Bioscience, Biotechnology, and Biochemistry

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Identification of methylglyoxal as a major

mutagen in wood and bamboo pyroligneous acids

Aya Onoda, Masaharu Asanoma & Haruo Nukaya

To cite this article: Aya Onoda, Masaharu Asanoma & Haruo Nukaya (2016) Identification
of methylglyoxal as a major mutagen in wood and bamboo pyroligneous acids, Bioscience,
Biotechnology, and Biochemistry, 80:5, 833-839, DOI: 10.1080/09168451.2015.1136880

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Bioscience, Biotechnology, and Biochemistry, 2016
Vol. 80, No. 5, 833–839
Identification of methylglyoxal as a major mutagen in wood and bamboo
pyroligneous acids
Aya Onoda1,*, Masaharu Asanoma1 and Haruo Nukaya2
1
Food Department, Nagoya City Public Health Research Institute, Nagoya, Japan; 2Graduate Division of Nutritional
and Environmental Sciences, University of Shizuoka, Shizuoka, Japan
Received October 28, 2015; accepted December 14, 2015
http://dx.doi.org/10.1080/09168451.2015.1136880
To identify the major mutagen in pyroligneous
acid (PA), 10 wood and 10 bamboo pyroligneous
acids were examined using the Ames test in
Salmonella typhimurium strains TA100 and TA98.
Subsequently, the mutagenic dicarbonyl compounds
(DCs), glyoxal, methylglyoxal (MG), and diacetyl in
PA were quantified using high-performance liquid
chromatography, and the mutagenic contribution
ratios for each DC were calculated relative to the
mutagenicity of PA. Eighteen samples were positive
for mutagens and showed the strongest mutagenicity
in TA100 in the absence of S9 mix. MG had the
highest mutagenic contribution ratio, and its pres-
ence was strongly correlated with the specific muta-
genicity of PA. These data indicate that MG is the
major mutagen in PA.
Key words: pyroligneous acid; mutagen; Ames test;
dicarbonyl compound; methylglyoxal
Pyroligneous acid (PA) is obtained by cooling the
smoke that is generated during the carbonization of
wood and is a predominant by-product of charcoal pro-
duction in Japan. It is known that PA has bactericidal,1)
antiviral,2) antifungal,3) and odor masking4) activities
because it contains multiple compounds, such as
organic acids, phenols, and carbonyl compounds.5,6)
Accordingly, PA is sold at gardening stores as an agri-
cultural antiseptic and soil improvement agent and is
sold at drugstores as a bath agent. In addition, PA is
used as a food additive to give a smoked flavor to
foodstuffs.7,8) However, several studies show that PA
was mutagenic in the Ames tests using Salmonella
typhimurium strains TA100 and TA98.9) Furthermore,
these studies have produced diverse results, and the pri-
mary mutagen in PA has not been sufficiently identi-
fied.10–13) Nonetheless, because wood smoke has been
shown to be mutagenic,14) it can be assumed that the
major mutagen in PA is formed during carbonization.
Similar to PA, roasted coffee beans are produced by
a high-temperature heating process and contain mainly
*Corresponding author. Email: onoda05-1@yahoo.co.jp
© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry
carbohydrate as raw materials. It had been reported that
the mutagens of coffee beans were produced during the
roasting process.15) Roasted coffee beans showed muta-
genicity in the TA100 in the absence of S9 mix,16) and
methylglyoxal (MG) was shown to be the primary
mutagen.17) MG is one of the several dicarbonyl com-
pounds (DCs) that are formed during decomposition of
carbohydrates.18,19) DCs have demonstrated mutagenic-
ity in TA10020) and are likely mutagenic components
of PA because the D-glucose polymer cellulose is a
major constituent of wood.
Most studies of PA mutagenicity in S. typhimurium
have been conducted on smoke condensates from
wood. In Japan, both wood pyroligneous acid (WPA)
and bamboo pyroligneous acid (BPA) are commercially
available and have been sold in equal proportions. In
this study, the mutagenicity of 10 WPAs and 10 BPAs
were examined using the Ames test in TA100 or TA98,
and furthermore, MG, glyoxal (G), and diacetyl (DA)
in WPAs and BPAs were quantified using high-perfor-
mance liquid chromatography (HPLC). The involve-
ment of each DC in the mutagenicity of PA was then
investigated, and the major PA mutagen was
determined.
Materials and methods
PA samples. WPAs and BPAs were purchased as
liquids from gardening or drug stores, and WPA and
BPA samples were denoted W-1–W-10 and B-1–B-10,
respectively.
Reagents. G and MG were purchased from Sigma-
Aldrich Corp. (St. Louis, MO, USA). DA, quinoxaline,
2-methyl-quinoxaline, 2,3-dimethyl-quinoxaline, and
1,2-phenylenediamine were purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan). HPLC
grade methanol and formic acid were purchased from
Kanto Chemical Co., Inc. (Tokyo, Japan). S9 was pur-
chased from Kikkoman Biochemifa Company (Tokyo,
Japan).
834 A. Onoda et al.
Instrument. The C18 cartridge Sep-Pak Plus was
purchased from Waters Corporation (Milford, MA,
USA), and the automatic colony counter MCL-2000
was purchased from SK-Electronics CO. Ltd. (Kyoto,
Japan). The HPLC system (Shimadzu Corporation,
Kyoto, Japan) was equipped with a system controller
(CBM-20A), a pump (LC-20AD), a diode array detec-
tor (SPD-M20A), an auto sampler (SIL-20AC), a col-
umn oven (CTO-20AC), and an Inertsil ODS-4 column
(5 μm particle size, 4.6 mm i.d. × 250 mm, GL
Sciences Inc., Tokyo, Japan).
Mutagenicity test. The Ames test was performed
according to a previously described preincubation
method21) using S. typhimurium strains TA100 and
TA98 in the absence and presence of S9 mix. Both
strains were kindly provided by Dr Bruce N. Ames,
University of California, Berkeley, California. The S9
mix contained 50 μL of S9, which was prepared from
the livers of male Sprague Dawley rats by treatment
with phenobarbital and 5,6-benzoflavone in a total vol-
ume of 500 μL. Revertants were counted using a col-
ony counter after incubation for 48 h at 37 °C, and
mutagenicity tests were performed in more than 2
plates. Distilled water was used as a negative control,
and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide or 2-
aminoanthracene was used as positive controls. PA
mutagenicity was recorded as positive when PA sam-
ples induced more than twice the number of negative
control revertants. Specific mutagenicity values of PA
represented net revertants per 1 μL of sample and were
calculated using dose–revertant response curves.
Determination of DCs in PA. G, MG, and DA in
PA were derivatized to quinoxaline, 2-methylquinoxa-
line, and 2.3-dimethylquinoxaline, respectively, using
1,2-phenylenediamine in a modified quinoxaline deriva-
tization method.22) Briefly, PA was diluted 10-fold with
0.2 M phosphate buffer solution (pH 8.0) and 2 mL of
diluted PA was added to 2 mL of 0.3 M 1,2-phenylene-
diamine solution containing 0.2 M phosphate buffer
(pH 8.0) and was allowed to react for 20 min at 37 °C.
After cooling to room temperature, distilled water was
added to a final volume of 10 mL. Subsequently, 5 mL
aliquots were loaded onto a Sep-Pak Plus C18 cartridge
that had been conditioned with 5 mL of methanol and
5 mL of distilled water and then washed with 5 ml of
distilled water. Subsequently, the cartridge was eluted
with 10 mL of 50% (v/v) methanol, and eluates were
analyzed using HPLC with a mobile phase of 50%(v/v)
methanol containing 0.1% (v/v) formic acid, a flow rate
of 1.0 mL/min, injection volume of 10 μL, column
temperature of 40 °C, and detection wavelength of
315 nm. A recovery test was performed 3 times using
B-1, and G, MG, and DA were added at 4.0, 2.4, and
0.4 μmol/mL, respectively.
Calculation of mutation incidence. The effect on
the mutation expression by the cytotoxicity of PA was
examined using the Ames tests. Tests of mutagenicity
were simultaneously performed in PA with (A) and
without (B) added 0.3 μmol MG for each dose using
TA100 in the absence of S9 mix. PA was prepared at
doses of 20, 40, 60, and 80 μL, and 0.3 μmol MG was
added to the same doses in parallel. As a control,
0.3 μmol MG (C) was used, and all sample solutions
were adjusted to 100 μL. The recovery rate of rever-
tants by added MG at each dose was calculated using
the following equation and was defined as the mutation
incidence of PA:
Mutation incidence ð%Þ ¼
f½the number of the net revertants of (A)
 the number of the net revertants of (B)
at the same dose of (A)]/the number of the net
revertants of (C)g 100
A regression line of mutation incidence of PAs was
obtained using the values calculated in each dose from
the above equation.
Calculation of mutagenic contribution ratios of DCs
in PA. Mutagenic contribution ratios were expressed
as percentages of mutagenicity from individual DCs
relative to the total mutagenicity of PA and were calcu-
lated for the PA dose that showed twice the number of
negative control revertants. The number of the net
revertants induced by DC contents of PA (D) and the
number of the net revertants of PA (Ec) were calculated
using dose-response curves from Ames tests. Ec values
were calculated by correcting the number of the net
revertants of PA (Em) with mutation incidence rate (F),
and Em values were the actual measured number of the
net revertants of PA. Mutagenic contribution ratios
were calculated under TA100 in the absence of S9 mix
as follows:
Mutagenic contribution ratio of DCð%Þ
¼ ðD=Ec Þ  100
Ec ¼ Em  ð100=F Þ
Statistical analysis. Differences were identified
using Student’s t-test and were considered significant
when p < 0.05.
Results and discussion
Mutagenicity of PA
In the Ames tests of PA in TA100 and TA98 (Tables
1 and 2), 18 (9 WPA and 9 BPA) of 20 samples had
positive mutagenicity. In TA100, 17 samples (8 WPA
and 9 BPA) were positive in the absence of S9 mix,
and 6 samples (2 WPA and 4 BPA) were positive in
the presence of S9 mix. In TA98, 10 samples (4 WPA
and 6 BPA) were positive in the absence of S9 mix,
and only B-8 was positive in the presence of S9 mix. It
was found that PA samples showed the strongest muta-
genicity in TA100 in the absence of S9 mix because
the values of specific mutagenicity in TA100 in the
absence of S9 mix were the highest in the Ames test
 Table 1. Mutagenicity tests of pyroligneous acids in Salmonella typhimurium TA100.

–S9 mix +S9 mix

Revertants Revertants

Dose (μL) Dose (μL)

Negative AF-2b Specific mutagenicity (revertants/ Negative 2AAc Specific mutagenicity (revertants/
Sample controla (0.01 μg) 5 10 25 50 100 μL) control (1.0 μg) 5 10 25 50 100 μL)
W-1 111 411 154 148 275 410 94* 6.0 126 805 152 150 191 281 365* 3.0
W-2 111 411 169 207 597 1522 3426 31.4 126 805 141 149 149 150 209 －
W-3 111 411 126 117 151 222 378 2.5 126 805 129 126 144 146 228 －
W-4 124 605 121 131 175 215 88* (1.8)d 123 1196 126 147 156 182 266 1.4
W-5 124 605 132 143 157 207 292 1.7 123 1196 118 131 170 168 166 －
W-6 124 605 122 122 118 131 144 －e 123 1196 125 125 130 148 150 －
W-7 124 605 128 139 167 218 399 2.5 123 1196 120 124 154 169 175 －
W-8 124 605 120 135 162 169 250 1.2 123 1196 121 130 132 157 163 －
W-9 124 605 136 145 170 287 100* 2.9 123 1196 133 168 166 170 201* －
W-10 128 734 123 142 219 374 956 7.4 124 984 104 106 125 121 131 －
B-1 111 411 182 227 410 75* 0* 12.0 126 805 142 161 242 260* 90* 3.1
B-2 124 605 121 131 138 143 150 － 123 1196 117 111 108 121 112 －
B-3 111 605 107 106 107 152 325 1.8 126 805 131 133 142 154 200 －
B-4 124 605 126 143 157 178 254 1.3 123 1196 119 120 164 150 201 －
B-5 124 605 137 164 211 269 410 2.9 123 1196 135 160 158 191 251 1.3
B-6 124 605 141 176 221 286 100* 3.4 123 1196 129 140 151 210 170* －
B-7 124 605 129 153 171 219 409 2.6 123 1196 123 153 161 188 281 1.5
Methylglyoxal as mutagen in pyroligneous acids

B-8 111 411 109 141 195 466 724 6.1 126 805 140 149 171 216 391 2.4
B-9 124 605 127 147 168 305 84* 3.2 123 1196 119 140 180 198 196* －
B-10 111 411 132 140 180 375 1031 8.1 126 805 124 121 113 140 169 －
Notes: Numbers of revertants for samples, AF-2 and 2AA are presented as the average of 2 plates, and the number of the negative control revertants is presented as the average of 5 plates. Specific mutagenicity values represent the number of the net
revertants per 1 μL of sample, and were calculated from dose–revertant response curves.
a
Distilled water;
b
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide;
c
2-Aminoanthracene;
d
Mutagenicity was pseudo-positive;
e
Mutagenicity was negative;
*Growth inhibition was observed.
835
836 A. Onoda et al.
Table 2. Mutagenicity tests of pyroligneous acids in Salmonella typhimurium TA98.

–S9 mix +S9 mix

Revertants Revertants

Dose (μL) Specific Dose (μL) Specific

Negative AF-2b mutagenicity Negative 2AAc mutagenicity
Sample controla (0.1 μg) 5 10 25 50 100 (revertants/μL) control (0.5 μg) 5 10 25 50 100 (revertants/μL)
W-1 19 520 21 29 34 47 16* 0.6 26 227 31 31 26 35 35* −
W-2 19 520 19 24 39 82 37 1.1 26 227 27 30 31 38 31 −
W-3 19 520 24 19 27 44 48 0.3 26 227 29 32 32 40 51 −
W-4 25 501 27 32 29 42 25* −d 30 327 32 31 42 46 43* −
W-5 25 501 26 29 33 35 46 − 30 327 26 33 37 33 36 −
W-6 25 501 26 29 26 33 33 − 30 327 29 31 32 35 37 −
W-7 25 501 30 37 45 53 51 0.6 30 327 32 41 41 40 51 −
W-8 25 501 26 37 38 40 45 − 30 327 31 35 36 38 35 −
W-9 25 501 26 37 36 32 21* − 30 327 29 49 34 35 31* −
W-10 25 453 29 22 22 31 45 − 23 245 25 28 26 25 29 −
B-1 19 520 23 33 30 7* 0* − 26 227 35 32 34 24* 8* −
B-2 25 501 24 22 22 27 25 − 30 327 26 39 32 34 32 −
B-3 19 520 21 21 28 36 61 0.4 26 227 32 27 33 33 41 −
B-4 25 501 25 32 37 37 47 − 30 327 27 40 33 49 42 −
B-5 25 501 25 38 41 44 53 0.3 30 327 34 38 37 37 48 −
B-6 25 501 29 37 38 53 26* 0.6 30 327 33 42 45 41 28* −
B-7 25 501 24 29 34 41 54 0.3 30 327 31 34 38 41 40 −
B-8 19 520 24 38 43 102 89* 1.5 26 227 28 30 40 52 60 0.4
B-9 25 501 27 36 33 47 25* − 30 327 33 39 36 39 20* −
B-10 19 520 24 22 24 37 58 0.4 26 227 25 30 23 25 37 −
Notes: Numbers of revertants for samples, AF-2 and 2AA are presented as the average of 2 plates, and the number of the negative control revertants is presented as
the average of 5 plates. Specific mutagenicity values represent the number of the net revertants per 1 μL of sample, and were calculated from dose–revertant response
curves.
a
Distilled water;
b
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide;
c
2-Aminoanthracene;
d
Mutagenicity was negative;
*Growth inhibition was observed.

under various conditions. In particular, W-2 and B-10

were highly mutagenic in TA100 in the absence of S9
mix and led to the generations of more than 1000 Table 3. Contents of dicarbonyl compounds in pyroligneous
revertants. In agreement with these observations, previ- acids.
ous studies showed stronger mutagenicity of PA in
Content (μmol/mL)
TA100 than in TA98 but showed differing effects of
metabolic activation.10–12) In this study, the mutagenic- Sample Ga MGb DAc Total (mg/mL)
ity of PA was strongest in TA100 in the absence of S9 W-1 5.2 3.7 0.4 0.60
mix, although mutagenicity was reduced by metabolic W-2 0.2 0.5 trd 0.05
activation in both TA100 and TA98. Similarly, Nakama W-3 0.9 1.0 0.1 0.13
et al. and Kim Oanh et al. reported that the mutagenic- W-4 0.1 1.3 0.3 0.13
ity of smoke generated by burning wood was strongest W-5 0.6 0.6 tr 0.07
W-6 NDe ND ND −f
in TA100 in the absence of S9 mix.11,14)
W-7 tr 0.4 ND 0.03
W-8 0.2 0.5 tr 0.05
W-9 0.5 2.4 0.1 0.21
Determination of DCs in PA W-10 0.3 0.2 ND 0.03
DC contents of PA (Table 3) were determined with a B-1 5.4 6.5 0.6 0.83
detection limit of 0.03 μmol/mL and a quantitation limit B-2 tr ND tr −
of 0.1 μmol/mL. Mean recovery rates ± coefficients of B-3 0.7 0.6 tr 0.08
variation of G, MG, and DA were 98.5 ± 1.4, 90.1 B-4 0.7 0.5 tr 0.08
B-5 2.3 1.5 0.1 0.25
± 2.1, and 96.1 ± 1.8%, respectively. HPLC chro-
B-6 0.9 1.0 ND 0.12
matograms of W-1 and B-1 were shown in Fig. 1. The B-7 0.4 0.7 0.1 0.08
highest DC contents were observed in sample B-1 and B-8 1.1 1.2 0.3 0.18
were present at a total of 0.83 mg/mL. DC contents and B-9 0.6 2.6 0.2 0.24
composition ratios of the 3 DCs, G, MG, and DA in PA B-10 tr tr ND −
were quite different between samples. Some among the Note: Values are the average of 2 times measurement.
20 samples, e.g., a pair of W-1 and B-1, W-9 and B-9, a
Glyoxal;
b
and W-10 and B-10, are produced by the same maker. c
Methylglyoxal;
Diacetyl;
With regard to W-1 and B-1, although the composition d
0.03 μmol/mL ≤ tr < 0.1 μmol/mL;
ratios of DCs were different, the total DC content was e
ND < 0.03 μmol/mL;
the highest value in the group of WPAs or BPAs. With f
Not calculated.
 Methylglyoxal as mutagen in pyroligneous acids
Fig. 1. HPLC chromatograms of W-1 and B-1 after derivatization.
Notes: Glyoxal, methylglyoxal, and diacetyl in W-1 and B-1 were
quantified as quinoxaline (Q), 2-methylquinoxaline (2-MQ), and 2.3-
dimethylquinoxaline (2.3-DMQ), respectively, by HPLC after quinox-
aline derivatization.
regard to W-10 and B-10, DC contents were lower val-
ues or less than the quantification limit, although these
samples showed relatively strong mutagenicity and the
equivalent specific mutagenicity values in TA100 in the
absence of S9 mix. With regard to W-9 and B-9, DC
contents were comparatively high, and the composition
ratios of DCs were similar. It was suggested that DC
contents and the composition ratios of DCs were similar
in WPA and BPA of the same maker. Therefore, differ-
ing DC contents and the composition ratios of DCs in
the present 20 PA samples may be caused by different
production heating temperature and time for each
maker. In agreement, previous studies showed that the
composition ratios of DCs in coffee beans and cookies
fluctuated with changes in heating times,23,24) and MG
production during heating varied with temperatures and
heating times.18) Therefore, differing contents and com-
position ratios of DCs in the present PA samples likely
837
reflect differences in heating temperatures and times
during manufacture. However, no significant differences
in DC contents were observed between WPA and BPA
samples by statistical analysis. DCs are the decomposi-
tion products of carbohydrate combustion and are the
major components of common WPAs and BPAs. Previ-
ous reports also show no large differences in composi-
tion of most volatile and odor components between
WPA and BPA,5,25) suggesting similar compositions of
these PAs.
Mutagenic contribution ratios of DCs from PA in
TA100 in the absence of S9 mix
Most PA samples contained DCs and were muta-
genic. Thus, in TA100 in the absence of S9 mix, the
mutagenic contribution ratios of DCs were calculated
as percentages of total PA mutagenicity. We calculated
the mutagenic contribution ratios of DCs not only
positive PA samples but also W-4, because W-4 was
regarded as pseudo-positive, showing 1.5–2 times the
number of negative control revertants. These analyses
showed the mutagenic contribution ratios of MG in
W-1, W-9, B-1, and B-9 exceed 100% (data are not
shown), and these PA samples showed clear cytotoxic-
ity in Ames tests (Table 1). The presence of
bactericidal components in PA has been previously
reported,1,5–7) and the cytotoxicity of these has been
shown in S. typhimurium.10–12) The present MG muta-
genic contribution ratios exceeding 100% may reflect
the underestimation of the mutagenicity of PA due to
PA cytotoxicity. Therefore, to examine the effects on
mutation expression by various components including
toxicity to test bacteria, we performed the Ames test of
PAs with and without added MG. Specifically, the
recovery rate of the number of net revertants derived
from MG that added to PA was calculated, and the
recovery rates of MG were regarded as the mutation
incidence. In a previous report, MG was identified as
the most mutagenic of the 3 DCs in TA100 in the
absence of S9 mix (about 18 times the mutagenicity
of G).26) Following similar observations (data are not
shown), we used MG for recovery tests and confirmed
that 0.3 μmol MG was optimum mutagenic dose but
not cytotoxic. Under these conditions, the mutation
incidence rates decreased with increasing PA doses,
except in the case of the W-5 (Fig. 2). The mutation
incidence rates of W-1, W-4, W-9, B-1, B-6, and B-9
particularly decrease compared with those of other sam-
ples, and these PA samples showed marked growth
inhibition (Fig. 2, Table 1). Therefore, it was found that
the mutation expression was decreased with increasing
PA doses because components toxic to test bacteria
increase with increasing PA doses. In addition, it was
suggested that toxic components and amounts were dif-
ferent between samples because the mutation incidence
showed a significantly different decreasing trend
between samples. Therefore, to calculate the mutagenic
contribution ratios, we determined to correct the num-
ber of the net revertants of PA by the mutation inci-
dence rates with using a method for adjusting the
decrease in the mutation expression level with the val-
ues of the mutation incidence rates. For the consistency
of mutagenic contribution ratios of DCs, calculations
838 A. Onoda et al.

Fig. 2. Mutation incidence rates in methylglyoxal recovery tests in Salmonella typhimurium TA100 in the absence of S9 mix.
Notes: Methylglyoxal (MG) recovery tests were conducted for all mutagenic pyroligneous acids (PA) using the Ames test. PAs were prepared at
doses of 20, 40, 60, and 80 µL, and simultaneous Ames tests were performed in PA with and without addition of 0.3 µmol MG; 0.3 µmol MG
was used as a reference material. Recovery rates were calculated as percentages after subtracting the number of the net revertants of PA from the
number of the net revertants of PA added 0.3 µmol MG and dividing by the number of the net revertants of 0.3 µmol MG. Hence, the recovery
rates of MG represent mutation incidence rates of PAs.

were performed at doses that corresponded with 2-fold MG contributed more than 70% of the mutagenicity.
increases in the number of the revertants compared These data suggest that MG is the major mutagen in
with the negative control, according to the threshold of PA. However, despite the strong mutagenicity of W-2,
positive PA mutagenicity. Mutagenic contribution ratios W-10, and B-10, MG contents were low and showed
ranged 0.0–8.8% for G and 0.0–93.0% for MG, and mutagenic contribution ratios of less than 1.7%. Corre-
they ranged 1.7–101.8% among all DCs (Table 4). lations between specific mutagenicity and MG contents
With regard to DA, mutagenic contribution ratio was among all 20 samples and after the exclusion of W-2,
not calculated because DA showed negative mutagenic- W-10, and B-10 showed coefficient (r) values of 0.17
ity due to the cytotoxicity in this study. DCs con- and 0.90, respectively. Consequently, with the excep-
tributed more than 25% of the mutagenicity of 12 PA tion of W-2, W-10, and B-10, MG was a major muta-
samples (60% of all samples), and MG accounted for gen in TA100 in the absence of S9 mix. On the other
the highest mutagenic contribution ratio in all PA sam- hand, the mutagenic activities of W-2, W-10, and B-10
ples. In particular, W-1, W-9, B-1, and B-9 contained indicate the presence of other mutagens, and these are
MG at concentrations of more than 2.4 μmol/mL, and currently under investigation.
In TA100 in the presence of S9 mix, MG showed
the strongest mutagenicity among 3DCs, and showed
Table 4. Mutagenic contribution ratios of dicarbonyl compounds the mutagenicity in a dose-dependent manner at higher
to the mutagenicity of pyroligneous acids in Salmonella typhimurium level than 0.75 μmol by dose-response curve (data are
TA100 in the absence of S9 mix.
not shown). B-1 contains the highest concentration of
Mutagenic contribution ratio (%) MG between samples and showed the mutagenicity
positive; however, the amount of MG was in low level
Sample Ga MGb DAc Total (%) to induce the mutation. Asita et al.10) reported that in
W-1 8.8 93.0 − d
101.8 the TA100 or TA98 in the presence of S9 mix, the con-
W-2 0.0 1.7 − 1.7 tribution of the polycyclic aromatic hydrocarbons
W-3 2.0 52.6 − 54.6 (PAHs) to the mutagenicity of the wood smoke conden-
W-4 0.1 31.6 − 31.7 sates could not be determined because of the concentra-
W-5 3.3 54.0 − 57.3
W-6 − − − −
tions of the PAHs was much lower level for inducing
W-7 0.0 16.3 − 16.3 the mutation. These results suggested that many more
W-8 0.2 17.3 − 17.5 mutagens contained in PA might contribute to the
W-9 0.4 70.4 − 70.8 mutagenicity of PA in the presence of S9 mix.
W-10 0.1 1.6 − 1.7 In this study, we showed that MG is the major muta-
B-1 3.1 78.4 − 81.5 gen of WPAs and BPAs in TA100 in the absence of S9
B-2 − − − − mix. However, although PA has been used in various
B-3 2.0 32.4 − 34.4
B-4 1.6 24.4 − 26.0
applications such as a bath agent and food additive
B-5 6.1 61.5 − 67.6 whereby it is in direct contact with the human body, it
B-6 0.7 17.4 − 18.1 appeared that PA has a serious safety issue because of
B-7 0.7 28.8 − 29.5 the mutagenicity. Therefore, it is important to explore
B-8 1.1 28.9 − 30.0 and evaluate the mutagenic substance in PA to ensure the
B-9 0.4 84.0 − 84.4 safety of PA. The mutagens of PA are produced in the
B-10 0.0 0.0 − 0.0
manufacturing process; so when the production condi-
a
b
Glyoxal; tions of PA are examined in a way the generation of MG
Methylglyoxal;
c is controlled or the formed MG is removed, PA might
Diacetyl;
d
Not calculated. become safe. In this study, no significant differences in
 Methylglyoxal as mutagen in pyroligneous acids
DCs contents were observed between WPA and BPA
samples; however, it appears that there is great variability
among quality of PA because of differences in the mate-
rials and production method based on the maker. At the
present day, with respect to the components of PA, stan-
dard values of PAHs including benzo[a]pyrene have
been established by JECFA (Joint FAO/WHO Expert
Committee on Food Additives). However, the risk of
these mutagenic substances has not been managed in
Japan. Therefore, the setting of standard values of harm-
ful substances and mutagenic substances such as MG in
PA is desired in future. The present data will contribute
to further studies of the risks associated with multiple
harmful compounds that are present in consumables and
the environment.
Author Contribution
A.O., M.A. and H.N. conceived and designed the
experiments, the results, and wrote the manuscript.
A.O. and M.A. performed the experiments and ana-
lyzed the data.
Disclosure statement
No potential conflict of interest was reported by the authors.
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