doi:10.1093/toxsci/kfh173
Advance Access publication May 24, 2004
Genotoxicity of Motorcycle Exhaust Particles InVivo and InVitro
Yu Wen Cheng,* Wen Wha Lee,† Ching Hao Li,† Chen Chen Lee,† and Jaw Jou Kang,†
,1*School of Pharmacy, Taipei Medical University, Taipei, Taiwan, and †Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan Received on March 2, 2004; accepted on May 7, 2004
We studied the genotoxic potency of motorcycle exhaust particles
(MEP) by using a bacterial reversion assay and chromosome
aberra-tion and micronucleus tests. In the bacterial reversion assay
(Ames test), MEP concentration-dependently increased TA98,
TA100, and TA102 revertants in the presence of
metabolic-activating enzymes. In the chromosome aberration test, MEP
concentration-dependently increased abnormal structural
chromo-somes in CHO-K1 cells both with and without S9. Pretreatment with
antioxidants (a-tocopherol, ascorbate, catalase, and NAC) showed
varying degrees of inhibitory effect on the MEP-induced mutagenic
effect and chromosome structural abnormalities. In the in vivo
micronucleus test, MEP dose-dependently induced micronucleus
formation in peripheral red blood cells after 24 and 48 h of treatment.
The increase of micronucleated reticulocytes induced by MEP was
inhibited by pretreatment with
a-tocopherol and ascorbate. The
fluorescence intensity of DCFH-DA–loaded CHO-K1 cells was
increased upon the addition of MEP. Our data suggest that MEP
can induce genotoxicity through a reactive oxygen species–(ROS-)
dependent pathway, which can be augmented by metabolic
activa-tion. Alpha-tocopherol, ascorbate, catalase, and NAC can inhibit
MEP-induced genotoxicity, indicating that ROS might be involved
in this effect.
Key Words: motorcycle exhaust particles; Ames test; reactive
oxygen
species;
chromosome
aberration;
micronucleus;
genotoxicity.
Recent prospective cohort studies have shown that long-term
exposures to particulate air pollution might be associated with
increases in the rates of morbidity and mortality from respiratory
and cardiovascular diseases in the general population (Abbey
et al., 1995; Dockery et al., 1993; Pope et al., 1995). In addition
to systemic toxicity, the possible genotoxicity of small
particu-late matter has been investigated in recent years. On metabolism,
activation, or accumulation, pollutants can become extremely
toxic to vital organs, and this is often related to a strong
genotoxic effect (Baulig et al., 2003; Harris et al., 1978).
Air-borne particles have been shown to induce chromosome
aberra-tions (Seemayer et al., 1994), sister chromatic exchanges
(Hornberg et al., 1996), the formation of DNA adduct (Gallagher
et al., 1990), tumorigenicity (Heussen et al., 1996), and
embryo-toxicity (Matsumoto and Kashimoto, 1986). In some industrial
cities, the exhaust of diesel vehicles is considered a major source
of air pollution that is ‘‘probably carcinogenic’’ (Aoki et al.,
2001; Baeza Squiban et al., 1999). Diesel exhaust particles
(DEP) are composed of carbon nuclei, absorbed organic
com-pounds, and trace heavy metals including iron and copper
(Schuetzle and Lewtas, 1986; Schuetzle et al., 1981). The
absorbed organic compounds consist of some highly mutagenic
chemicals including polycyclic aromatic hydrocarbons (PAHs)
and nitroaromatic hydrocarbons (Grimmer et al., 1987; Lewtas,
1988) and were shown to cause pulmonary tumors (Mauderly
et al., 1987).
In Taiwan, motorcycles are widely used; more than 11 million
motorcycles were registered in 2000 (Ministry of Transportation
and Communications, 2000). The use of motorcycles, especially
those with two-stroke engines, introduce about 16,000 and
15,000 tons of total suspended particles and particulate matter
of 10
mm (PM10), respectively, per year in Taiwan
(Environ-ment Protection Agency, 2000). However, the impact of
motor-cycle exhaust on the environment and its biological effects are
relatively unknown. Motorcycle exhaust particles (MEP) have
been found to impair endothelium-dependent relaxation in
the rat aorta (Cheng and Kang, 1999) with short-term treatment
(10 min), enhance vasoconstriction in rat smooth muscle cells
with long-term treatment (18 h; Tzeng et al., 2003), induce
apoptosis in macrophages (Lee and Kang, 2002), and affect
metabolic enzyme activities in rat tissues (Ueng et al., 1998).
Also, particles collected from scooter or motorcycle exhaust
were shown to cause a certain degree of DNA damage
(Kuo et al., 1998; Zhou and Ye, 1997), similar to that reported
with DEP (Casellas et al., 1995; Li et al., 1996).
Although both DEP and MEP come from vehicles’
combus-tion, there might be some difference between the chemical
com-ponents in the exhausts due to the different engine structures and
the fuel used. For example, many motorcycles use two-stroke
engines, which require the mixing of lubricant with the fuel. It
has been shown that the addition of lubricant will lead to the
incomplete combustion and mass exhaust of submicrometer
carbonaceous particles (Lewtas, 1983). Chemical analysis
stu-dies demonstrated that MEP contained higher levels of benzene,
xylene, and toluene than did gasoline and diesel engine exhausts
(Chan et al., 1993; Jemma et al., 1995). Although both DEP and
1
To whom correspondence should be addressed at Institute of Toxicology, College of Medicine, National Taiwan University, No. 1 Jen-Ai Road, Section 1, Taipei, Taiwan. Fax: 1886 2-23410217. E-mail: jjkang@ha.mc.ntu.edu.tw.
Toxicological Sciences vol. 81 no. 1#Society of Toxicology 2004; all rights reserved.
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MEP contain PAHs, their compositions are different; the major
PAH components found in DEP are phenanthrenes (Barfknecht
et al., 1982). Therefore, it is possible that DEP and MEP might
exert different biological effects.
The aim of this study was to contribute to a better
under-standing of the genotoxic effect of MEP and the possible
involvement of reactive oxygen species (ROS) in the induction
of genotoxicity using the short-term in vitro mutagenicity
bio-assay (Ames Salmonella bio-assay), the chromosome aberration test
with CHO mammalian cells, and the in vivo rodent micronucleus
test. We found that MEP contains chemicals that can induce
genotoxicity both in vitro and in vivo and that ROS are involved
in this pathway.
MATERIALS AND METHODS
Chemicals. Fetal bovine serum (FBS), penicillin/streptomycin, Dulbecco’s modified Eagle medium (DMEM), F-12K medium, and trypsin were obtained from Gibco BRL (Grand Island, NY). Ascorbate, D (1)-glucose, and sodium chloride were obtained from Merck (Darmstadt, Germany). Acridine orange, N-acetyl-L-cysteine (NAC), catalase, glucose-6-phosphate (G6P),b-nicotinamide adenine dinucleotide phosphate (b-NADP), a-tocopherol, colcemid, 3-methyl-chlolanthrene, and the positive control chemicals for the Ames test, 9-aminoa-cridine, 4-nitroquinoline-N-oxide (4-NQO), 2-aminoanthracene (2AA), streptozotocin (STZ), benzo(a)pyrene (BaP), and mitomycin c, were obtained from Sigma Chemical Co. (St. Louis, MO).
For the micronucleus assay in mice, MEP agents were dissolved in corn oil and mitomycin c was dissolved in PBS to give solutions with different concentrations of the tested compound for intraperitoneal (ip) injections at 10ml/g body weight of mice. For the in vitro Ames/Salmonella test, the compounds were dissolved in DMSO.
Collection and preparation of MEP. MEP were collected on a 0.5-mM quartz fiber filter from a 50-cm3Yamaha two-stroke engine using 95% octane
unleaded gasoline. The sampling apparatus consisted of, in sequence, a 40-(long) 3 2.2-cm (diameter) stainless still dilution tube, a filter holder, and a vacuum pump. The engine was running at 150 rpm on an empty load, and the pump was set at a flow rate of 20 l/min to collect particles for 1 h, four times daily. The filter with particulate matter was left to dry and repeatedly extracted with methanol, four times under sonication. The washed particles and organic com-ponents absorbed on the filter were pooled, and the methanol was removed in a vacuum evaporator. The final residues, the MEP and methanol extract, were collected and kept desiccated at20C. Approximately 32.7mg of final residue
(MEP) could be derived from 1 l of motorcycle exhaust. In our extraction, metha-nol was used to wash and bring down the particles from the filter. After removing the methanol, the final MEP residue should contain the chemicals as well as the particles absorbed on the filter. MEP were then dissolved in DMSO or corn oil before being added to the culture medium or intratracheally instilled solution.
Experimental animals. For the Ames test, Wistar rats (200 g) were used for preparation of the liver microsomal (S9) fraction. For the micronucleus test, male ICR mice, aged 8 to 9 weeks and weighing 30–40 g, were employed. All animals were purchased from the Animal Center of the College of Medicine, National Taiwan University. The animals were allowed at least a 1-week acclimation period in their animal quarters with air conditioning and a 12-h light/dark cycle. All animal treatments were approved by the Institution Animal Care and Use Committee (IACUC) of the College of Medicine, National Taiwan University, which follows the Animal Welfare Protection Act of the Department of Agriculture, Executive Yuan, Taiwan.
S9 fraction preparation. Rat liver S9 used for metabolic activation was prepared as described previously (Maron and Ames, 1983; Matsuoka et al., 1979). To obtain the liver microsomal fraction, each of the Wistar
rats was injected ip with 3-methylcholanthrene (30 mg/kg body weight) every day, and 4 days later the rats were killed by cervical dislocation. The livers were homogenated, diluted 1:4 with 0.15 M KCl, and centrifuged for 10 min at 9000 3 g. The supernatant was pulled and diluted (giving a protein concentration of 30 mg/ml), frozen in small aliquots, and stored at70 to 80C until use.
The final preparation of the metabolizing system (S9 mixture) was made in accordance with the protocol of Ames et al. (1975). The composition and final concentrations of the S9 mix used for the CHO-K1 cell chromosome aberration test were as follows: glucose-6-phosphate, 4.4 mM; NADP, 0.84 mM; KCl, 30 mM; NaHCO3,0.032%; and S9 fraction, 10%.
Ames Salmonella/microsome test. The method we used followed the recommendations of Maron and Ames (1983) and Organization for Economic Cooperation and Development (OECD) guidelines (1997). The Salmonella typhimurium bacteria and histidine auxotrophic strains TA98, TA100, and TA102 were obtained from MOLTOX (Molecular Toxicology, Annapolis, MD) and grown for 14 h at 35 6 2C with continuous shaking. Bacteria were grown to a density of 1 to 2 3 109cells/ml with OD600 absorbance of 0.2–0.3. Top agar, containing 2 ml of heated agar, 0.1 ml of test chemical, 0.1 ml of bacteria, and 0.5 ml of S9 solution, was mixed up and added to three different minimal glucose agar plates. All plates were incubated at 37C for 48 h, and the number of bacteria colonies was determined. The entire experiment was repli-cated again on a different day with a total of six plates for each concentration of MEP with and without S9. S9 liver cell extracts contain enzymes that may activate the potential mutagen. Each tester strain was routinely checked to con-firm its features for optimal response to known mutagenic chemicals as follows: 4-NQO (0.5mg/plate), mitomycin c (0.5 mg/plate), and 2AA (5 mg/plate). BaP (10mM) and STZ (0.5 mM) were used as ROS-dependent positive controls with and without S9, respectively. A test compound was judged to be mutagenic in the plate test if it produced, in at least one concentration and one strain, a response equal to twice (or more) of the control incidence with a dose-response relation-ship considered to be positive (De Serres and Shelby, 1979; Suter et al., 2002). The only exception was strain TA102, which has a relatively high spontaneous revertant number, where an increase by a factor of 1.5 above the control level was taken as an indication of a mutagenic effect.
Chromosomal aberrations. Chinese hamster ovary epithelial cells (CHO-K1, ATCC: CCL-61) were plated into 6-cm dishes at 5 3 105cells/plate for the
24-h treatment group. After overnight incubation, cells were treated with DMSO (solvent), mitomycin c (1mg/ml), BaP pyrene (5 mg/ml), and various concentra-tions of MEP for 3 h with or without S9. Then, 3 h after the end of the treatment time, 0.1mg/ml colcemid was administered, and metaphase chromosomes were prepared as described (Tsutsui et al., 1983). After trypsinization, cells were treated with 0.9% sodium citrate at 37C for 10 min, fixed in Carnoy’s solution
(methanol:acetic acid, 3:1), and spread on glass slides by the air-drying method. Specimens were stained with a 3% Giemsa solution in 0.07 M phosphate buffer (pH 6.8) for 30 min. For determination of both chromosome aberrations, 100 metaphases per experimental group were scored. Structural chromosome aberrations observed in each experimental group were classified into seven types as follows: chromosome-type gap (G), chromosome-type break (B), chromo-some-type ring (R), chromochromo-some-type dicentric (D), chromatid-type gap (g), chromatid-type break (b), and chromatid-type exchange (e). Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, it was recorded as a break.
Micronucleus assay. The micronucleus assay from peripheral blood cells was performed as previously described (Hayashi et al., 1990). The number of micronucleated cells was counted in 1000 reticulocytes (RETs) per animal. Slides were analyzed using a fluorescent microscope with a combination of a blue excitation (e.g., 488 nm) and a yellow-to-orange barrier filter (e.g., 515 nm long pass), with a 3100 objective for RETs.
Analysis of ROS production by flow cytometry. Intracellular ROS genera-tion was measured by a flow cytometer with an oxidagenera-tion-sensitive DCFH-DA fluoroprobe (Rothe and Valet, 1990). DCFH-DCFH-DA (20,70-dichlorofluorescin diacetate) is a nonfluorescent compound that is freely taken up into cells and
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hydrolyzed by esterase by removing the DA group. This nonfluorescent molecule (DCFH) is then oxidized to fluorescent dichlorofluorescin (DCF) by the action of cellular oxidants. When DCFH-DA was deacetylated and oxidized to form the fluorescent compound, fluorescence intensity was increased in a concentration-dependent manner. We stained 2 3 106 CHO-K1 cells with
20mg/ml DCFH-DA for 30 min at 37C in the dark. Cells were then collected
after PBS washing for fluorescence measurement. The level of intracellular ROS was determined with a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. For each treatment, 10,000 cells were counted and the experiment was performed in triplicate.
Statistical analysis. Data are expressed as the mean 6 SEM for the number of experiments indicated. Statistical analysis of the data was performed by Student’s t-test, and p 5 0.05 was considered significantly different. The values of IC50were calculated and obtained from five regression lines; each regression
line was constructed of at least five points. The values of inhibition of these points ranged from 20 to 80%.
RESULTS
Mutagenicity of MEP
The mutagenicity of MEP was examined by the Ames method
(Maron and Ames, 1983). The assay was carried out in vitro using
three histidine-requiring strains of Salmonella typhimurium
(TA98, TA100, and TA102) with and without a
metabolic-activating enzyme (S9). Each strain of Salmonella was treated
with MEP at 1, 10, 100, 1000, and 2000
mg/plate, respectively.
Our data showed that MEP did not increase colony formation in
strains TA98, TA100, and TA102 without the S9 mix (Table 1) at
concentrations of up to 2 mg/plate. On the other hand, in the
presence of S9, the number of revertants in MEP-treated plates
was concentration-dependently increased in TA98, TA100, and
TA102 strains (Table 1). Pretreatment with antioxidants,
includ-ing
a-tocopherol (3.5 mM/plate), ascorbate (100 mM/plate), NAC
(1 mM/plate), and catalase (1000 U/plate), showed different
potencies, but all attenuated the colony formation induced by
MEP (1000
mg/plate) in TA98, TA100, and TA102 strains with
the S9 mix (Table 2). In particular,
a-tocopherol significantly
exerted attenuation of MEP-induced colony formation, even up
to 30–40% inhibition of these testing strains. Two well-known,
ROS-dependent mutagens, STZ and BaP, were used as positive
controls (Table 1). The effects of antioxidants on these positive
compounds have been widely reported. STZ can induce
ROS-dependent mutagenesis without metabolic activation
(Bedoya et al., 1996; Nukatsuka et al., 1988; Takasu et al.,
1991); BaP is a promutagen (Kim and Wells, 1996; Wells
et al., 1997; Winn and Wells, 1997).
TABLE 1
Revertants in Three Strains of Salmonella typhimurium Treated with Different Concentrations of MEP in the Absence and Presence
of a Metabolic-Activating Enzyme (S9)
Without S9
His1/plate (S9)
MEP (mg/plate)
Strain Negative control Positive control STZ (0.5 mM) 1 10 100 1000 2000
TA98 20 6 2 251 6 34a 98 6 10a 20 6 1 21 6 2 24 6 2 31 6 6 33 6 5 TA100 79 6 1 852 6 59a 3989 6 252a 76 6 5 86 6 0 101 6 8 79 6 7 81 6 17 TA102 150 6 7 1264 6 96a 805 6 60a 167 6 5 179 6 16 231 6 6 175 6 1 172 6 5 With S9 His1/plate (1S9) MEP (mg/plate)
Strain Negative control Positive control BaP (10mM) 1 10 100 1000 2000
TA98 28 6 2 3802 6 129a 684 6 50a 37 6 6 54 6 13 250 6 53b 387 6 42a 407 6 73a
TA100 92 6 6 3473 6 385a 976 6 42a 92 6 1 107 6 13 287 6 13c 291 6 52b 459 6 105b
TA102 250 6 12 1699 6 207a 857 6 61a 248 6 14 283 6 22 310 6 22 478 6 89c 637 6 68b
Note. Values are presented as the mean 6 SE (n 6). The negative control consisted of 2 ml DMSO/plate. The positive ontrol in the S9 plate consisted of TA98, 2mg/plate 4-nitro-o-phenylenediamine; TA100 and TA102, 0.5 mg/plate mitomycin C. The positive control in the 1S9 plate consisted of 5 mg/plate 2-aminoanthrene. The ROS-dependent positive controls were streptozotocin (STZ, 0.5 mM without S9) and benzo(a)pyrene (BaP, 10mM with S9).
a
p5 0.005 versus negative control.
b
p5 0.01 versus DMSO.
c
p5 0.05 versus DMSO.
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Induction of Chromosome Aberrations by MEP in CHO Cells
The in vitro effect of MEP on chromosomes was studied with
CHO-K1 cells, and the results of CHO-K1 cell chromosome
analysis are given in Table 3. The incidence of CHO-K1 cells
with structural chromosomal aberrations significantly increased
in cells treated with BaP (5
mg/ml; 13.1 6 2.2%, p 5 0.001;
Table 3) and mitomycin c (1
mg/ml; 9.8 6 2.2%, p 5 0.001;
Table 3) with and without S9, respectively. Both in the absence
and presence of S9, MEP (0.5, 5, and 50
mg/ml)
concentration-dependently increased structural chromosomal aberrations at 3 h
of treatment compared to the solvent control (0.1% DMSO,
Table 3). We also found that MEP- (5 and 50
mg/ml) induced
chromosomal aberration was potentiated up to 2-fold by the
addition of S9 (Table 3). Pretreatment with the antioxidants
a-tocopherol (3.5 mM), ascorbate (100 mM), catalase
(1000 U/ml), and NAC (1 mM) yielded different potencies,
but all significantly attenuated the increase in aberrant cells
induced by MEP (50
mg/ml) in the S9-treated group, though
not in the group without S9 (Table 4).
In Vivo Induction of Micronuclei by MEP
Results of the micronucleus assay of ICR mice treated with
different doses of MEP and the positive control are shown in
Table 5. Mice injected (ip) with mitomycin C (1 mg/kg body
weight) showed a significant increase in the frequency of
RETs at 24 (12.2 6 1.4) and 48 h (36.5 6 1.6) of treatment
compared to control (1.2 6 0.2 and 1.4 6 0.5, respectively).
Intratracheal instillation with various doses of MEP (160, 200,
and 240 mg/kg body weight) in mice significantly increased
the formation of RETs at both 24 and 48 h, with a return to
basal level at 72 h after treatment (Table 5). Cotreatment
with antioxidants
a-tocopherol (75 mg/kg body weight) and
ascorbate (1 g/kg body weight) inhibited the MEP- (200 mg/kg
body weight) induced increase of RETs formation only at 48 h
(Table 5).
MEP Induced ROS Formation in CHO-K1 Cells
ROS generation induced by MEP was examined using a
DCFH-DA fluorescence probe. The fluorescence intensity of
oxidative DCF in reaction with various concentrations of MEP
was determined in CHO-K1 cells. We found that MEP increased
the fluorescence intensity as ROS level in a
concentration-dependent manner (Fig. 1). MEP- (50
mg/ml) induced
fluo-rescence intensity was potentiated up to 2-fold compared to
control.
DISCUSSION
Large numbers of people in the world continue to be exposed
to pollutant mixtures containing known or suspected
carcino-gens. Epidemiologic studies over the last 50 years suggest rather
consistently that general ambient air pollution, mainly due to the
incomplete combustion of fossil fuels, may be responsible for
increased rates of lung cancer (Cohen and Pope, 1995). These
substances are present as components of complex mixtures,
which may include carbon-based particles that absorb organic
compounds, oxidants such as ozone, and sulfuric acid in aerosol
form (Cass et al., 1984; Dockery et al., 1993). In Taiwan, the
combustion of fossil fuels for power generation and
transporta-tion (especially motorcycles), also the primary source of many
organic and inorganic compounds, oxidants, and acids,
contri-butes heavily to particulate air pollution. In this study, we found
MEP extract was mutagenic in both in vitro and in vivo
experi-mental assays.
TABLE 2
The Effect of Antioxidants on the Revertants in Salmonella typhimurium TA98, TA100, and TA102 Treated with MEP in the Presence
of Metabolic-Activating Enzymes
His1/plate (1S9)
Treatment group TA98 TA100 TA102
Negative control 22 6 5 81 6 2 249 6 15
BaP (10mM) 684 6 50a 976 6 42a 857 6 61a
MEP (1000mg/ml) 397 6 35a 317 6 64a 444 6 67a
1 1 mM NAC 285 6 47a,b(28.2) 245 6 22a(22.7) 329 6 31a(25.9)
1 100mM ascorbate 256 6 55a,b(35.5) 221 6 23a(30.3) 289 6 39a,b(34.9)
1 3.5mM a-tocopherol 244 6 61a,b(38.6) 164 6 12a,b(48.3) 293 6 25a,b(34.0)
1 1000 U catalase 280 6 63a(29.5) 239 6 14a(24.6) 339 6 47a(23.6)
Note. Values are presented as the mean 6 SE (n 6). The negative control consisted of 2 ml DMSO/plate. The ROS-dependent positive control was 10 mM BaP. The percentage of inhibition (% In) 5 100 [ (number of revertants per plate in the presence of antioxidant)/(number of revertants per plate in the absence of antioxidant) 3 100].
a
p5 0.05 versus negative control.
b
p5 0.05 versus MEP (1000mg/ml).
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Over the years, the Ames test has been used worldwide as an
initial screening tool to determine the mutagenic potential of
new chemicals and drugs, because there is a high predictive
value for rodent carcinogenicity when a mutagenic response
is obtained (McCann et al., 1975; Sugimura et al., 1976; Zeiger
et al., 1990). Our data showed that MEP contains chemicals that
are mutagenic to Salmonella strains TA98, TA100, and TA102
upon metabolic activation. These results indicated that the active
forms of MEP are their metabolites. Some carcinogenic
chemi-cals, such as aromatic amines and PAHs, are biologically
inac-tive unless they are metabolized to their acinac-tive forms. Several
mutagens, such as PAHs, nitrofluorene, and various aromatic
nitroso derivatives of amine carcinogens (Isono and Yourno,
1974), have been found in DEP (Li et al., 2000; Schuetzle
et al., 1981) and MEP (Ueng et al., 2000). It is interesting to
note that while MEP (this study) and gasoline engine exhaust
particulate extracts (Crebelli et al., 1991) showed a
promuta-genic effect, scooter exhaust contained chemicals that were
directly acting mutagens (Zhou and Ye, 1997).
We do not know, at this point, the chemical compounds that are
responsible for the mutagenicity seen in our study. Many reports
in recent years have focused on the toxic effect of the extracts of
DEP. They have been shown to generate intracellular ROS,
lead-ing to a variety of cellular responses (Hiura et al., 2000; Ma and
Ma, 2002; Yang et al., 2001). The organic component of DEP has
also been shown to generate ROS that produce
8-hydroxydeox-yguanosine (8-OHdG) in cell culture (Tsurudome et al., 1999).
Our data showed the mutagenic effect on TA102, providing proof
of the involvement of ROS. Strain TA102 was developed
containing AT base pairs at the hisG428 mutant site, and the
mutation is reverted by mutagens that cause oxidative damage
(Myriam et al., 2000; Niittykoski et al., 1995). The involvement
of ROS in MEP-induced mutation was further evidenced by the
alleviation of mutation with the use of antioxidants including
a-tocopherol, ascorbate catalase, and NAC. Alpha-tocopherol
and ascorbate have similar effects in preventing MEP-induced
increases of revertants in TA98 (38.6% inhibition with S9) and
TA100 (48.3% inhibition with S9), as did ascorbate in TA102
(34.9% inhibition with S9). Although the antioxidants can
attenuate the mutagenic effect, however, the inhibition is not
complete. This might suggest that there are other chemicals
that are involved in the mutagenic effect but independent of ROS.
DNA breaks and the formation of clastogens could be detected
by in vitro CHO cells in the chromosome aberration test and
in vivo micronucleus assay. In the chromosome aberration test,
MEP concentration-dependently increased the number of
abnor-mal structural chromosomes in the absence of metabolic
activa-tion (10.0 6 2.0, p 5 0.001 at 50
mg/ml) in CHO cells. However,
TABLE 3
Chromosome Aberrations of CHO-K1 Cells Treated with MEP
Without S9
Number of aberrations/100 cells
Treatment group Aberrant cells (%) G B D R G b e
Solvent control (DMSO) 3.1 6 0.1 1.6 6 0.3 0 0 0 0 1.4 6 0.3 0
MMC (1mg/ml) 9.8 6 2.2a 0 5.3 6 0.3 0.3 6 0.3 0 0.2 6 0.2 3.4 6 1.8 0 MEP (mg/ml) 0.5 6.0 6 2.2 2.7 6 1.5 0.5 6 0.3 0.2 6 0.2 0.2 6 0.2 0.8 6 0.8 1.5 6 1.3 0.2 6 0.2 5 6.2 6 1.9b 1.3 6 0.9 0.2 6 0.2 0.2 6 0.2 0 3.7 6 1.9 0.8 6 0.4 0 50 10.0 6 2.0a 1.3 6 1.3 0.7 6 0.3 1.0 6 0.5 2.0 6 1.0 2.3 6 2.3 2.0 6 0.5 0 With S9
Number of aberrations/100 cells
Treatment group Aberrant cells (%) G B D R g b e
Solvent control (DMSO) 3.4 6 1.0 1.6 6 0.6 0 0 0.1 6 0.1 1.6 6 0.8 0.2 6 0.2 0 BaP (5mg/ml) 13.1 6 2.2a 3.8 6 1.3 0.5 6 0.2 0.1 6 0.1 0.8 6 0.4 4.0 6 1.8 0.5 6 0.3 1.0 6 0.2 MEP (mg/ml)
0.5 7.0 6 2.9c 1.5 6 0.7 1.2 6 1.2 0.2 6 0.2 0.5 6 0.5 3.0 6 1.5 0.3 6 0.3 0.3 6 0.3
5 13.3 6 2.0a 3.2 6 1.6 1.3 6 1.3 1.7 6 1.4 0.8 6 0.8 3.8 6 2.0 1.0 6 0.6 1.5 6 1.5
50 19.8 6 3.5a 3.8 6 2.0 3.0 6 2.5 2.0 6 2.0 2.0 6 1.0 5.7 6 3.0 1.0 6 0.6 2.3 6 1.9
Note. Values are presented as the mean 6 SE (n 5 3). MEP was dissolved in DMSO, and the solvent control did not exceed 0.1%. Mitomycin C (MMC) was the positive control in the medium without S9, and BaP was the positive control in the medium with S9. G, chromosome gap; B, chromosome break; D, dicentric; R, ring; g, chromatid gap; b, chromatid break; e, exchange.
a p5 0.001 versus DMSO. b p5 0.01. c p5 0.05.
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the addition of S9 greatly enhanced the effect of the
MEP-induced aberrations (19.8 6 3.5, p 5 0.001 at 50
mg/ml;
Table 3). These data suggested that MEP contains chemicals
that can induce chromosome aberrations both with and without
metabolic activation in CHO cells. Previously, Kuo et al. (1998)
also showed that MEP induced genotoxicity in a ROS-dependent
manner by using cell-junction communication as an
experimen-tal model. Pretreatment with antioxidants selectively attenuated
MEP-induced aberration of cells in the S9-treated group
(Table 4). This result indicated that MEP might contain
chemicals that exert a mutagenic effect independent of ROS
and bioactivation in CHO cells. The mechanistic detail still
requires further investigation.
The clastogenic effects of MEP were also detected with the
in vivo micronucleus test in mice peripheral red blood cells.
Micronuclei scoring is based on the observation that displaced
chromatin, resulting from chromosome loss or breakage, may
fail to be incorporated into daughter nuclei as a cell divides. The
resulting micronucleus is found in the cytoplasm. During
erythropoiesis, an erythroblast expels its main nucleus to
become a reticulocyte (RET), while the micronuclei remain
in the cytoplasm. The newly formed RET is then released from
the bone marrow into the circulating bloodstream, where it
develops into a normochromatic erythrocyte (NCE). Elevations
in the frequency of micronuclei are indicative of genotoxic
activity (Hayashi et al., 2000). MEP dose-dependently increased
the micronucleus formation at 24 and 48 h after treatment, and
this effect was slightly reversed to basal level at 72 h. Obviously,
the in vivo study showed that the effect is reversible or can be
repaired after 72 h. Intraperitoneal injection with ascorbate
(1 g/kg in water) and
a-tocopherol (75 mg/kg in corn oil),
30 min before MEP (200 mg/kg) treatment, greatly reduced
the micronucleus formation at 48 h in the treated group.
Micronuclei can arise from acetric fragments induced by a
substance causing chromosomal breakage (clastogens) as well as
aneuploidy (aneugens). However, with the conventional
micro-nucleus test, it is impossible to distinguish between these two
events. We cannot rule out the possibility that MEP might be an
aneugen in this study. The reason the antioxidants showed the
most obvious protecting effect at 48 h might be because the
micronucleus showed the most dramatic change at 48 h. It is
also possible that the antioxidants only have a partial effect, as
seen with the Ames and chromosome tests. The basal
micro-nucleus observed at all time points, which was not inhibited
by the antioxidants, might be caused by some chemicals in
MEP that damage the chromosome independently of ROS
formation.
There are some possible sources that can account for the
origination of ROS generation. They include the following:
1. Among the 4100 chemicals that are present in MEP (Chan
et al., 1993; Zhou and Ye, 1997), such as PAH, nitroderivatives
of PAH, and oxygenated PAH derivatives (ketones, quinines,
and diones) are candidate chemicals that may contribute to ROS
generation (Alsberg et al., 1985; Anderson et al., 1998; Li et al.,
1996; Schuetzle et al., 1981).
2. Quinone
is
reduced
to
semiquinone
radicals
by
microsomally localized cytochrome P450 reductases (Chesis
TABLE 4
Chromosome Aberrations of MEP-Treated CHO-K1 Cells in the
Presence of Antioxidants
Aberrant cells (%) Treatment group 3 h without S9 3 h with S9 Solvent control (DMSO) 3.3 6 0.3 4.4 6 0.7 Positive control 10.7 6 0.9a 16.3 6 1.2a MEP (50mg/ml) 10.0 6 2.0a 20.3 6 1.5a
1a-tocopherol (3.5 mM) 7.0 6 1a 8.5 6 0.5a,b 1 ascorbate (100mM) 9.0 6 0a 9.0 6 1.5a,b 1 catalase (1000 U/ml) 8.0 6 1a 14.3 6 1.7a,b 1 NAC (1 mM) 9.5 6 0.5a 10.7 6 2.0a,b Note. Values were presented as the mean 6 SE (n 5 3). MEP was dissolved in DMSO, and the solvent control did not exceed 0.1%. MMC was the positive control in the medium without S9, and BaP was the positive control in the medium with S9. G, chromosome gap; B, chromosome break; D, dicentric; R, ring; g, chromatid gap; b, chromatid break; e, exchange.
a
p5 0.05 versus control.
b
p5 0.05 versus 50mg/ml MEP.
TABLE 5
Micronucleus Formation in Peripheral Blood Cells of Mice
Treated with MEP and the Effects of Antioxidants In Vivo
Time (h) Treatment group 24 48 72 Control 1.2 6 0.2 1.4 6 0.5 1.0 6 0.2 MMC (1 mg/kg) 12.2 6 1.4a 36.5 6 1.6a 9.3 6 1.2a MEP (160 mg/kg) 1.8 6 0.5 2.7 6 0.8b 2.0 6 0 MEP (200 mg/kg) 2.8 6 0.6b 4.8 6 0.5a 2.2 6 0.7 MEP (240 mg/kg) 5.0 6 0.3a 5.9 6 0.8a 2.4 6 0.8 a
p5 0.005 versus the control.
bp5 0.01 versus the control. cp5 0.05 versus the control.
Time (h) Treatment group 24 48 72 Control 1.2 6 0.2 1.4 6 0.5 1.0 6 0.2 MEP (200 mg/kg) 2.7 6 0.5d 5.6 6 0.4d 2.1 6 0.5 MEP (200 mg/kg) 1 ascorbate (1 g/kg) 2.0 6 0.8 2.0 6 0.2e 1.5 6 0.5 MEP (200 mg/kg) 1 a-tocopherol (75 mg/kg) 3.1 6 1.5d 2.6 6 0.6d,e 1.7 6 0.1
Note. Values are presented as the mean 6 SE (n 3). The control consisted of 1% Tween 80 in DMSO:PBS (1:9). Ascorbate (1 g/kg in water) anda-tocopherol (75 mg/kg in corn oil) were used for pretreatment by an ip injection for 30 min.
a
p5 0.05 versus the control.
b
p5 0.05 versus MEP (200 mg/kg).
at Taipei Medical University Lib. on March 25, 2011
toxsci.oxfordjournals.org
et al., 1984). These semiquinones group together, thereby
initiating a futile redox cycle (Chesis et al., 1984; Monks
et al., 1992).
3. PAH may contribute to further ROS production during
cytochrome P4501A1-dependent transformation.
4. In addition to the contribution of organic chemical
com-pounds, that of metal ions, such as Fe
21, may relate to ROS
generation.
5. Mitochondria have been implicated in the induction of
apoptosis by a growing list of pro-oxidative chemicals, including
redox-cycling quinines and PAH (Segura-Aguilar et al., 1998;
Yamaguchi et al., 1996; Zoratti and Szabo, 1995).
ROS generation has been linked to the mutagenic effects of
DEP chemicals (Ichinose et al., 1997). Although these
MEP-associated chemicals need to be identified, we know that MEP
contain PAHs, which have potentially harmful effects on
humans. The PAH components can react with nitrous oxides
in the air, such as NO
2and NO, and form nitro-PAH. Recent
studies demonstrated that nitro- and dinitropyrene play a minor
role in air particulate mutagenicity (Crebelli et al., 1991).
Our study provides evidence of the potentially genotoxic
effects of MEP extract both in vitro and in vivo. Although
bioac-tivation and ROS generation were shown to play major roles in
the genotoxic effects that we observed, MEP also contains
directly acting chemicals and chemicals that act independently
of ROS. The impact of this toxic effect of MEP on the health
of people in Taiwan is worthy of emphasis and further
investigation.
ACKNOWLEDGMENTS
This study was supported in part by a grant (NSC-91-2320-B038-039) from the National Science Council, Taiwan.
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