Genotoxicity of low dose N-nitroso propoxur to human gastric cells

Article abstract of DOI:10.1016/j.fct.2008.01.014

Propoxur is among the most popular insect control agents in subtropical countries such as Taiwan. As a member of the N-methylcarbamate insecticide group, propoxur is notorious for its potential for conversion into highly genotoxic N-nitroso derivatives. D

Full text of DOI:10.1016/j.fct.2008.01.014

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Food and Chemical Toxicology 46 (2008) 1619–1626
www.elsevier.com/locate/foodchemtox
Genotoxicity of low dose N-nitroso propoxur to human gastric cells
H.H. Kuo, S.S. Shyu, T.C. Wang *
Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan
Received 22 December 2006; accepted 3 January 2008
Abstract
Propoxur is among the most popular insect control agents in subtropical countries such as Taiwan. As a member of the
N-methylcarbamate insecticide group, propoxur is notorious for its potential for conversion into highly genotoxic N-nitroso derivatives.
Due to the fact that the stomach has been identified as the major target for N-nitroso N-methylcarbamates, this investigation used a
human gastric cell line, SC-M1, in order to obtain results pertinent to the authentic adverse effects of this compound on human health.
This report reveals that at dose levels inhibiting 610% cell growth, a 2-h pulsed treatment of N-nitroso propoxur induced significant
amounts of DNA damage. Most of the damaged DNA was repaired within 24 h after treatment removal, such that an outcome with
a significant induction of chromosomal aberrations was not observed. Gene mutations and anchorage independence, on the other hand,
were significantly induced by this same treatment. In conclusion, exposure to low doses of N-nitroso propoxur is not cytotoxic nor clas-
togenic, nevertheless, has the potential to increase genetic instability and, possibly as a result, to enhance the malignant potential of trea-
ted cells. We suggest that although the damaged DNA was repaired during the transient G2/M arrest period, it was probably not done in
an appropriate way which would preserve the original genetic stability.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Carbamate insecticide; Human gastric cell; Malignant potential
1. Introduction
we demonstrated their genotoxicities in Chinese hamster
V79 lung cells and preneoplastic transformation in primary
rat tracheal epithelial cells (Wang et al., 1998a,b). Tumor-
igenesis experiments in a rodent model indicated that
N-nitroso N-methylcarbamate insecticides mainly target
the forestomach (Lijinsky, 1992). In view of this, the results
would be more relevant if cells originating from the target
tissue could be used for the investigation. Therefore a
human gastric cell line, SC-M1, isolated from a local
patient in Taiwan was employed in the present study to
explore the adverse effects induced by N-nitroso propoxur,
an N-nitroso derivative of a major N-methylcarbamate
insecticide used in Taiwan.
N-Methylcarbamate esters are inhibitors of enzyme cho-
linesterase in insects. They are insecticides of economic
importance, manufactured in very large quantities, and
widely used for controlling agricultural and household
insect pests. Due to its lower acute toxicity to mammals,
N-methylcarbamate is gaining popularity over organo-
phosphate insecticides, another cholinesterase inhibitor,
especially for the control of insects in household areas,
where contamination of the human environment can easily
occur. N-Methylcarbamate insecticides are converted to N-
nitroso metabolites by N-nitrosation, under conditions of
stomach physiology. N-Nitroso methylcarbamates are not
cholinesterase inhibitors and are less toxic to mammals
than the parent N-methylcarbamate insecticides. They are
nonetheless quite carcinogenic and mutagenic. Previously,
2. Materials and methods
2.1. Cell culture
SC-M1, a gastric adenocarcinoma cell line, kindly provided by
Dr. C-W Chi of Veterans General Hospital, Taipei, Taiwan, was previ-
ously established from fresh tumor tissue removed from a 72-year-old
*
Corresponding author. Tel.: +886 2 27899540; fax: +886 2 27858059.
E-mail address: tcwang@sinica.edu.tw (T.C. Wang).
0278-6915/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.01.014

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H.H. Kuo et al. / Food and Chemical Toxicology 46 (2008) 1619–1626
male patient at Triservice General Hospital, Taiwan (Tzeng et al., 1991). It
was cytogenetically analyzed at the 200th passage, revealing a near-trip-
loid modal karyotype in the cell line.
NaOH; 1% N-lauroylsarcosine, 1% Triton X-100, and 10% dimethyl
sulfoxide were added immediately before use) at 4 °C overnight. Slides
were then denatured in an electrophoresis tank containing 0.3 M NaOH
(pH 13.4) and 1 mM sodium EDTA for 20 min. Electrophoresis was
carried out at 25 V, 300 mA for 30 min. Slides were washed, blotted, and
then transferred to 0.4 M Tris–HCl at pH 7.5 before being stained with
YOYO-1 (Molecular Probes; 50 ll of a 20 lM solution prepared in glyc-
erol/Na2EDTA 0.4 M at pH 7.4 (50/50), containing 0.1% 8-hydroxy-
quinoline). The COMET image of 100 cells per treatment was recorded
with a fluorescence microscope (shortwave pass filter 450–490 nm, chro-
matic beam splitter 510 nm, and longwave pass filter 520 nm) and a digital
camera (DCS-420; Kodak, Rochester, NY, USA). Migration of DNA
from the nucleus in each cell was expressed by the parameter of the tail
moment and measured with Comet Assay III software (available from
www.perceptive.co.uk).
2.2. N-Nitrosation of propoxur insecticides
Preparation of N-nitroso derivatives of propoxur (O-isopropoxy-
phenyl methyl carbamate, CAS 114-26-1) followed the method described
by Blevins et al. (1977a,b). Propoxur (98.7%, Chem Service, West Chester,
PA) was dissolved in glacial acetic acid (Merck, Darmstadt, Germany),
cooled in ice, and added with sodium nitrite (Merck). The solution was
kept cold for 1 h and then at room temperature for another hour before
being added with ether and ddH2O. The yellow ether layer was separated
off and dried using anhydrous sodium sulfate. The residual N-nitroso
propoxur was confirmed by checking its maximum intensity at 420, 400,
and 385 nm on a Hitachi U2000 spectrophotometer.
2.7. Flow cytometry for cH2AX
2.3. Measurement of cell kinetics
The cell preparation for cH2AX staining followed that described by
Olive (2004). Treated cells were fixed in 70% ethanol overnight, and then
were permeabilized and rehydrated in a cold TST solution (0.25% Triton
X-100 and 4% FBS in PBS) for 15 min. Samples were centrifuged, and the
pellet was resuspended in 100 ll mouse monoclonal anti-cH2AX (Upstate
Biotechnology) diluted 1:500 in TST at room temperature for 3 h. At the
end of incubation, cells were washed by repeatedly centrifuging and
resuspending them in 3–4 ml PBST (0.05% Tween 20 in PBS). The cell
pellet then was resuspended in 50 ll anti-mouse-IgG-FITC (Jackson
Immuno Research) for 30 min at room temperature before being washed
and centrifuged in PBST. The cell pellet was further resuspended in a PI
solution (containing 0.2 ml of 20 lg/ml PI, 0.2 ml of 5% Triton X-100,
0.1 ml of 2 mg/ml RNase A, and 0.5 ml PBS).
Cells were cultivated in relative low densities as described previously
(Kalweit et al., 1999), which allowed the colony size to be measured.
Briefly, 1000 cells were plated into each grid of a 60-mm dish. Numbers of
individual cells in each colony were counted 24, 44, 48, and 68 h after the
initial plating using a dissecting microscope. Based on the distribution of
the colony sizes, a proliferation index was derived according to the fol-
lowing formula: Proliferation index = [(c1 Â 1) + (c2 Â 2) + (c4 Â 3) +
(c8 Â 4) + (c16 Â 5)]/n, where c1, c2, c4, c8, and c16 each indicates the
number of single-cell, 2-cell, 4-cell, 8-cell, and 16-cell colonies, respec-
tively, and n is the total number of cell colonies.
2.4. Measurement of growth inhibition
2.8. Induction of chromosome aberrations
The method using UV absorption as an approximation for the cell
number proposed by Chang (1991) was used to measure the growth
inhibition induced by N-nitroso propoxur. Briefly, 3 Â 105 SC-M1 cells
were plated onto each 60-mm Petri dish for overnight. After treatment
with N-nitroso propoxur in a series of different doses for 2 h, cells were
trypsinized and collected in a centrifuge tube, washed twice with cold
calcium magnesium-free phosphate-buffered saline (CMF-PBS), extracted
twice with cold methanol: acetic acid (3: 1) to remove free nucleosides and
nucleotides, and dissolved at 37 °C in a 0.2 N NaOH solution at 106 cells/
ml for 24 h. The absorption at 260 nm for each cell solution was deter-
mined using a Hitachi U2000 spectrophotometer. The percent growth
inhibition of each treatment was estimated by dividing the absorbance of
the treatment by that of the control.
Cultures were set up by plating 5 Â 105 cells into each 60-mm Petri dish
and then incubating them overnight. Cells were treated with N-nitroso
propoxur for 2 h, then incubated for 24 or 48 h. Two hours prior to the
end of incubation, colcemid (0.2 lg/ml) was added to the culture. Mitotic
cells were harvested by trypsinization, and slides were prepared by an air-
dried technique. Slides were stained in a 3% Giemsa solution, coded, and
blindly scored using an Olympus New Vanox photomicroscope. At least
100 metaphases were randomly sampled for the assay of chromosome
aberrations.
2.9. HPRT gene mutations
SC-M1 cells (at 105) were grown in complete medium containing
100 lM hypoxanthine, 2 lM aminopterin, and 30 lM thymine (HAT
medium, Sigma) for 3 days to reduce the number of preexisting HPRT-
deficient cells. Cells (at 106/100-mm Petri dish) were plated and incubated
overnight. Cultures were exposed to 0, 2, and 4 lg/ml N-nitroso propoxur
for 2 h. At the end of treatment, the cultures were washed twice with PBS
and re-fed with fresh medium. Cells were maintained in their proliferative
growing state for an 8-day expression period before mutant selection.
Afterward, 2 Â 106 cells were divided among twenty 100-mm Petri dishes,
fed complete media containing 10 lg/ml 6-thioguanine (6TG, Sigma), and
incubated for 7 days. Plating efficiency was also determined at the same
time by plating 100 cells in each 60-mm Petri dish with complete medium.
The mutation frequency was determined as the number of 6TG-resistant
colonies induced per 106 surviving cells.
2.5. Apoptosis assay
After treatment removal and at the indicated times, 0.5 ml of
approximately 1 Â 106 cells/ml were supplemented with 10 ll Media
Bonding Reagent and 1.25 ll Annexin-V-FITC (Apoptosis Detection Kit,
CalbiochemÒ). The mixture was then incubated for 15 min at room tem-
perature in the dark and then centrifuged at 1000g for 5 min. The cell
pellet was re-suspended in 0.5 ml cold 1X binding buffer. Immediately
before analysis with a Coulter Epics XL-MCL flow cytometer, 10 ll PI
was added to the suspension. Experiments were performed in triplicate.
2.6. Single-cell alkaline electrophoresis (comet assay)
Fifty millilitre of a cell suspension containing 2 Â 105 cells in PBS and
250 ll of an agarose solution were thoroughly mixed, and 65 ll of the
mixture was pipetted onto the slide pre-coated with 1% normal-melting
point agarose. Seventy millilitre of 1% low-melting point agarose was
applied as the third layer of agarose. The slides were immersed in an ice-
cold lysis solution (2.5 M NaCl, 100 mM sodium ethylenediaminetetra-
acetic acid (EDTA), and 10 mM Tris, with the pH adjusted to 10.0 with
2.10. Soft-agar colonization
The soft agar assay followed procedures described previously by other
authors (Koi et al., 1989). Cells (104–105) with or without N-nitroso
propoxur treatment were suspended in 0.3% Difco bactoagar in normal
growth media containing 10% FBS and 0.1% bactopeptone. All

H.H. Kuo et al. / Food and Chemical Toxicology 46 (2008) 1619–1626
1621
experiments were performed three times. The suspended cells were plated
in 0.3% agar in 60-mm dishes above a layer of 0.6% agar containing
normal media with 10% FBS and 0.1% bactopeptone. Dishes were incu-
bated at 37 °C in 5% CO2 for 10–14 days and then scored for colony
growth.
SC-M1 cells in the logarithmic phase doubled their popula-
tion about every 24 h.
3.2. Growth inhibition
The dose–growth inhibition curve of N-nitroso propo-
xur-treated SC-M1 cells is shown in Fig. 2. A 2-h pulse
treatment at doses of 64 lg/ml showed growth inhibition
of <10%. As the dose increased to 8 lg/ml, inhibition
increased to about 20%. When treatments with N-nitroso
propoxur were raised to 16 and 32 lg/ml, there was still
about 50% growth observed. A J-shaped regression model
better fit the dose–response curve than a linear one, with an
r-value of 0.97 vs. 0.85, respectively. Determining whether
this implies a hormetic-like relationship [Calabrese, 2003
#5] in which the cell growth rate exceeds the control value
when the N-nitroso propoxur treatment dose becomes
more dilute is worthy of further investigation.
2.11. Cell cycle analysis
SC-M1 cells (at 3 Â 105) were plated in each 60-mm dish overnight
before being treated with N-nitroso propoxur (2 lg/ml) for 2 h. Treated
cells were then washed with 10 ml PBS twice and cultured for a series of
different durations after treatment removal. Upon the end of each post-
treatment culture, cells were trypsinized, fixed in 70% ethyl alcohol, and
centrifuged at 1000 rpm for 5 min. The cell pellets were stained in a PI
mixture containing 550 ll CMF-PBS, 200 ll of 5% Triton X-100, 200 ll of
20 lg/ml PI, and 50 ll of 2 lg/ml RNase A for 15 min in the dark at room
temperature. Stained cells were then passed through a 40-lm nylon mesh
and analyzed for cell cycle progression using a Coulter Epics XL-MCL
flow cytometer with excitation at 488 nm and emission at 610 nm.
3. Results
3.3. Apoptosis
3.1. Cell doubling time
Whether the growth inhibition caused by N-nitroso
propoxur treatment in SC-M1 cells was due to the induc-
tion of apoptosis was investigated using staining with
Annexin-V in conjunction with PI (Fig. 3). It has been
demonstrated that Annexin-V can bind to phosphatidylser-
ine, which makes it a very useful tool for detection of apop-
tosis in the early stages of the process (Darzynkiewicz et al.,
1997; van England et al., 1998). The frequency of SC-M1
cells positively stained for Annexin-V only, an indication
of apoptosis, was less than 1% in both the control and
N-nitroso propoxur-treated group up to 8 lg/ml. The posi-
tive control assay performed twice with 0.5 lM actinomy-
cin D showed approximate 21% apoptotic cells. A 24-h
The proliferation index of SC-M1 cells, which signifies
the rounds of cell duplication completed within an indi-
cated duration, was calculated by plating individual cells
for 24, 44, 48, and 72 h as previously described above
(Fig. 1). A regression equation was calculated from the log-
arithmically growing SC-M1 cells as: f(x) = 0.0425
x + 0.9781, where f(x) corresponds to the proliferation
index after the duration, x(h). According to this equation,
Fig. 1. Cytokinetics of human gastric SC-M1 cells. One thousand cells
were plated into each grid 60-mm dish. Numbers of individual cell in each
colony were counted 24, 44, 48 and 68 h after the initial plating using a
dissecting microscope. Based on the distribution of the colony sizes, a
proliferation index (PI) was derived according to the formula described in
the text.
Fig. 2. Growth inhibition of N-nitroso propoxur to SC-M1 cells. Number
of cells survived after treatment was approximated using UV absorption at
260 nm. Percent survival was calculated by dividing the absorbance of the
treatment with that of the concurrent control.

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H.H. Kuo et al. / Food and Chemical Toxicology 46 (2008) 1619–1626
Fig. 3. Flow cytometric analysis of cell death induced by N-nitroso propoxur in SC-M1 cells. Cells were stained for propium iodine and Annexin-V after a
2 h pulsed-treatment with N-nitroso propoxur. Upper row indicates the results of assay immediately after the treatment, while the bottom row is those
after a 24-h post-treatment incubation. Data shown in the figure were one example among three different experiments.
post-treatment incubation increased the frequency of
Annexin-V staining, however, not significantly. The overall
cell mortality, including that caused by apoptosis and
necrosis, was less than 2.5% with treatment using N-nitroso
propoxur at up to 4 lg/ml. This is consistent with the
results of the growth inhibition assay described previously
above. The portion of apoptotic cells which was positively
stained by Annexin-V and negatively with PI never
exceeded 4% of the total population in the treatment with
N-nitroso propoxur at 6 4 lg/ml. Therefore, the growth
inhibition caused by N-nitroso propoxur in our investiga-
tion was unlikely due to the induction of apoptosis.
3.4. DNA damage
N-nitroso propoxur-induced DNA damage was esti-
mated using assays for single-cell electrophoresis
(COMET) and the expression of phosphorylated histone
H2AX (Banath and Olive, 2003). The DNA migration
caused by N-nitroso propoxur treatment was measured
Fig. 4. COMET analysis of DNA damage induced by N-nitroso prop-
oxur. Data shown are results of two independent experiments on COMET
by the tail moment in the COMET assay (Fig. 4). In 2 inde-
pendent studies, treatments with 2 and 4 lg/ml N-nitroso
assay performed either immediately after a 2 h-treatment (open circle) or
propoxur greatly enhanced DNA migration. When an
after a 24-h post-treatment incubation (open rectangular). Every individ-
assay was performed immediately after treatment removal,
ual comet recorded are shown in the figure. The horizontal bar indicates
the tail moments induced were around 66–95 times as high
as the control treatment, respectively. The assay performed
24 h later, on the other hand, showed a great reduction in
the tail moment in the treatment groups, implying that sig-
nificant DNA repair had occurred during this period.
DNA damage was also analyzed using a flow cytometric
method. SC-M1 cells were stained for DNA and cH2AX
after N-nitroso propoxur treatment. A gate was drawn to
exclude >95% of the cells of the untreated population,
the medium in per 100 observations of each treatment.
and the fraction of cells within the gate indicated the spe-
cific level of cH2AX expression for each treatment. A sig-
nificant dose–response induction of DNA strand breaks,
mainly occurring in the S phase, was observed immediately
after N-nitroso propoxur treatment (Fig. 5). The incidence
of DNA damage investigated by this method also showed a
significant reduction after the 24-h post-treatment incuba-

H.H. Kuo et al. / Food and Chemical Toxicology 46 (2008) 1619–1626
1623
Fig. 5. Flow cytometric analysis of DNA damage induced by N-nitroso propoxur. SC-M1 cells stained for propidium iodine and cH2AX after a 2h-
treatment with N-nitroso propoxur were shown in (A). An overall results of three different experiments were shown in (B). Upper row indicates the results
of assay immediately after the treatment, while the bottom row is those after a 24-h post-treatment incubation.
tion, which is similar to the results found with the COMET
assay. Therefore, N-nitroso propoxur treatment at 2 and
4 lg/ml was capable of damaging the DNA of SC-M1 cells.
Most of the DNA damage, however, could be repaired
within 24 h, i.e. within one replication cycle of SC-M1 cells.
induction of chromosome aberration (Table 1). Only when
the treatment was increased up to 8 lg/ml did the induction
of chromosome aberrations become significant. The
delayed expression of chromosome aberrations was not
found until the dose reached 8 lg/ml, with those incubated
for 48 h showing higher chromosome aberrations than
those incubated for 24 h. In treatment with 16 lg/ml N-
nitroso propoxur, no mitotic cells were available for anal-
ysis in those cells harvested 24 h after the end of treatment,
while significant chromosome aberrations were observed in
those incubated for 48 h after treatment removal. The
major type of chromosome aberration induced by N-
nitroso propoxur was chromatid exchanges.
3.5. Chromosome aberrations
A 2-h pulse treatment of N-nitroso propoxur with doses
64 lg/ml was unable to induce significant chromosome
aberrations in SC-M1 cells, although a previous investiga-
tion on COMET and cH2AX expression showed it caused
significant DNA damage at those dose levels. N-nitroso
propoxur is recognized as one of the potential compounds
which produce O6-methylguanine adducts in mammalian
cells in culture conditions (Wang et al., 1998b). These com-
pounds were reported to yield chromosome aberrations
with a delayed expression (Bean and Galloway, 1993; Gal-
loway, 1994; Kaina et al., 1997). Therefore, we investigated
the chromosome aberrations in cells harvested both 24 and
48 h after treatment removal, which are approximately
equal to completion of 1 and 2 cell cycles in SC-M1 cells,
respectively. However, neither of them showed significant
3.6. Hprt mutation
Induction of hprt gene mutation was expressed as the
number of cells resistant to 6-thioguanine per 106 cells
(Table 2). In two independent experiments, the frequency
of 6-thioguanine resistance was <10À6 in control SC-M1
cells. A 2-h treatment with 2 lg/ml N-nitroso propoxur
increased the resistance frequency 10–20 times. The muta-
tion frequency of the hprt gene in treatments with 4 lg/
ml N-nitroso propoxur was 40–60 times as high as that
of the control group.
Table 1
Induction of chromosome aberrations in SC-M1 cells by N-nitroso
propoxur
Table 2
N-nitroso propoxur-induced 6-thioguanine (6-TG) resistance in SC-M1
cells
N-Nitroso propoxur
(lg/ml)
Percent (%) aberrant cells,a
Post-treatment incubation for
Dose
(lg/ml)
Plating efficiency
(%)
Number of 6-TG-resistant cells
2 Â 106 cells
Per 106 surviving cells
24 h
48 h
0
2
4
8
0
0
Experiment 1
1
1
0
2
4
102.3
99.3
100
2
21
121
0.97
10.6
60.5
1
4
9**
15**
53**
b
16
**
–
Experiment 2
Indicates a significant difference at the p < 0.01 level according to a
0
2
4
80.7
148.3
91
1
71
71
0.6
23.9
39.1
binomial distribution.
a
Cells with only a gap were not included.
Analysis failed due to a lack of mitotic cells.
b

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H.H. Kuo et al. / Food and Chemical Toxicology 46 (2008) 1619–1626
3.7. Anchorage independence
Results of three independent studies showed that SC-M1
cells in the control and N-nitroso propoxur-treated (2 lg/ml,
2 h) groups did not significantly differ in plating efficiencies
on plastic (Table 3). Their ability of soft-agar colonization,
on the other hand, almost doubled after N-nitroso propoxur
treatment. This indicated that the anchorage dependence in
SC-M1 cells was significantly decreased by N-nitroso prop-
oxur treatment at 2 lg/ml, which is a dose without apparent
cytotoxic effects (p < 0.01, according to Student’s t-test).
3.8. Perturbation of cell cycle progression
After 2 h of N-nitroso propoxur treatment (2 lg/ml),
the logarithmically growing SC-M1 cells were incubated in
N-nitroso propoxur-free normal media for a series of differ-
ent time periods ranging from 2 to 24 h. In the first 6 h of
the post-treatment period in normal media, cells from the
control and treated groups showed similar patterns of cell
cycle progression, with about 65–68% of cells remaining
in the G1 stage, and 13% and 20% remaining in the S and
G2/M stages, respectively (Fig. 6). Starting from around
8 h after N-nitroso propoxur removal, the effect of the
treatment on cell cycle progression became apparent. The
N-nitroso propoxur-treated cell population in the G1 stage
gradually decreased, while that of the control group was
still maintained at the previous level. The G1 population of
cells collected during 12–16 h after N-nitroso propoxur
removal was 20% less than that collected from the control
group at the same period. After that, the G1 population in
N-nitroso propoxur-treated cells gradually increased again
and returned to the same level as the control group 24 h after
N-nitroso propoxur removal. During the period when the G1
population in N-nitroso propoxur-treated cells decreased,
i.e. between 8 and 16 h after N-nitroso propoxur removal,
the G2/M population was found to have concomitantly
increased. In treated cells collected 16 h after N-nitroso prop-
oxur removal, 50% of them stayed in the G2/M phase. In the
control treatment, only 20% of G2/M cells were collected in
the same period. Therefore, N-nitroso propoxur treatment
induced transient cellcycle arrestat the G2/M stage, and con-
secutively caused the cell population to decrease in the G1
stage. Deviations also found in S-phase cells between the con-
trol and N-nitroso propoxur treatments, however, were not
as apparent.
Fig. 6. Perturbation of cell cycle progression by N-nitroso propoxur. SC-
M1 cells were treated with control media (open circle) or 2 lg/ml N-
nitroso propoxur for 2 h. The cells were then harvested for cell cycle
analysis after incubated in normal media for a series of different period.
4. Discussion
N-Methylcarbamate insecticides hold a special position
among many pesticides used in pest control programs.
They serve as possible precursors of N-nitroso compounds,
and thus they probably play key roles in the development
of gastric cancer in humans (Palli et al., 2001). The sugges-
tion that the formation of mutagenic metabolites should be
included as a parameter in the toxicological evaluation of
carbamate insecticides was proposed as early as the 1970s
(Uchiyama et al., 1975) when the interaction of the N-
methylcarbamate insecticide, carbaryl, with sodium nitrite
was demonstrated to produce N-nitroso carbaryl (Elespuru
and Lijinsky, 1973; Siebert and Eisenbrand, 1974). In the
following years, numbers of N-methylcarbamate insecti-
cides that were shown to convert into N-nitroso metabo-
lites have accumulated. Among N-methylcarbamate
insecticides, propoxur is one of the most extensively used
pest insect control agents in Taiwan. The possibility of it
contaminating food, water, and air, thereby producing
adverse effects in humans, is inevitable. In vitro studies on
Chinese hamster V79 and primary rat tracheal epithelial
cells demonstrated solid evidence that the N-nitroso deriv-
ative of propoxur is highly genotoxic to mammalian cells
Table 3
N-nitroso propoxur-induced anchorage independence in gastric SC-M1
cells
N-nitroso propoxur Plating efficiency
(lg/ml)
In soft agar On plastic
Per 100 surviving cells
in soft agar
0
2
14.8 2.8
77.1 12.3 19.1 0.9
29.0 4.0** 83.0 12.7 36.2 1.1**
** Indicates a significant difference from the control at p < 0.01, according
to Student’s t-test.

H.H. Kuo et al. / Food and Chemical Toxicology 46 (2008) 1619–1626
1625
(Wang et al., 1998a,b). What we present in this report are
results of an investigations carried out on human gastric
cells. This should be more relevant to the elucidation of
its adverse effect since the stomach has been recognized
as the major target for the N-nitroso N-methylcarbamate
insecticides (Lijinsky, 1992).
properly preserved. Therefore, exposure to low doses of
N-nitroso propoxur has the potential to accelerate sponta-
neous genetic changes in human gastric cells without
showing an apparent growth inhibition effect or gross chro-
mosome alterations. The increase in genetic instability has
the potential to progress to cellular malignancy. Our results
in the soft-agar assay provide auxiliary evidence for elucida-
tion of this phenomenon. Soft-agar colonization, an indica-
tion of anchorage-dependent loss, is commonly recognized
as one of many phenomena characterizing the progression
to cellular malignancy (Allan et al., 2006; Gao et al.,
2005). After being treated with 2 lg/ml N-nitroso propoxur,
twice as many SC-M1 cells grew in soft-agar colonization
as in the controls. This implies that low-dose N-nitroso
propoxur exposure not only increased the genetic instability
but also pushed the development of malignancy forward in
SC-M1 cells. Contamination by propoxur in the environ-
ment, due to its potential to convert to N-nitroso deriva-
tives, is thus a great concern to human health.
In addition to using target cells of N-nitroso N-methyl-
carbamate to obtain pertinent results, this report revealed
the potential risks of low doses of N-nitroso propoxur to
human health which have not been disclosed in previous
investigations. Compared with Chinese hamster V79 and
primary rat tracheal epithelial cells, human gastric SC-M1
cells are much more resistant to N-nitroso propoxur. A 2-
h pulse treatment of N-nitroso propoxur at 2 lg/ml induced
a mortality of P50% in V79 and primary rat tracheal epi-
thelial cells (Wang et al., 1998b). In SC-M1 cells, on the
other hand, more than 90% of cells survived with the same
treatment. When the treatment dose of N-nitroso propoxur
increased to 4 lg/ml, 90% of SC-M1 cells continued to
grow, while 90% of V79 cells were killed. At these non-cyto-
toxic dose levels, i.e. doses 64 lg/ml, N-nitroso propoxur
nevertheless induced a considerable amount of DNA dam-
age in SC-M1 cells, which was identified by both COMET
and cH2AX staining assays. The DNA damage in SC-M1
cells induced by N-nitroso propoxur treatment at those dose
levels, however, did not seem likely to turn into aberrant
chromosomes. The cytogenetic assay indicated that at these
dose levels, both the regular and delayed harvest protocols
failed to detect significant chromosome aberrations. The
phenomenon that N-nitroso propoxur induced DNA dam-
age but not chromosome aberrations is probably due to
most of the DNA damage produced by low-dose N-nitroso
propoxur treatment being repaired before cells entered the
mitotic stage. This is true because both the COMET and
cH2AX staining assays showed that DNA damage signifi-
cantly decreased after a 24-h post-treatment incubation in
drug-free media. Cell cycle analysis further revealed that a
transient G2/M arrest was observed in N-nitroso propo-
xur-treated cells starting from the 8th hour after treatment
removal. The transient G2/M arrest provides a protective
mechanism to ensure repair of induced DNA damage and
prevented inappropriate mitotic entry so that we were
unable to find significant induction of chromosome aberra-
tions in low-dose treatments of N-nitroso propoxur.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
This research was supported by Grants from the Insti-
tute of Cellular and Organismic Biology, Academia Sinica
in Taiwan.
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