-
tion time of 15–17 min on the YMC-Pack ODS-AM
324 column, were evaporated to dryness and then dis-
solved in 0.4 ml of 30% acetonitrile. The solution was
further applied to the CAPCELL PACK C18 UG80
column. A mobile phase of 30% acetonitrile in 25 mM
Tris–HCl buffer (pH 6.5) was pumped in at a flow rate
of 1 ml/min. Fractions corresponding to PBTA-6 were
Scheme 1. Synthesis of PBTA-5 and -6.

112
T. Watanabe et al. / Mutation Research 498 (2001) 107–115
were observed at chemical shifts similar to those of
PBTA-1 [12] and PBTA-2 [9], i.e. three aromatic
protons at 7.19, 7.81, 8.35 ppm, acetyl protons at
2.27 ppm, methoxy protons at 3.96 ppm, amino pro-
tons at 4.41 ppm, and an amido proton at 11.12 ppm
(Fig. 1). Two triplets due to ethylene protons were
observed at 3.59 and 4.25 ppm, and a singlet at
2.04 ppm was assigned to the methyl protons of the
bis(2-acetoxyethyl)amino group. The 13C NMR spec-
trum of PBTA-5 (data described in Section 2) also
support the structure shown in Fig. 1.
In the presence of the S9 mix, both PBTA-5 and
-6 exerted stronger mutagenicity towards TA98
(PBTA-5, 56,000 revertants/␮g; PBTA-6, 17,900
revertants/␮g) than TA100. Similar phenomena were
observed in YG1024 and YG1029 strains with the
S9 mix, which are O-acetyltransferase overproducing
derivatives of TA98 and TA100 [17], respectively.
These findings indicate that both PBTA-5 and -6
exhibit more frame-shifts than base-substitution ac-
tivity. The mutagenic activities of PBTA-5 and -6 in
YG1024 (PBTA-5, 723,000 revertants/␮g; PBTA-6,
485,000 revertants/␮g) was 13- and 27-fold higher
than those in TA98, respectively, suggesting that
O-acetyltransferase is required for mutagenicity of
these two PBTA derivatives. In the absence of S9
mix, neither PBTA-5 nor PBTA-6 showed distinct
mutagenicity towards these four strains.
The hydrolyzed product, PBTA-6, was produced
from PBTA-5 by treatment with sodium hydrox-
ide. In the MS spectrum, a molecular ion peak
was observed at m/z: 512, indicating that the bis(2-
acetoxyethyl)amino group was changed to the bis(2-
hydroxyethyl)amino group by hydrolysis. As shown
1
in Fig. 1, the H NMR spectrum of PBTA-6 showed
a similar signal pattern to that of PBTA-5 except for
signals due to the alkyl amino group. Two methy-
lene protons appeared at 3.31 and 3.61 ppm, and
hydroxy protons were observed at 2.88 ppm as a
broad signal (2H), which was exchangeable by D2O
treatment. These findings confirm that the chemical
structure of this hydrolyzed product, PBTA-6 has a
bis(2-hydroxyethyl)amino group instead of the bis(2-
acetoxyethyl)amino group for PBTA-5.
3.3. Detection of PBTA derivatives in river waters
To clarify whether river waters are contaminated
with PBTA-5 and -6, the concentrates of waters were
collected using blue rayon from five rivers in Japan,
i.e. the Asuwa, Tobei, Nishitakase, Katsura, and Uji
Rivers, which flow through the areas of textile relating
industries. All the concentrates were collected at sites
downstream of the textile dyeing factories or munic-
ipal sewage plants treating effluents from the textile
dyeing plants. The blue rayon adsorbates were sepa-
rated by HPLC on a YMC-Pack ODS-A324 column.
The fractions corresponding to authentic PBTA-5 and
-6 were further purified using a CAPCELL PAK C18
column and finally analyzed by HPLC on a YMC-
Pack A-303 column with a photodiode array detector.
No peak, which corresponded to PBTA-5, was de-
tected in the concentrates from the five river waters.
However, single peaks corresponding to PBTA-6 were
detected at retention times of 18 min in all of the con-
centrates from five river waters. The UV absorption
spectrum of each peak fraction was identical to that
of authentic PBTA-6. Fig. 2 shows the HPLC chro-
matogram and UV absorption spectrum of PBTA-6 in
the water concentrates from the Asuwa River. Table 2
shows the amount of PBTA-6 in the concentrates of
river waters and ratios of contribution of PBTA-6
to the mutagenicity of the concentrates towards S.
typhimurium YG1024 with S9 mix. The amounts
of PBTA-6 were 3–134 ng/g of blue rayon-adsorbed
3.2. Mutagenicity of PBTA-5 and PBTA-6
The mutagenic potencies of PBTA-5 and -6 in S.
typhimurium tester strains are summarized in Table 1.
Table 1
Mutagenicities of PBTA-5 and -6 in four S. typhimurium strains
with S9 mixa
Compound
Revertants/␮g
TA98
TA100
YG1024
YG1029
PBTA-5
PBTA-6
56000
17900
387
146
723000
485000
1580
953
a The mutagenic activities of samples were calculated from
linear portions of the dose–response curves in two independent
experiments. The numbers of spontaneous revertants were as fol-
lows: TA98, 29 revertants (rev.) per plate; TA100, 139 rev. per
plate; YG1024, 118 rev. per plate; YG1029, 207 rev. per plate. The
numbers of revertant colonies of positive controls were as follows:
TA98, 302 rev./2.5 ␮g of benzo[a]pyrene (B[a]P); TA100, 633
rev./1.25 ␮g of B[a]P; YG1024, 985 rev./15 ng of acetylaminoflu-
orene; YG1029, 716 rev./15 ng of 2-aminoanthracene.

T. Watanabe et al. / Mutation Research 498 (2001) 107–115
113
Fig. 2. HPLC chromatogram (A) and UV absorption spectrum (B) of PBTA-6 in water from the Asuwa River on a YMC-Pack A-303
column. The fraction corresponding to authentic PBTA-6 was injected into the YMC-Pack A-303 column after purification with a YMC-Pack
ODS-AM 324 and a CAPCELL PACK C18 UG80 columns. The material was eluted with 45% acetonitrile in 25 mM H3PO4/NaH2PO4
(pH 2.0) at a flow rate of 1.0 ml/min. A chromatogram is presented as the UV absorption of eluate at 260 nm. The peak corresponding to
the retention time of authentic PBTA-6 is indicated by an arrow.
materials, and 0.6–13% of the mutagenicity of river
water samples were attributable to PBTA-6.
with flocculants under alkaline conditions to remove
dissolved and suspended substances prior to being
discharged into surface water. In the laboratory ex-
periment, PBTA-5 and -6 were extracted from water
to a similar extent, i.e. 41% for PBTA-5 and 54%
for PBTA-6, using blue rayon, and the recoveries of
PBTA-5 and -6 during the purification processes by
HPLC were 74 and 80%, respectively. The minimum
detection limits for PBTA-5 and -6 were 0.4 ng/g of
blue rayon with the photodiode array detector. The
coefficients of variation of HPLC analysis for PBTA-
5 and -6 were 5%. High levels of PBTA-6 were
detected in all river water samples examined, how-
ever, the amount of PBTA-5 in the samples was less
than the detection limit. These findings suggest that
the bis(2-acetoxyethyl)amino group of PBTA-5 was
hydrolyzed to the bis(2-hydroxyethyl)amino group in
In this study, PBTA-5 was successfully synthe-
sized from azo dye Disperse Blue 79:1, which is
prevailingly utilized in the textile dyeing industries
[19], by reduction, chlorination, and PBTA-6 was
obtained by hydrolysis of PBTA-5. The reducing
agents such as sodium hydrosulfite are generally
employed for discharge printing and bleaching in
textile dyeing factories. Sodium hypochlorite is com-
monly used for bleaching in textile production and
decolorization of wastewaters and is also mainly
utilized in sewage plants for disinfection purposes.
The bis(2-acetoxyethyl)amino group of PBTA-5 was
easily hydrolyzed in alkaline solution, and PBTA-6
was formed as a result with a high yield. In most
textile dyeing factories, the wastewaters are treated
Table 2
Amounts of PBTA-6 and the ratio of contribution to mutagenicity of river water samples in Salmonella typhimurium YG1024 with S9 mix
River
Sampling date
Amounta
(ng/g of blue rayon)
Mutagenicity
(revertants/g of blue rayon)
Contribution
ratio (%)
Tobei
22 April 1998
80
112
21
3
134
2390000
413000
407000
238000
515000
2
13
3
0.6
13
Asuwa
Nishitakase
Katsura
Uji
31 March 1998
15 February 1999
15 February 1999
15 February 1999
a Values were corrected for recoveries during the purification process.

114
T. Watanabe et al. / Mutation Research 498 (2001) 107–115
the textile dyeing and/or wastewater treatment pro-
cesses in the factories, therefore, the levels of PBTA-5
were relatively low in the river waters.
site in the Aberjona watershed, Environ. Health Perspect. 106
(1998) 1069–1074.
[7] P.A. White, J.B. Rasmussen, The genotoxic hazards of
domestic wastes in surface waters, Mutat. Res. 410 (1998)
223–236.
[8] H. Nukaya, J. Yamashita, K. Tsuji, Y. Terao, T. Ohe,
H. Sawanishi, T. Katsuhara, K. Kiyokawa, M. Tezuka, A.
Oguri, T. Sugimura, K. Wakabayashi, Isolation and chemical-
structural determination of a novel aromatic amine mutagen
in water from the Nishitakase River in Kyoto, Chem. Res.
Toxico1. 10 (1997) 1061–1066.
Recently, Ohe et al. [20] reported that the muta-
tion spectra produced by PBTA-1 and -2 in TA98
and TA100 strains were similar to those produced
by the heterocyclic amine food mutagen Glu-P-1 in
both strains. Furthermore, PBTA-2 was positive in
the in vitro micronucleus test using Chinese ham-
ster cell lines, CHL and V79-MZ, and polyploidy
was induced in both cells [21]. To estimate the risk
of PBTA-type mutagens including PBTA-6 to human
health and aquatic ecosystems, studies on other bio-
logical activities of the PBTA derivatives in vivo such
as carcinogenicity and the more detailed quantification
in environmental waters and drinking water will be
necessary.
[9] A. Oguri, T. Shiozawa, Y. Terao, H. Nukaya, J. Yamashita,
T. Ohe, H. Sawanishi, T. Katsuhara, T. Sugimura, K.
Wakabayashi, Identification of
a 2-phenylbenzotriazole
(PBTA)-type mutagen, PBTA-2, in water from the Nishitakase
River in Kyoto, Chem. Res. Toxicol. 11 (1998) 1195–
1200.
[10] H. Nukaya, T. Siozawa, A. Tada, Y. Terao, T. Ohe,
T. Watanabe, M. Asanoma, H. Sawanishi, T. Katsuhara,
T. Sugimura, K. Wakabayashi, 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, Mutat.
Res. 492 (2001) 73–80.
Acknowledgements
[11] T. Shiozawa, A. Tada, H. Nukaya, T. Watanabe, Y.
Takahashi, M. Asanoma, T. Ohe, H. Sawanishi, T. Katsuhara,
T. Sugimura, K. Wakabayashi, Y. Terao, Isolation and
identification of a new 2-phenylbenzotriazole-type mutagen
(PBTA-3) in the Nikko river in Aichi, Japan, Chem. Res.
Toxicol. 13 (2000) 535–540.
[12] T. Shiozawa, K. Muraoka, H. Nukaya, T. Ohe, H. Sawanishi,
A. Oguri, K. Wakabayashi, T. Sugimura, Y. Terao, Chemical
synthesis of a novel aromatic amine mutagen isolated from
water of the Nishitakase River in Kyoto and a possible
route of its formation, Chem. Res. Toxicol. 11 (1998) 375–
380.
This study was supported by Grants-in-Aids for
Cancer Research from the Ministry of Health and Wel-
fare of Japan, the Promotion and Mutual Aid Corpo-
ration for Private Schools of Japan, and funds under a
contract with the Environment Agency of Japan.
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