Subsequent products after antioxidant actions of beta-carotene and alpha-tocopherol have no Salmonella mutagenicity.

Article abstract of DOI:10.1271/bbb.66.363

Beta-carotene and alpha-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered. Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures o

Full text of DOI:10.1271/bbb.66.363

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Subsequent Products After Antioxidant Actions of
β-Carotene and α-Tocopherol Have No Salmonella
Mutagenicity
a
b
a
a
Mingzhou SUN , Ryo YAMAUCHI , Hitoshi ASHIDA & Kazuki KANAZAWA
a
Department of Life Science, Graduate School of Science and Technology, Kobe
University Rokkodai, Nada-ku, Kobe 657-8501, Japan
b
Department of Bioprocessing, Faculty of Agriculture, Gifu University 1-1 Yanagido, Gifu
City, Gifu 501-1193, Japan
Published online: 22 May 2014.
To cite this article: Mingzhou SUN, Ryo YAMAUCHI, Hitoshi ASHIDA & Kazuki KANAZAWA (2002) Subsequent Products After
Antioxidant Actions of β-Carotene and α-Tocopherol Have No Salmonella Mutagenicity, Bioscience, Biotechnology, and
Biochemistry, 66:2, 363-372, DOI: 10.1271/bbb.66.363
To link to this article: http://dx.doi.org/10.1271/bbb.66.363
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Biosci. Biotechnol. Biochem., 66 (2), 363–372, 2002
Subsequent Products After Antioxidant Actions of b-Carotene
and a-Tocopherol Have No Salmonella Mutagenicity
Mingzhou SUN,* Ryo YAMAUCHI,‡ Hitoshi ASHIDA,* and Kazuki KANAZAWA
*
,
†
*
Department of Life Science, Graduate School of Science and Technology, Kobe University,
Rokkodai, Nada-ku, Kobe 657-8501, Japan
‡
Department of Bioprocessing, Faculty of Agriculture, Gifu University, 1-1 Yanagido,
Gifu City, Gifu 501-1193, Japan
Received August 27, 2001; Accepted September 28, 2001
b
-Carotene and
a
-tocopherol are important antiox-
b-carotene instead increased the cancer incidence and
8)
idants biologically, but whether their oxidized products
are toxic or not remains to be discovered. Here, we
chromatographically separated 5 pure products or iso-
meric mixtures from reaction mixtures of
and reactive oxygens, and 17 lipid-radical scavenging
products of -tocopherol. The products were tested for
mutagenicity using Salmonella typhimurium TA98,
TA100, TA102, and TA104, in the presence and absence
of S9. None showed mutagenicity against any of the
four strains, or cytotoxicity that in‰uenced the survival
of the bacteria. Lipid-peroxides have been known to in-
crease the formation of mutagens from dietary procar-
cinogens such as heterocyclic amines. So, we also
measured the activity to increase 3-amino-1-methyl-
total mortality. Another study has found that
women with higher concentrations of -tocopherol
and carotenoids in plasma had higher levels of 8-oxo-
7,8-dihydro-2 -deoxyguanosine in lymphocytes,
correlating positively and signiˆcantly. Under high
oxygen tension, -carotene had been shown to cause
concentration-dependent DNA breakdown.
ˆnding may indicate that the subsequent products of
a
b-carotene
?
9
)
a
b
1
0)
The
carotenoids and
a-tocopherol formed after the an-
tioxidant actions have genotoxicity.
Here, we examined various oxidation products of
b
-carotene and a-tocopherol for mutagenicity using
Salmonella typhimurium TA98, TA100, TA102, and
TA104 strains. -Carotene has been recognized to
play an antioxidative role as a quenching and
scavenging reactive oxygen species and -tocopherol
b
5
H
-pyrido[4,3-
b
]indole (Trp-P-2) mutagenicity. The
products from
b
-carotene and -tocopherol did not in-
a
a
1,2)
crease, but rather several of them suppressed, the muta-
genicity of Trp-P-2. Thus, the products of -carotene
and -tocopherol formed after the antioxidant actions
as a trapper for lipid-peroxyl radicals. Then, we
tested major 5 reaction products of -carotene with
and 17 products from
tocopherol after the peroxidized lipids were
b
b
1
1,12)
a
reactive oxygen species
a-
were not genotoxic.
1
3–18)
trapped.
Since physiological levels have been
Key words: oxidized
b
-carotene;
oxidized
a-
estimated in healthy human blood to be around
1
9)
tocopherol; mutagenicity; antioxidant;
oxidative stress
0.12–0.89 nmol ml for
b
-carotene
and 16–
-tocopherol, the concentration of
each peroxidation product is considered to rise to
W
2
0)
31 nmol ml for
a
W
Carotenoids and tocopherols are among the im-
above 10 nmol ml. We then did experiments with a
concentration of 10 nmol plate of each peroxidation
product. On the other hand, it has been also reported
that lipid peroxides were cofactors in cytochrome
P450 (CYP)-mediated oxidation
W
1
,2)
portant biological antioxidants. They strongly sup-
press oxidative stress to quench singlet oxygen or to
scavenge radicals, and thereby contribute to suppress
the occurrence of several diseases such as protopor-
W
21)
and facilitated
3
)
4)
phyria, photocarcinogenesis, cardiovascular dis-
metabolic activation of dietary procarcinogens such
as heterocyclic amines to their ultimate carcinogenic
5
)
6,7)
ease, and atherosclerosis. In a human interven-
tion study, however, daily supplementation with
2
2,23)
form.
Also, peroxyl radicals generated from
2
0 mg
b
-carotene or 50 mg
a
-tocopherol did not
lipid-peroxides have been known to epoxidize polycy-
clic aromatic hydrocarbons and to produce ultimate
reduce the incidence of lung cancer in smokers, and
†
To whom correspondence should be addressed. Kazuki KANAZAWA, Fax: +81-78-803-5879; E-mail: kazuki
AAPH, 2,2 -azobis(2-amidinopropane)dihydrochloride; AMVN, 2,2 -azobis(2,4-dimethylvaleronitrile); CYP,
cytochrome P450 monooxygenases; DMSO, dimethylsulfoxide; HPLC, high-pressure liquid chromatography; ML, methyl linoleate; 13-
MLOOH, methyl (9 , 11 )-( )-13-hydroperoxy-9,11-octadecadienoate; PLPC, 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine; 13-
PLPCOOH, 1-palmitoyl-2-[(9 ,11 )-( )-13-hydroperoxy-9,11-octadecadienoyl]-3-sn-phosphatidylcholine; Trp-P-2, 3-amino-1-methyl-
-pyrido[4,3- ]indole
＠
kobe-u.ac.jp
Abbreviations
:
?
?
Z
E
S
Z
E
S
5
H
b

3
64
M. SUN et al.
2
4)
carcinogenic forms.
products of -carotene and
the mutagenicity of 3-amino-1-methyl-5
]indole (Trp-P-2), a typical dietary procarcino-
We examined whether the
-tocopherol increased
-pyrido
carotene. Each of the products was free of other
products on HPLC and did not further decompose in
b
a
H
chloroform ethanol (2:1, v v) at „20
9
C before it
W
W
[
4,3-2
b
was used at around day 10.
5)
gen. None of the oxidation products showed muta-
genicity, and interestingly, several of them inhibited
the mutagenicity of Trp-P-2.
Oxidation products of
idation products of -tocopherol were prepared in six
diŠerent systems. First, -tocopherol was photooxi-
dized in the presence of methylene blue as a pho-
tosensitizer and the resulting 8a-hydroperoxy-
a-tocopherol. Seventeen ox-
a
a
Materials and Methods
a
-
1
3)
Chemicals
from Wako Pure Chemical Industries (Osaka,
Japan) and recrystallized from benzene. 2 ,4 ,8
-Tocopherol was from Sigma Chemical Co. (St.
.
All-trans
-
b
-carotene was obtained
tocopherone (T1) was puriˆed by HPLC. Second,
-tocopherol was oxidized in phosphatidylcholine
liposomes, and three products, -tocopherylquinone
(T2), 2,3-epoxy- -tocopherylquinone (T3), and
5,6-epoxy- -tocopherylquinone (T4), were ob-
a
R
?R
?R
-
a
a
a
Louis, MO) and was puriˆed by reversed-phase
HPLC with methanol as the solvent. Methyl linoleate
ML) was obtained from Tokyo Kasei Co., Tokyo,
Japan, and was puriˆed to be peroxide-free by silica
a
7,28,29)
1
tained.
Third, the spirodiene dimer of a-
(
tocopherol (T5) was synthesized following the proce-
3
0)
dure of Nelan and Robeson. Fourth, a-tocopherol
15)
gel column chromatography. Linoleic acid (Wako
Pure Chemical) was oxidized with soybean lipox-
ygenase-1 (Sigma) and the resulting hydroperoxide
was converted to its methyl ester with dia-
was left to stand in an autoxidation system of methyl
linoleate under air-insu‹cient conditions. This
yielded two isomeric products of
methyl linoleate radicals, methyl (10
-tocopheroxy)-10,12-octadecadienoate (T6) and
methyl (9 ,11 )-13-( -tocopheroxy)-9,11-octadeca-
dienoate (T7). Alternatively, under air-su‹cient
conditions, eight isomeric products of -tocopherol
a
-tocopherol with
E
,12 )-9-
Z
1
6)
zomethane.
)-13-hydroperoxy-9,11-octadecadienoate (13-
MLOOH) was about 90 , the remainder being the
-isomer. 1-Palmitoyl-2-linoleoyl-3-sn-phosphatidyl-
The purity of methyl (9
Z
, 11
E
)-
(a
(
S
Z
E
a
1
5,31)
z
9
a
1
4,15,31)
choline (PLPC) was synthesized by 2-acylation
of 1-palmitoyl-3-sn-glycerophosphocholine with
linoleic acid anhydride according to the method of
with ML-peroxyl radicals were obtained:
methyl (9
pherone]-9,11-octadecadienoate
(9 ,11 )-( )-13-[( )-8a-dioxy-a-tocopherone]-
Z
,11
E
)-(
S
)-13-[(
S
)-8a-dioxy-
a
-toco-
(T8a),
methyl
26)
Gupta et al
.
1-Palmitoyl-2-[(9
Z
,11
E
)-(
S
)-13-
Z
E
R
R
hydroperoxy-9,11-octadecadienoyl]-3-sn-phospha-
9,11-octadecadienoate (T8b), methyl (9
13-[( )-8a-dioxy- -tocopherone]-9,11-octadeca-
dienoate (T8c), methyl (9 ,11 )-( )-13-[( )-8a-
dioxy- -tocopherone]-9,11-octadecadienoate (T8d),
methyl (10 ,12 )-( )-9-[( )-8a-dioxy- -toco-
pherone]-10,12-octadecadienoate (T9a), methyl
(10 ,12 )-( )-9-[( )-8a-dioxy- -tocopherone]-
10,12-octadecadienoate (T9b), methyl (10 ,12 )-
)-9-[( )-8a-dioxy- -tocopherone]-10,12-octadeca-
dienoate (T9c), and methyl (10 ,12 )-( )-9-[( )-
8a-dioxy- -tocopherone]-10,12-octadecadienoate
(T9d). Fifth, -tocopherol was reacted with 13-
MLOOH in an iron-catalyzed reaction. This yielded
a mixture of methyl ( )-(12 ,13 )-9-(8a-dioxy-
tocopherone)-12,13-epoxy-9-octadecenoate and
methyl ( )-(12 ,13 )-11-(8a-dioxy- -tocopherone)-
12,13-epoxy-9-octadecenoates (T10). Sixth, similar
products of -tocopherol were obtained from the
reaction with 13-PLPCOOH in the presence of an
iron-chelate, Fe(III)-acetylacetonate, at 37 C in
Z,11E )-(R)-
tidylcholine (13-PLPCOOH) was prepared with soy-
S
a
2
7)
bean lipoxygenase-1 by the method of Brash et al
.
Z
E
S
R
Alkylperoxyl radical generators, a lipid-soluble
a
2
a
,2?
-azobis(2,4-dimethylvaleronitrile)(AMVN) and
E
Z
S
S
a
water-soluble 2,2 -azobis(2-amidinopropane)di-
?
hydrochloride (AAPH), were obtained from Wako
Pure Chemical. For Salmonella tests, agar and ex-
tracts from beef and yeast were purchased from
Difco Laboratory (Detroit, MI). Trp-P-2 was from
Wako Pure Chemical. All other chemicals were of
the highest grade commercially available.
E
Z
R
R
a
E
Z
(
R
S
a
E
Z
S
R
a
a
Oxidation products of
b
-carotene.
b
-Carotene was
C for 2 h in
E
S
S
a-
incubated with AMVN in benzene at 37
9
1
1)
the dark under air. The subsequent products were
separated and collected through HPLC with an Iner-
E
S
S
a
16)
tsil Prep ODS column (20
×
250 mm, GL Sciences,
a
Tokyo, Japan) developed with methanol ethyl
W
acetate (8:2, v v) at a ‰ow rate of 15 ml min. Then
9
W
W
1
1,12)
they were identiˆed as mentioned previously:
a mixture of 12-formyl-11-nor- -carotene and 15
formyl-15-nor- -carotene; C2, 19-oxomethyl-10-
nor- -carotene; C3, 5,6-epoxy-5,6-dihydro-
carotene; C4, a mixture of 13,15 -epoxyvinyleno-
3,15 -dihydro-14,15-dinor- -carotene and 15 ,13-
epoxyvinyleno-13,15 -dihydro-14,15-dinor-
carotene; and C5, 11,15 -dihydrooxepin-
C1,
?-
ethanol. The reaction products were separated
through HPLC with an Inertsil Prep ODS column
b
,b
b
,b
(20
×
250 mm) developed with methanol ethanol
W
b
,b
b,b-
(2:3, v v) at a ‰ow rate of 15 ml min. The HPLC
W
W
?
gave a single peak, which was characterized chemi-
cally as a mixture of 1-palmitoyl-2-[12,13-epoxy-11-
1
?
b
,
b
?
b
b,b
?
,
b
-
-
(8a-dioxy-
a-tocopherone)-9-octadecanoyl]-3-sn-
?
phosphatidylcholine and 1-palmitoyl-2-[12,13-epoxy-

Toxicity of Oxidized
b
-Carotene and
a
-Tocopherol
365
9
-(8a-dioxy-
a
-tocopherone)-10-ocatadecenoyl]-3-sn
-
)
0.1 nmol of Trp-P-2 in 0.1 ml of water at 379C for
phasphatidylcholine (T11): UV (ethanol)
l
max
(
e
20 min in the presence of the S9 mix. After the culti-
+
2
40 nm (11800); and FABMS (glycerol as the matrix)
vation, the number of His revertant colonies was
+
+
m z 1235 (MH ), 806 ([MH„429] ), 702 ([790„
counted and compared to that of revertant colonies
without products. The tests were done two times with
three plates for each product.
W
+
+
choline] , 430 ([
CH
a
-tocopherone] ), 224 ([CH
＝
CH-
+
2
-O-phosphocholine] ), and 184 ([phosphocho-
+
line] ).
The
of other products on the respective HPLC and were
almost completely stable in ethanol at „20
used.
a
-tocopherol products thus obtained were free
Results
9
C until
Evaluation of the products of
genicity
b-carotene for muta-
We separated three pure products (C2, C3, and
C5) and two isomer-mixtures (C1 and C4) from the
Mutagenicity test. Salmonella strains TA102 and
TA104 are superior for detecting mutations caused
by active oxygen species such as aldehydes and
reaction mixture of b-carotene and peroxyl radicals
generated by AMVN. They were added to the
3
2,33)
34)
peroxide,
as is TA100. We used these three
Salmonella strains at concentrations of 1, 2, 5, and
strains as well as strain TA98. Tests were done essen-
10 nmol plate. Table 1 shows the results at the
W
3
5)
tially as described previously except for the mixing
of products with bacteria. The S9 mix was prepared
from the S9 fraction obtained from the liver of
highest concentrations of 10 nmol plate (12.5
m
M
in
W
the bacterial solution), because the products gave
similar results at every concentration. Generally, the
tested chemicals have been judged to be mutagenic
when could give two-fold of revertant numbers or
more compared to that of the control (vehicle alone).
Sprague-Dawley
rats
given
intraperitoneally
3
0 mg kg day phenobarbital for 3 days, accompa-
W W
nied by 80 mg kg day methylcholanthrene plus
W W
0 mg kg day phenobarbital for another 2 days.
3
6)
3
None of the products or the intact b-carotene
W W
Bacterial strains were grown overnight in a liquid
broth medium at 37 C. A part of the bacterial sus-
pension (0.1 ml) was mixed with peroxidation
products of -tocopherol or -carotene in 0.1 ml of
increased the revertant numbers of any Salmonella
strains or in‰uenced cell numbers. In the same
strains, positive controls greatly increased the
revertant numbers. For example, 0.5 nmol of 1-
9
a
b
dimethylsulfoxide (DMSO), and sonicated for 10 sec
in order to enter the cells. Sonication for less than
nitropyrene (Wako Pure Chem. Ind.) gave 4684
266 revertants for TA98 and „S9; 0.1 nmol of Trp-
P-2, 1423 87 for TA98 and +S9; 10 nmol of
methyl- -nitro-nitrosoguanisine (Tokyo Kasei Co.),
536 for TA100 and „S9; 0.5 nmol of a‰atox-
(Makor Chem. Ltd., Israel), 1661 272. Cu-
±
1
20 sec did not in‰uence the ratios of cells. For exam-
±
N-
7
ple, the surviving cell numbers (
×
10 ) of TA98,
N?
TA100, TA102, and TA104 after 10-sec sonication
4454
in B
±
1
were 303
respectively, compared to 165
and 796
pension was incubated at 37
presence or absence of the S9 mix (0.5 ml, containing
.052 nmol of CYP). The incubation mixture was
added to 2 ml of molten top agar, poured onto
minimal-glucose agar medium, and cultured at 37
±
52, 241
±
17, 623
±
11, and 817
±
±
12,
14,
±
±
5, 202 13, 616
±
mene hydroperoxide has been recognized to be muta-
genic and gives 246 10 revertants for TA102 and
„S9, 321 4 for TA102 and +S9, 583 7 for TA104
and „S9 at 11 nmol of the concentration.
Then, the reaction mixture of -carotene and
±
11 for untreated ones. The bacterial sus-
C for 20 min in the
±
9
±
±
34)
0
b
AMVN was directly tested with four strains
(Table 2). The mixture was not mutagenic, regardless
of the presence or absence of S9. These results clearly
9
C
for 2 days. To detect cytotoxicity of the products,
surviving cells were enumerated simultaneously. A
part of the mixture was washed with 0.4 ml of
showed that no products of
b-carotene after the
reaction with peroxyl radicals were mutagenic or
cytotoxic.
0
.1 m
M
sodium phosphate buŠer (pH 7.4) by cen-
trifugation at 3000 rpm for 10 min, twice. The bac-
6
teria were resuspended in the buŠer and diluted 10 or
1
Evaluation of the products of
mutagenicity
a-tocopherol for
7
0 -fold with saline solution. A 0.1-ml portion was
cultured on minimal medium together with top agar
containing a 5 m+ excess of histidine. After the culti-
vation, the His colonies and surviving colonies
a-Tocopherol was incubated with ML under air in
M
the presence of AMVN or AAPH as an initiator, and
the incubation mixture was assayed for mutagenicity
(Table 2). The mixtures of ML with AMVN and ML
„
(
His ) were enumerated. The tests were done two
times with two plates for each product.
plus
revertant numbers compared to AMVN alone. We
separated 5 oxidation products of -tocopherol (T1-
T5) and 12 products of -tocopherol with lipid-
a-tocopherol with AMVN slightly increased the
EŠects on mutagenicity of Trp-P-2. For the
a
3
7)
antimutagenicity test, Salmonella TA98 was used.
a
The bacteria (0.1 ml) was mixed with the products of
-carotene or -tocopherol, and then incubated with
peroxyl or lipid-alkyl radicals (T6-T11). Table 3
shows the eŠects of these products on the four
b
a

3
66
M. SUN et al.
Table 1. The EŠects of Subsequent Products of
b
-Carotene after the Antioxidant Action on Four Salmonella Strains
TA98
TA100
„S9 +S9
Revertant number (Mean
TA104
a
Product (10 nmol plate)
W
Surviving
cells (
„
S9
+S9
„S9
+S9
7
b
×
10 )
±
Vehicle (DMSO)
17
18
±
±
3
6
24
19
±
±
6
3
121
98
±
±
27 132
±
±
44
2
196
183
±
9
±
26 156
30 136
±
±
25
29
796
817
±
±
11
12
b
-Carotene
16
93
±
16 160
±
C1
C2
19
19
±
±
4
6
22
20
±
±
4
3
109
103
±
±
6
89
±
±
6
188
±
3
168
±
±
23 148
±
17
40
66
52
±
±
21
794
778
±
±
14
16
27
87
6
6
179
172
±
15 167
17 161
±
3
C3
C4
15
16
±
±
4
3
22
21
±
±
5
3
118
113
±
±
12
16
76
92
±
±
±
4
5
169
176
±
±
9
66
50
±
12
2
59
38
±
±
9
9
815
793
±
±
11
18
11 186
±
22
±
C5
18
±
4
21
±
6
98
±
34
84
±
10 189
±
10 166
±
22
56
±
8
84
±
20
786
±
11
a
The tests were done at a concentration of 1, 2, 5, or 10 nmol plate, each of which gave similar results.
Surviving cell numbers remained unchanged under conditions of „ and +S9, and in TA98, TA100, and TA102, compared to vehicle alone.
W
b
Table 2. EŠects of Peroxidizing Mixtures of
b
-Carotene and
a
-Tocopherol on Four Salmonella Strains
a
Reaction component
TA98
TA100
„S9
TA102
TA104
„
S9
+S9
+S9
„S9
+S9
±
SD)
„S9
+S9
Revertant number (mean
Vehicle (DMSO)
20
13
24
20
15
28
21
7
±
±
±
±
±
±
±
±
4
4
4
6
4
5
4
3
34
21
±
±
±
±
±
±
±
±
16
99
104
98
110
76
93
±
±
±
±
±
±
±
±
12
5
107
114
117
115
100
120
114
97
±
±
±
±
±
±
±
±
22
7
15
7
28
33
21
6
214
206
185
221
213
182
250
227
±
±
±
±
±
±
±
±
41
17
14
17
18
15
11
27
217
229
211
241
254
267
232
261
±
±
±
±
±
±
±
±
48
13
9
406
±
±
±
±
±
±
±
±
129
179
382
±
±
±
±
±
±
±
±
161
AMVN
AMVN
AAPH
8
2
5
4
4
3
5
366
378
392
206
247
301
723
295
276
258
356
713
448
746
15
11
93
49
96
40
66
＋
b
-Carotene
25
23
22
21
14
20
16
6
15
20
19
5
54
33
30
37
25
51
9
AAPH+ML
AMVN+ML
AAPH+ML
19
24
10
23
＋
＋
a
-Tocopherol
-Tocopherol
81
95
AMVN+ML
a
a
The mixtures in a benzene solution were incubated under air at 37
9
C for 1 h, dried, resuspended in DMSO, and then added to Salmonella cells. The ˆnal
-tocopherol, 10 nmol.
dose-concentrations plate: AAPH and AMVN, 1 nmol; ML, -carotene and
b
a
W

Toxicity of Oxidized
b
-Carotene and
a
-Tocopherol
367
Table 3. The EŠects of Subsequent Products of
a
-Tocopherol, after the Antioxidant Action, on Four Salmonella Strains
TA98
TA100
+S9
Revertant number (Mean
TA102
TA104
„S9 +S9
SD, n
Product (10 nmol plate)
W
Surviving
„
S9 +S9 „S9
„S9 +S9
7
a
cells (
×
10 )
±
＝
4)
Vehicle (DMSO)
ML
17
17
17
±
±
±
3 24
3 22
3 25
±
±
±
5 123
2 109
±
±
±
22 117
15 106
29 90
±
±
±
16 260
258
10 248
±
±
±
12 271
235
22 312
±
±
±
19 167
11 145
15 162
±
±
±
21 173
20 83
24 105
±
23 729
±
11 672
±
14 703
±
25
±
30
±
18
8
9
1
3-MLOOH
7
95
PLPC
PLPCOOH
17
18
±
±
4 22
3 21
±
±
4 105
77
±
±
4
83
±
±
9
6
239
245
±
±
5
224
±
±
21 142
28 84
±
±
32 54
11 58
±
±
7
8
750
724
±
8
3
24 82
30 221
±
11
a
-Tocopherol
20
28
±
±
5 24
7 28
±
±
6 108
±
±
36 83
±
4
5
235
237
±
±
14 297
±
±
19 152
18 147
±
±
12 92
±
25 734
±
24
20
T1
7
5
88
98
10 90
±
29 333
10 89
±
19 733
±
T2
T3
T4
T5
11
12
20
16
±
±
±
±
2 21
2 21
4 20
3 21
±
±
±
±
±
±
±
±
15 114
19 115
14 104
18 96
±
±
±
±
7
212
292
±
±
±
±
20 296
32 216
23 312
33 312
±
±
±
±
25 175
26 186
37 171
20 155
±
±
±
±
14 130
12 167
25 85
15 67
±
31 652
±
13 747
±
11 636
±
20 628
±
±
±
±
19
4 105
6
5
4
5
66
62
10 244
29
31
4
244
247
T6
15
±
2 19
±
3
98
±
21 88
±
7
±
23 324
±
28 190
±
11 88
±
14 691
±
25
Contd.

3
68
M. SUN et al.
Table 3. Contd
TA98
TA100
TA102
„S9 +S9
TA104
„S9 +S9
Product (10 nmol plate)
W
Surviving
„
S9 +S9 „S9
+S9
7
a
cells (
×
10 )
29
T7
28
±
5 25
±
6
68
±
17 71
±
3
270
±
12 273
±
14 189
±
35 70
±
23 673
±
T8a-d
T8a
T8b
T8c
T8d
T9a-d
16
17
27
27
±
±
±
±
4 32
±
10 82
3 106
±
±
±
±
11 81
90
13 87
28 93
±
±
±
±
9
258
±
±
±
±
18 331
27 279
31 366
13 281
±
±
±
±
19 166
22 157
36 169
20 172
±
±
±
±
18 96
19 133
13 80
10 101
±
26 648
±
28 626
±
20 880
±
26 689
±
±
±
±
28
23
26
32
2 21
5 24
6 23
±
5
10 293
11 239
6
±
±
5
4
76
86
211
T9a
T9b
T9c
T9d
T10
23
13
20
13
22
±
±
±
±
±
4 25
3 23
5 30
2 18
4 16
±
±
±
±
±
5 104
4 108
±
±
±
±
±
19 90
34 100
19 114
11 90
30 120
±
±
±
±
±
11 244
±
±
±
±
±
15 309
31 267
22 258
28 300
19 261
±
±
±
±
±
10 157
26 178
23 190
45 170
25 191
±
±
±
±
±
9
141
±
±
±
±
±
16 657
±
±
±
±
±
23
13
18
22
44
6
7
6
267
235
239
20 53
15 89
24 76
23 249
9
727
8
3
74
90
20 632
19 687
45 646
2 110
11 227
T11
18
±
4 21
±
5 102
±
13 109
±
4
225
±
16 252
±
22 107
±
16 150
±
3
734
±
23
a
Surviving cell numbers remained unchanged under conditions of „ and +S9, and in TA98, TA100, and TA102, compared to vehicle alone.

Toxicity of Oxidized
b
-Carotene and
a
-Tocopherol
369
Fig. 1. EŠects of Products of
Against a mutagenicity of 0.1 nmol Trp-P-2, the suppressive eŠects of the oxidation products (10 nmol) were measured. Data are the
mean SD for 6 plates tested. No products in‰uenced cell survival compared to the vehicle, as shown in Tables 1 and 3.
b-Carotene and a-Tocopherol after the Antioxidant Actions on Trp-P-2 Mutagenicity.
±
Salmonella strains. None of the products increased
Trp-P-2 for 10 min, and with S9 for another 10 min,
and then incubated with bacterial cells for 20 min. In
design C, products were ˆrst incubated with S9, and
with Trp-P-2, and then with bacteria. In design D,
Trp-P-2 was ˆrst activated with S9 following a 10-sec
boiling to inactivate the S9 enzymes, and incubated
with the products, and then mixed with bacteria. In
design E, TA98 cells were ˆrst mutated by incubation
with Trp-P-2 and S9, and then incubated with the
products, as an assay to detect bio-antimutagenici-
ty. Compared to design A, 13-MLOOH showed
similar suppression in designs C and D, suggesting
that it aŠected S9 and the activated Trp-P-2.
PLPCOOH and C3 were more suppressive in design
D, indicating that they acted on the activated Trp-P-
2. None of the four products used here showed any
bio-antimutagenicity.
the revertant numbers or in‰uenced cell survival
compared to vehicle, ML, PLPC, or
a
-tocopherol
alone. Thus, the products of
a-tocopherol that
formed after the antioxidant action had no mutagen-
icity or cytotoxicity. The slight increases of revertant
numbers in Table 2 were considered to be attributed
to reaction products of AMVN and ML but not to
oxidation products of
a
-tocopherol.
3
8)
EŠects of products of
on Trp-P-2 mutagenesis
b
-carotene and
a
-tocopherol
Trp-P-2 becomes mutagenic after being metaboli-
cally activated by CYP 1A enzymes, which can be de-
3
6)
tected with Salmonella TA98. The 5 products of
carotene and 17 products of -tocopherol were added
to the mutagenesis system and the eŠects on Trp-P-2
mutagenicity assayed (Fig. 1). All of the -carotene
products suppressed mutagenicity to some extent; C2
and C3 inhibited it by 29 at 10 nmol compared to
Trp-P-2 at 0.1 nmol. Among the 17 -tocopherol
b-
a
b
Discussion
z
a
This study showed that none of the products of
b-
products, 7 suppressed the mutagenicity partly. Lipid
hydroperoxides, 13-MLOOH and PLPCOOH,
showed marked suppression.
Then, we examined the mechanisms of suppression
by 13-MLOOH, PLPCOOH, C2, and C3, on mixing
with Trp-P-2, S9 and TA98, which were set as ˆve ex-
perimental designs (Fig. 2). In design A, the peroxi-
dation products were incubated with all three factors,
which is the same method used for the test in Fig. 1.
In design B, the products were ˆrst incubated with
carotene and -tocopherol after acting as antiox-
a
idants had mutagenicity against Salmonella TA98,
TA100, TA102, or TA104. These two biological an-
tioxidants are well known to suppress oxidative stress
that is closely associated with degenerative diseases
including cancer. The absence of genotoxicity indi-
cates that
b-carotene and a-tocopherol should be
favored as biological antioxidants.
One question remaining about the method for
testing mutagenicity was whether the substances had

3
70
M. SUN et al.
Fig. 2. Elucidation of the Suppressive Mechanism of Salmonella Mutation Induced by Trp-P-2.
Products (10 nmol) were added to the 0.1-nmol Trp-P-2-mutagenicity system, in ˆve diŠerent mixing orders (experimental design
A-E) and incubated at 37
revertants obtained with Trp-P-2 (2610
tained with both the products and Trp-P-2, and
9
C. The percentage of suppression was calculated as: [(
254), is the number of spontaneous revertants (20
is the number of revertants obtained with the products.
X
-
Y
)-(
Z
-
W
)] (
X
-
Y
)
×
100, where
X is the number of
W
±
Y
±
3), Z is the number of revertants ob-
W
been incorporated into the bacterial cells su‹ciently.
All the products of -carotene and -tocopherol
facilitate their incorporation. The results in Tables
1–3 were obtained following a 10-sec sonication, and
were similar to the results without sonication (data
b
a
tested here are lipophilic. Generally, lipophilic sub-
stances can easily pass through the bacterial cell
membrane, which is constructed of phospholipids
but hydrophilic substances require a transporter pro-
3
4)
not shown). Among the tester strains, Dillon et al
.
who tested several lipophilic aldehydes and peroxides
such as benzoyl peroxide and veratraldehyde,
described how Salmonella TA100, TA102 and TA104
were better for detecting mutagenicity. Therefore, we
3
9,40)
tein.
Further, in this study, we used sonication to
mix the products with bacterial cells in order to

Toxicity of Oxidized
b
-Carotene and
a
-Tocopherol
371
believe that the products of
tocopherol entered the bacterial cells and that they
had no mutagenicity.
b
-carotene and
a
-
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)
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7
)
Iwatsuki, M., Niki, E., Stone, D., and Darley-
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2
1–23)
of
and
mutagenicity of Trp-P-2. The results in Fig. 1,
however, showed that most products of -carotene
and -tocopherol did not increase but rather sup-
N
-hydroxyl.
Thus, the products of b-carotene
a
-tocopherol had been predicted to increase the
1
029–1035 (1994).
b
9
)
Bianchini, F., Elmstaåhl, S., Martinez-Garci aá , C.,
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N-hydroxylation of Trp-P-2. Other hydro-
peroxides, 13-MLOOH and PLPCOOH, suppressed
the mutagenicity, and showed obvious activity when
1
b
previously incubated with S9 or
Fig. 2). Both CYP and -hydroxy-Trp-P-2 have
been recognized to be sensitive to reactive oxygen
N
-hydroxy-Trp-P-2
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(
N
1
1
1) Yamauchi, R., Miyake, N., Inoue, H., and Kato, K.,
4
1,42)
Products formed by peroxyl radical oxidation of b-
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W
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m
M
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3) Yamauchi, R., Kato, K., and Ueno, Y., Reaction of
8
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4
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