N-Nitrosocarbofuran, but not Carbofuran, induces apoptosis and cell cycle arrest in CHL cells

Article abstract of DOI:10.1016/S0300-483X(01)00502-9

Carbofuran (CF) is one of the most widely used carbamate pesticides in the world applied for insect and nematode control. Due to its widespread use in agriculture and households, contamination of food, water, and air has become serious, and consequently a

Full text of DOI:10.1016/S0300-483X(01)00502-9

Toxicology 169 (2001) 153–161
www.elsevier.com/locate/toxicol
N-Nitrosocarbofuran, but not Carbofuran, induces
apoptosis and cell cycle arrest in CHL cells
Ji-Young Yoon a, Seon-Hee Oh a, Su-Mi Yoo a, Sang-Jun Lee a, Hye-Suk Lee a,
Sung-Jin Choi a, Chang-Kiu Moon b, Byung-Hoon Lee a,*
a College of Pharmacy and Medicinal Resources Research Center, Wonkwang Uni6ersity, Iksan, Jeonbuk 570-749, South Korea
b College of Pharmacy, Seoul National Uni6ersity, Seoul, South Korea
Received 3 August 2001; received in revised form 3 August 2001; accepted 21 August 2001
Abstract
Carbofuran (CF) is one of the most widely used carbamate pesticides in the world applied for insect and nematode
control. Due to its widespread use in agriculture and households, contamination of food, water, and air has become
serious, and consequently adverse health effects are inevitable in humans, animals, wildlife and fish. It has been
reported that CF alone or in combination with other carbamate insecticides influences the level of reproductive and
metabolic hormones such as thyroxine and corticosterone, and results in impairment of endocrine, immune and
behavioral functions. In this study, we evaluated the effects of CF and its metabolite, the N-nitroso derivative
N-nitrosocarbofuran (NOCF), on genotoxicity, cell growth, cell cycle and apoptosis of Chinese hamster lung
fibroblast (CHL) cells. NOCF, but not CF, induced genotoxicity determined by Ames test. NOCF inhibited the
growth of Chinese hamster lung fibroblast (CHL) cells with an IC50 of 12.8 mM. NOCF induced apoptosis of CHL
cells, which was demonstrated by morphological changes, DNA fragmentation and flow cytometric analysis.
Treatment of CHL cells with NOCF induced significant G2/M cell cycle arrest. Caspase-3, an executioner of apoptosis
was also activated by the treatment of CHL cells with NOCF. These results suggest that NOCF, that is an important
metabolite of CF, leads to the induction of cell cycle arrest and apoptosis in CHL cells. © 2001 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: N-methylcarbamate; Carbofuran; N-Nitrosocarbofuran; O6-Methylguanine; Apoptosis; Cell cycle arrest
Abbre6iations: Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide; CF, carbofuran; CHL, Chinese hamster lung fibrob-
last; MNNG, N-methyl-N%-nitro-N-nitrosoguanidine; MTT, 3-(4-,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide;
O6MeG, O6-methylguanine; PBS, phosphate buffered saline; PI, propidium iodide; TUNEL, terminal deoxynucleotydyl transferase-
mediated dUTP nick-end labeling.
* Corresponding author. Tel.: +82-63-850-6806; fax: +82-63-850-7309.
E-mail address: bhlee@wonkwang.ac.kr (B.-H. Lee).
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0300-483X(01)00502-9

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J.-Y. Yoon et al. / Toxicology 169 (2001) 153–161
1. Introduction
tion, micronucleus formation and aneuploidy in
vitro and in vivo (Gonzalez Cid et al., 1990;
Soderpalm-Berndes and Onfelt, 1988; Chauhan et
al., 2000). Like most of the N-nitrosoamide com-
pounds, N-nitrosocarbamates are potent direct
acting mutagens and carcinogens. N-Nitrosocar-
bofuran (NOCF) is mutagenic (Nelson et al.,
1981) and induces DNA single strand breaks in
human skin fibroblasts (Blevins et al., 1977).
Apoptosis is a highly regulated process by
which an organism eliminates unwanted cells
without eliciting an inflammatory response. Some
abnormal physiological and chemical injuries,
therefore, induce apoptosis (Pallardy et al., 1999).
Apoptosis is also triggered in response to various
forms of stress and DNA damages (Smith and
Fornace 1996; Park et al., 1998). Apoptosis in-
duced by mutagenic or genotoxic carcinogens is
vital because it eliminates cells harboring muta-
genic DNA damage from the body. In fact, hu-
man cancers contain cells that have been
potentially compromised in their ability to un-
dergo apoptosis (Hollstein et al., 1991). Only a
limited number of papers deal with the genotoxic
mechanism of NOCF. Wang et al. (1998a,b) re-
ported recently that N-nitrosation greatly in-
creased the cytotoxicity and mutagenicity of
N-methylcarbamates at the hprt locus in Chinese
hamster V79 cells. They showed that the major
mutational change is a transition from GC to AT,
which is similar to the results reported for N-
Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzo-
furanyl methylcarbamate; CF) is one of the most
widely used N-methylcarbamate esters applied for
insect and nematode control. Acute toxic effects
induced by N-methylcarbamate pesticides are
caused by the inhibition of acetylcholine esterase
in the nervous tissue; the inhibition is labile, of
short duration, and reversible compared with that
induced by organophosphate compounds (Gupta
1994; Maroni et al., 2000) Due to its widespread
use in agriculture and household, contamination
of food, water and air has become serious, and
consequently adverse health effects are inevitable
in humans, animals, wildlife and fish. A growing
body of epidemiological studies suggests that
long-term exposure to relatively safe pesticides,
including CF, may be associated with increased
risk of gastrointestinal, neurological and cardiac
dysfunction as well as retinal degeneration (Cole
et al., 1998; Peter and Cherian 2000; Kamel et al.,
2000). CF has been reported to be detected re-
peatedly in drinking water supplies with a typical
concentration range between 1.0 and 5.0 mg/l
(Hallberg, 1987). Therefore, the contamination of
human environment with CF can easily occur and
there is an increasing risk of a long-term low dose
exposure to this pesticide. Moreover, it has been
reported that CF alone or in combination with
other carbamate insecticides influences the level of
reproductive and metabolic hormone, resulting in
impairment of endocrine, immune and behavioral
functions (Cranmer et al., 1978; Rawlings et al.,
1998).
methyl-N%-nitro-N-nitrosoguanidine
(MNNG).
We report in the present study on the cellular
response in Chinese hamster lung fibroblast
(CHL) treated with NOCF in terms of the induc-
tion of cell cycle arrest and apoptosis.
Carbamate pesticides are readily converted to
N-nitroso metabolites in the presence of nitrites
or nitrogen oxides in the stomach under acidic
conditions or in the colon by intestinal bacteria
(Elespuru and Lijinsky, 1973; Regan et al., 1976;
Hughes et al., 2001). It is reported that 0.5–2% of
the carbamate dose were isolated as the nitroso
derivative from the guinea pig stomach (Rickard
and Dorough, 1984). The nitroso derivatives of
carbamate pesticides lose the cholinesterase in-
hibiting properties of the parent compounds and
are less toxic to mammals. However, they induce
sister chromatid exchange, chromosomal aberra-
2. Materials and methods
2.1. Chemicals
CF was purchased from Aldrich Chemical Co
(Milwaukee, WI) and NOCF was synthesized by
nitrosating CF in an acidic condition in the pres-
ence of sodium nitrate (Fig. 1) (Blevins et al.,
1977). Stock solutions (100 mM) of CF and
NOCF were prepared by dissolving the com-
pounds in dimethyl sulfoxide.

J.-Y. Yoon et al. / Toxicology 169 (2001) 153–161
155
2.2. Cell culture
2.5. Morphological analysis and TUNEL assay
CHL cells were maintained in minimum essen-
tial medium supplemented with 5% fetal bovine
serum (GIBCO BRL, Grand Island, NY). Cells
were harvested using 0.2% trypsin and seeded
onto 100 mm culture dishes. The cells were then
allowed to grow at 37 °C in a 5% CO2-95% air
humidified incubator.
Cells were treated with NOCF for 48 h, washed
gently three times with phosphate buffered saline
(PBS) and fixed with paraformaldehyde for 30
min. The cells were washed with PBS and then
stained with propidium iodide (PI) solution (50
mg/ml of PI with 100 mg/ml of RNase). The
morphology of the cells was examined using the
Leitz phase-contrast microscope or Olympus Flu-
oview laser scanning confocal microscope. Apop-
tosis was also detected by the terminal
2.3. Ames test
The preincubation method of Maron and Ames
(1983) was carried out using S. typhimurium
TA100 in the presence and absence of S9 mixture.
S9 fraction was prepared from the pooled livers of
Aroclor 1254-induced Sprague–Dawley rats.
deoxynucleotidyl
oxyuridine
(TUNEL) method using the Boehringer in situ
death detection kit according to the instruction of
the supplier (Mannheim, Germany).
transferase-mediated
nick-end
de-
labeling
triphosphate
2.4. Cell proliferation assay
2.6. DNA fragmentation analysis
Cell proliferation was determined by the 3-(4-,
5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide (MTT) assay. CHL cells were seeded into
a 96-well plate at a concentration of 3×104 cells/
ml in 100 ml of medium and left overnight to
adhere. The chemicals in 100 ml volumes were
added and incubated for different time periods as
indicated in the figure legend. General viability of
cultured cell was determined by reduction of
MTT to formazan following 96 h of incubation
(Monks et al., 1991). The concentration required
for 50% inhibition of the growth (IC50) was deter-
mined by non-linear regression analysis using the
GraphPad PRISM™ statistics software package
(Ver. 2.0; San Diego, CA).
The cells grown at a density of 2×106 cells/ml
in 100 mm culture dishes were treated with appro-
priate concentrations of the chemicals as de-
scribed in the figure legends. Genomic DNA was
prepared with Wizard Genomic DNA Purification
Kit (Promega, Madison, WI). DNA was precipi-
tated with isopropanol, separated in 1.5% agarose
gel and visualized by UV illumination after ethid-
ium bromide staining.
2.7. Flow cytometry and cell cycle analysis
Exponentially growing cells were diluted and
2×106 cells were seeded in 10 ml of culture
medium in 100 mm culture dishes. They were
Fig. 1. The formation of NOCF from CF in acidic condition.

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J.-Y. Yoon et al. / Toxicology 169 (2001) 153–161
Fig. 2. Mutagenicity and antiproliferative effect of CF and NOCF. Genotoxicity of CF (ꢀ) and NOCF (ꢁ) was determined by
Ames test in the absence of metabolic activation system (A). Mutation rate was calculated by dividing the mean number of test
reverent colonies by that of spontaneous reverent colonies. Cells were incubated with CF or NOCF (ꢂ, control; ꢃ, 1 mM; ꢄ, 10
mM; ", 30 mM; ꢁ, 50 mM) and the time-course of proliferation was determined by MTT assay as described in Section 2 (B). Each
point represents the mean and standard error from three experiments.
exposed to CF or NOCF for 48 h and centrifuged
at 800×g for 5 min. The pellets were mixed with
1:1 (v/v) mixture of PBS and 0.2 M Na2HPO4-0.1
M citric acid (pH 7.5) and fixed with ice-cold
ethanol at 4 °C for 1 h. The cells were washed
twice with PBS and resuspended in 1 ml of a
staining solution containing 10 mg/ml PI contain-
ing 100 mg/ml DNase-free RNase A. The cell
suspensions were incubated at room temperature
for 1 h and 20 000 cells were analyzed on a
FACScalibur flow cytometer (Becton–Dickinson)
dose dependent manner up to a concentration of
20 mg per plate, which is in accordance with the
previous results (Fig. 2, A). Neither CF nor
NOCF was mutagenic in the presence of
metabolic activation system (data not shown).
The effects of CF and NOCF on the proliferation
of CHL cells were tested. Cells were treated with
the compounds for 48 h and the number of viable
cells was determined by MTT assay. The treat-
ment of CHL cells with NOCF significantly inhib-
ited the growth of the cells in a dose dependent
manner. The IC50 at 48 h of incubation was 12.8
mM. On the other hand, CF did not influence the
growth rate of CHL cells at concentrations be-
tween 1 and 50 mM (Fig. 2, B).
2.8. Caspase-3 acti6ity
Substrate
(N-acetyl-Asp-Glu-Val-Asp-p-ni-
troanilide; Ac-DEVD-pNA) was added to the cell
lysates in assay buffer (50 mM HEPES; 100 mM
NaCl; 0.1% CHAPS; 10 mM dithiothreitol; 1 mM
EDTA; 10% glycerol; pH 7.4) and incubated for 1
h at 37 °C. The cleavage of the peptide substrate
was monitored at 405 nm.
3.2. Induction of apoptosis by CF and NOCF in
CHL cells
During the course of the cytotoxicity experi-
ment we observed that NOCF induced morpho-
logical changes that are characteristic of
apoptosis. Although untreated cells exhibited typ-
ical adherent and well-spread morphology, cells
exposed to NOCF frequently displayed round
shape and condensation of chromatin. To clarify
the mode of cell death induced by NOCF, DNA
fragmentation analysis, confocal microscopy of
nuclear morphology and TUNEL staining were
performed. Fig. 3A, depicts the results of the
agarose gel electrophoresis of cellular DNA show-
ing that NOCF treatment induced an oligosomal
DNA ladder. NOCF exposure induced substantial
3. Results
3.1. Genotoxicity and cytotoxicity of CF and
NOCF
We have tested the genotoxicity of CF and
NOCF using the Ames test with S. typhimurium
TA100. NOCF was mutagenic in the absence of
metabolic activation system, while CF was not.
The mutagenicity of NOCF was increased in a

J.-Y. Yoon et al. / Toxicology 169 (2001) 153–161
157
nuclear condensation, shrinkage and a loss of
internal nuclear structure. Staining of apoptotic
nuclear buds and fragments was also observed
(Fig. 3, B-c). TUNEL staining shows an intense
fluorescence in NOCF treated cells (Fig. 3, B-f).
Treatment of CF did not alter the morphology
and TUNEL staining of the cells (Fig. 3, B-b,
B-e).
by agarose gel electrophoresis. The histogram of
the cells treated with 30 mM NOCF shows an
increase in the G2/M phase and a decrease in the
G1 phase. The quantitative analysis of the his-
tograms to determine the percentage of cells in the
G1, S, and G2/M phases for the control cells and
the cells treated with NOCF for 48 h is shown on
Fig. 4C. The data clearly indicate that NOCF
causes a significant increase in the percentage of
G2/M phase cells and a significant decrease in the
percentage of G1 phase cells, whereas the percent-
age of S phase remains unchanged.
3.3. Flow cytometry and cell cycle analysis
Flow cytometric analysis was performed to
quantify the degree of apoptosis by NOCF and to
determine the effect of NOCF on the cell cycle
distribution of the CHL cells. NOCF, but not CF,
resulted in the progressive generation of cells with
hypodiploid DNA content (Fig. 4A) that is char-
acteristic of apoptosis. Quantitation of the apop-
totic cells shows a dose-dependent response to
NOCF (Fig. 4B), and this result is in good accor-
dance with that of the DNA fragmentation assay
3.4. Caspase-3 Acti6ity
Caspase-3 activation is a late signal that accom-
plishes apoptosis in mammalian cells. To deter-
mine whether caspase activation is involved in the
NOCF-induced cell death, CHL cells were ex-
posed to NOCF and assayed for caspase-3 activ-
ity. Treatment of the cells with NOCF activated
Fig. 3. Morphological analysis and TUNEL assay CHL cells were exposed to CF or NOCF for 48 h. For DNA fragmentation
analysis, genomic DNA was extracted, electrophoresed in 1.5% agarose gel and visualized by UV illumination after ethidium
bromide staining (A). For morphological analysis, control (B-a), CF (B-b) and NOCF treated cells (B-c) were fixed with
paraformaldehyde and stained with PI. NOCF exposure induced substantial nuclear condensation, shrinkage and a loss of internal
nuclear structure. Staining of apoptotic nuclear buds and fragments were also observed. For TUNEL analysis, control (B-d), CF
(B-e) and NOCF treated cells (B-f) were stained with Boehringer in situ death detection kit according to the supplier’s manual.
TUNEL staining shows an intense fluorescence in NOCF treated cells.

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J.-Y. Yoon et al. / Toxicology 169 (2001) 153–161
Fig. 4. Flow cytometric analysis of apoptosis and cell cycle distribution. CHL cells were treated with CF or NOCF for 48 h, stained
with PI and analyzed on a FACScalibur flow cytometer (A). Percentage of apoptotic cells (B) and dose-dependent changes in CHL
cell cycle induced by NOCF (C) is shown at the indicated concentrations. Representative diagrams are presented and the deviation
was within the range of 5% in all three independent experiments.
caspase-3 in a dose dependent manner (Fig. 5).
The dose-dependent increase in the caspase-3 ac-
tivity is in good accordance with that in the
percentage of apoptotic cells determined by flow
cytometric analysis (Fig. 4). CF did not affect the
caspase-3 activity at a concentration of 30 mM.
N-Nitroso methylcarbamates induced sister
chromatid exchange and micronuclei formation in
human lymphocytes (Gonzalez Cid et al., 1990).
They are more cytotoxic than their parent com-
pounds, and they induce chromosome aberration
and hprt gene mutation in V79 cells, and cell
transformation in rat tracheal epithelial cells
(Wang et al., 1998a,b). N-Methylcarbamates do
not induce cytotoxicity or genotoxicity, but they
inhibit gap-junctional intercellular communication
in the cells at doses that produce little
cytotoxicity.
4. Discussion
In the present study, we demonstrated that
exposure of CHL cells to NOCF induced cell
cycle arrest and apoptotic death of CHL cells.
The parent compound CF was neither genotoxic
nor cytotoxic in bacterial as well as in mammalian
cell systems.
It is now generally recognized that total en-
dogenous nitrosation processes contribute signifi-
cantly to a genotoxic burden for the general
population who are not known to have suffered

J.-Y. Yoon et al. / Toxicology 169 (2001) 153–161
159
excessive exposure to any environmental methy-
lating agents (Georgiadis et al., 2000). The obser-
vation of a possible protective effect of intake of
ascorbic acid and green tea consumption on nitro-
sation supports the hypothesis that endogenous
nitrosation play an important role in the total
human exposure to genotoxic methylating agents
(Vermeer et al., 1999).
According to our not yet published results, treat-
ment of calf thymus DNA with NOCF, but not
with CF, induced the formation of O6MeG DNA
adduct.
We observed that the treatment of CHL cells
with NOCF caused a profound alteration in cell
cycle progression. An arrest in G2/M phase of the
cell cycle was induced already with 10 mM of
NOCF. Cell cycle arrest is a common response to
DNA damaging agents, and such arrest frequently
precedes the onset of apoptosis (Meikrantz and
Schlegel 1995). DNA damage caused by numer-
ous alkylating agents or ionizing radiation in var-
ious cell types has been associated with a G2
phase delay or arrest (Tobey 1975; Maity et al.,
1994; Lin et al., 1999). Activation of p53 can
occur in response to a number of cellular stresses,
including DNA damage and it controls cell cycle
progression and cell survival through apoptosis.
However, many commonly used Chinese hamster
cell lines, such as Chinese hamster ovarian cell,
are defective in p53 function. CHL cells have a
spontaneously induced high level of p53 pheno-
type not inducible by X-irradiation (Hu et al.,
1999). Therefore, it seems unlikely that the signal
for apoptosis initiated by treatment with NOCF is
transmitted by the p53 tumor suppressor protein.
We can not, however, exclude the possibility that
the p53 protein is mutated by treatment of
NOCF, because a mutation at codon 211 of the
p53 gene in the Chinese hamster ovarian cell line
has been reported to be responsible for the cells
losing ability to arrest in the G1 phase after
radiation (Lee et al., 1997; Hu et al., 1999), rather
they show G2/M arrest, as was shown in the
NOCF treated cells. Studies are currently under
way to address the molecular mechanisms under-
lying the effects of NOCF on the induction of
apoptosis and G2/M arrest.
Similarly to the results reported for MNNG
(Lukash et al., 1991; Yang et al., 1991), the hprt
mutation analysis by N-nitrosopropoxur revealed
that the major mutational change is a transition
from GC to AT. Since the GC to AT transition
occurs mainly as a result of the O6MeG adduct
formation, Wang et al. (1998b) have postulated
that NOCF might produce this type of DNA
adduct, if any. O6MeG is the major mutagenic
base derivative formed in DNA on exposure of
cells to DNA methylating agents. This modified
base mispairs with thymine during DNA replica-
tion, and as a result methylating agents are potent
mutagens and carcinogens. It is now generally
accepted that alkylation-induced cell killing is
largely attributable to apoptosis and the O6MeG
acts as a trigger of this toxic response (Kaina et
al., 1997; Meikrantz et al., 1998; Ochs and Kaina
2000). Therefore, we postulated that NOCF in-
duces apoptosis, which is triggered by O6MeG.
Organophosphate or chlorinated hydrocarbon
insecticides have been previously reported to in-
duce apoptosis (Carlson et al., 2000; Kannan et
al., 2000; Rought et al., 2000). Due to the toxicity
and persistence in the environment, pesticides of
these classes are not frequently used. N-Methyl-
carbamates, on the other hand, are regarded as
relatively safe chemicals because extensive toxico-
logical research demonstrated that these com-
Fig. 5. Activation of caspase-3. Extracts of control cells and
cells treated with CF or NOCF were analyzed for caspase-3
activity as a function of concentration. Ac-DEVD-pNa was
used as the substrate for caspase-3 and the cleavage of the
peptide was monitored at 405 nm. The caspase activity of
control cells was set to 100% and the relative change in the
activity was shown (*PB0.01). Each data represents the mean
and standard deviation in three independent experiments.

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Hu, T., Miller, C.M., Ridder, G.M., Aardema, M.J., 1999.
Characterization of p53 in Chinese hamster cell lines CHO-
K1, CHO-WBL, and CHL: implications for genotoxicity
testing. Mutat. Res. 426, 51–62.
Hughes, R., Cross, A.J., Pollock, J.R.A., Bingham, S., 2001.
Dose-dependent effect of dietary meat on endogenous
colonic N-nitrosation. Carcinogenesis 22, 199–202.
Kaina, B., Ziouta, A., Ochs, K., Coquerelle, T., 1997. Chro-
mosomal instability, reproductive cell death and apotosis
induced by O6-methylguanine in Mex−, Mex+ and methy-
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pounds produced only transient, short-term toxic-
ity following acute administration. Our study
clearly demonstrates that the N-nitroso metabo-
lite of CF is genotoxic and induces cell cycle
arrest and apoptosis in CHL cells.
Acknowledgements
This work was supported by the grant (KOSEF
1999-2-21800-002-3) from the Korea Science and
Engineering Foundation.
Kamel, F., Boyes, W.K., Gladen, B.C., Rowland, A.S., Ala-
vanja, M.C., Blair, A., Sandler, D.P., 2000. Retinal degen-
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Kannan, K., Holcombe, R.F., Jain, S.K., Alvarez-Hernandez,
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