Journal of Environmental Protection, 2010, 1, 314-323
doi:10.4236/jep.2010.13037 Published Online September 2010 (http://www.SciRP.org/journal/jep)
Apoptosis Induced by N-Nitrosamines in Two
Cancer Cell Lines
Paloma Morales, Nuria Arranz, Ana I. Haza
Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de
Madrid, Madrid, Spain.
Received May 20th, 2010; revised July 1st, 2010; accepted July 3rd, 2010.
ABSTRACT
In the present study, we investigated the induction of apoptosis by N-nitrosopyrrolidine (NPYR) and N-nitrosodimethy-
lamine (NDMA) in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by:
1) chromatin condensation, 2) flow cytometry analysis and 3) poly (ADP-ribose) polymerase cleavage. Both cell lines
exhibited morphological changes consistent with apoptotic events following treatment with N-nitrosamines. Flow cy-
tometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-
manner. NPYR was stronger than NDMA, since it induced a significant apoptotic cell death after 72 h starting from a
concentration of 10 mM, whereas NDMA was effective at 27 mM. Furthermore, NPYR and NDMA caused the cleavage
of PARP in HL-60 cells whereas no PARP cleavage was detected in HepG2 cells. However, NPYR- and NDMA-
induced cell death in HepG2 cells was prevented by specific caspase inhibitors. Caspase-8 mediated main pathway and
was responsible for 76% (NPYR) and 64% (NDMA) inhibition of apoptosis. The data demonstrate that NPYR and
NDMA induce apoptosis in HL-60 and HepG2 cell lines via caspase-dependent pathway.
Keywords: N-nitrosopyrrolidine, N-nitrosodimethylamine, Apoptosis, Caspases, HL-60 Cells, HepG2 Cells
1. Introduction
The N-nitroso compounds (NNC) are recognized as one
of the most potent chemical mutagens and carcinogens
present in environment and in food [1]. N-nitrosamines
are NNC found in foodstuffs, drinking water, rubber
products, drug formulations, tobacco and tobacco smoke
[2]. Their precursors, nitrites and secondary amines,
contained in many common foods, can react under acidic
conditions of the stomach and also are produced in vivo
by reduction of nitrates by bacteria [3].
The majority of N-nitrosamines tested has been shown
to cause cancer at different organs in a variety of animal
species and may be causative agents in human cancer [4].
N-nitrosopyrrolidine (NPYR) induces mainly liver tu-
mours in rats [5] and is a weak pulmonary carcinogen in
mice [6]. N-Nitrosodimethylamine (NDMA), the sim-
plest and most widely occurring nitrosamine, has been
shown to be a potent liver, lung and kidney carcinogen
[7]. The metabolic activation of NPYR and NDMA to
reactive intermediates by cytochrome P450 is required
for the expression of their toxic potential [8]. The key
activation pathway is cytochrome P450-catalyzed hy-
droxylation of the carbon α to the nitroso group [9].
Toxic effect of chemicals can lead to passive cell death
or necrosis, or result in the active mechanism of apop-
tosis. Necrotic cell death is an unregulated process re-
sulting from severe damage to the cell and is character-
ized by ATP depletion, cell swelling, lysis, and the re-
lease of intracellular contents resulting in tissue inflam-
mation [10]. Apoptosis, often described as cell suicide
[11], is important in carcinogenesis [12], atherogenesis
[13] and a number of other diseases [14]. Apoptotic cell
death is a complex process characterized by biochemical
events and definite morphologic changes [15]. One of the
earliest and most consistently observed features is the
induction of a series of cytosolic cysteine proteases,
known as caspases [16]. Activation of the caspase cas-
cade leads to changes in the plasma-membrane, mito-
chondria and nucleus [17]. Among the family of ten or
more different caspases, already described, caspase-8 and
-9 are involved in receptor mediated and intracellular
(mitochondrial) pathways of apoptotic cascade, respec-
tively. Different reports indicate that caspase-3, -6 and -7

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines 315
are the major effector caspases of apoptosis [18].
It is now evident that many environmental chemicals
exert their toxicity via apoptotic cell signalling [19]. For
instance, it has been demonstrated that numerous food
mutagens [20-22] and tobacco specific N-nitrosamine
[23] induce apoptosis. Moreover, apoptosis induced by
carcinogens seems to have an important role in cancer
development. In general, apoptosis of cells exposed to
carcinogen compounds allows the removal of cells with
extensive DNA damage. However, the removal of these
cells may give survival and proliferating signals to the
surrounding cells with less DNA damage. This may cause
a selection of preneoplasic cells that have become more
resistant to carcinogen-induced cell death [24]. NDMA
causes the apoptosis of rat neutrophils in vivo [25] and
their reactive intermediate metabolites cause cell death in
P450 2E1-expressing cells by triggering apoptosis [26].
However, to our knowledge, there are not available data
about NPYR induced apoptosis.
Thus, the aim of the present study was to investigate
the induction of apoptosis by NPYR and NDMA in two
human cell lines. The HepG2 hepatoma cell line has been
reported to retain some of the drug metabolizing enzyme
activities of normal hepatocytes and to increase mRNAs
for specific P450 enzymes [27]. Since NPYR and NDMA
require metabolic activation catalyzed by the cytochrome
P450 for its mutagenicity and carcinogenicity, their ac-
tions have been well studied in the liver but there have
been few studies in the immune system (blood circula-
tory system). Moreover, NDMA can influence the activ-
ity of the immune system [25]. For this reason, in addi-
tion to HepG2 cells, the leukemia cell line HL-60 was
tested. This cell line has proven to be a good model for
studying the apoptotic process induced by chemicals in
lymphoid organs [28] and express relatively high levels
of enzymatic isoforms of cytochrome P450 [29].
This study also addresses the role of caspases in
N-nitrosamines-induced apoptosis in HL-60 and HepG2
cells.
2. Material and Methods
2.1. Chemicals
N-nitrosopyrrolidine (NPYR), N-nitrosodimethylamine
(NDMA), dimethyl sulfoxide (DMSO), etoposide and
acridine orange, were purchased from Sigma-Aldrich, Inc.
(St. Louis, MO). The caspase inhibitors, Z-DEVD-FMK
(caspase-3 inhibitor), Z-VEID-FMK (caspase-6 inhibitor),
Z-IETD-FMK (caspase-8 inhibitor) and Z-LEHD-FMK
(caspase-9 inhibitor) were obtained from BD Pharmigen
(USA). For western blot analysis, polyclonal poly (ADP-
ribose) polymerase (PARP) antibody was purchased
from Alexis Biochemicals (Lausen, Switzerland) and
secondary goat anti-rabbit conjugated to peroxidase was
obtained form Chemicon (Temecula, CA). All other che-
micals and solvents were of the highest grade commer-
cially available.
Standards solutions of NPYR and NDMA (500 mM)
were prepared in mili Q water (Millipore, Japan). N-nitr-
osamines are potent carcinogenic agents, safety precau-
tions were taken for proper handling and disposal of the
chemicals.
2.2. Cell Lines and Culture Conditions
Human hepatocellular carcinoma cells (HepG2) and hu-
man peripheral blood promyelocytic leukemia cells
(HL-60) were obtained from the Biology Investigation
Center Collection (BIC, Madrid, Spain). HepG2 cells
were cultured as monolayer in Dulbecco´s Modified Ea-
gle’s Medium. HL-60 cells were maintained in RPMI
1640 Medium. The media were supplemented with 10%
v/v heat-inactivated fetal calf serum, 50 mg/ml strepto-
mycin, 50 UI/ml penicillin and 1% v/v L-Glutamine.
Culture medium and supplements required for the growth
of the cell lines were purchased from GIBCO Laborato-
ries (Life Technologies, Inc., Gaithersburg, MD 20884-
9980). Controls included a medium control without N-
nitrosamines as a negative control. Etoposide has been
extensively studied [30] and was used in this study as a
positive control (5 μM, HL-60 cells; 100 μM, HepG2 cells)
of apoptosis.
2.3. Chromatin Condensation Assay
To examine the effect of N-nitrosamines on nucleus
chromatin condensation, HL-60 and HepG2 cells (1 ×
106/ml) were treated with NPYR (10-50 mM) or NDMA
(27-135 mM) at different incubation times (24-72 h).
After treatments, the cells were stained with acridine
orange (5 µg/ml) for 10 minutes and observed under a
UV-visible fluorescence microscope (Axiostar plus mi-
croscope, Zeiss) as described by Gregory et al. [31].
Cells exhibiting brightly fluorescent condensed or frag-
mented nuclei were considered apoptotic. A total of 200
cells were counted in multiple randomly selected fields,
and the percentage of apoptotic cells was then calculated.
2.4. TdT-dUTP Terminal Nick-End Labeling
(TUNEL) Assay
Apoptotic cell death was also measured by the In Situ
Cell Death Detection Kit, Fluorescein according to the
manufacturer’s protocol (Roche, Indianapolis, USA).
HL-60 and HepG2 cells were treated with NPYR (10, 30
and 50 mM) or NDMA (27, 68 and 135 mM) for 24, 48
and 72 h. Briefly, 3 × 106 cells were washed with PBS
and fixed in 2% formaldehyde in PBS (1 ml) for 1 hour

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines 316
at room temperature. Cells were washed with PBS and
incubated with permeabilization solution (0.1% Triton
X-100 in 0.1% sodium citrate) for 2 min on ice. Subse-
quently, the cells were incubated with the TUNEL reac-
tion mixture [50 μl of enzyme solution (TdT) and 450 μl
of label solution (fluorescein-dUTP)] for 1 hour at 37℃
in the dark in a humidified atmosphere. During this in-
cubation period, TdT catalyses the addition of fluo-
rescein-dUTP to free 3’-OH groups in single- and double
stranded DNA. Omission of TdT from the staining pro-
tocol constituted the negative control. After the cells
were washed with PBS, the label incorporated into the
damaged sites of DNA was detected using a FACS Cali-
bur flow cytometer (Becton and Dickinson) and the
CellQuest software. For each experiment 104 cells were
analyzed. To examine the effect of N-nitrosamines on
nucleus chromatin condensation, HL-60 and HepG2 cells
(1 × 106/ml) were treated with NPYR (10-50 mM) or
NDMA (27-135 mM) at different incubation times (24-72
h). After treatments, the cells were stained with acridine
orange (5 µg/ml) for 10 minutes and observed under a
UV-visible fluorescence microscope (Axiostar plus mi-
croscope, Zeiss) as described by Gregory et al. [31].
Cells exhibiting brightly fluorescent condensed or frag-
mented nuclei were considered apoptotic. A total of 200
cells were counted in multiple randomly selected fields,
and the percentage of apoptotic cells was then calculated.
2.5. Western Blot
After incubation of cells with NPYR (10, 30 and 50 mM)
or NDMA (27, 68 and 135 mM) for 24-72 h (HepG2),
protein extracts were obtained with Nucbuster Protein
Extraction Kit (Novagen, Darmstadt, Germany). Equal
amounts of protein cell extracts (30 μg) measured by the
Non Interfering Protein Assay Kit (Calbiochem) were
used for western blot analyses. Samples were resus-
pended in a buffer containing 63 mM Tris-HCl, pH 6.8,
10% glycerol, 1% 2-mercaptoethanol, 2% sodium dode-
cyl sulfate (SDS) and 0.025% bromophenol blue and
boiled for 15 min. Proteins were resolved on a 10% so-
dium dodecyl sulfate-polyacrylamide gel and electroblo-
tted onto an immune-blot PVDF membrane (Bio-Rad
Laboratories) at 119 V for 1 hour and 15 min in Tris gly-
cine buffer (25 mM Tris, 192 mM glycine, 20% metha-
nol, pH 8.3). Equal protein loading and the integrity of
transfer were confirmed by Blot-Fast-Stain (Chemicon,
Temecula, CA). Subsequently, the membranes were
blocked overnight in milk block buffer (PBS, 0.2%
Tween, 10% non fat dry milk) and then incubated with
polyclonal poly (ADP-ribose) polymerase (PARP) anti-
body diluted 1:1000 in milk block buffer on a plate
shaker for 1 h at room temperature. The membranes were
then washed three times (10 min each) in milk block
buffer, and goat anti-rabbit peroxidase conjugated diluted
1:3000 in milk block buffer was applied to the blots for 1
h at room temperature with shaking. Then, the blots were
washed as described above and one more time with
PBS-Tween. Blots were developed using the super signal
substrate (Pierce, Rockford, IL) and chemiluminiscence
was directly detected using Bio-Rad Fluor S instrument
and analysed used the Bio-Rad quantity one software
package.
2.6. Caspase Activity
To address the significance of caspases activation in N-
nitrosamines-induced apoptosis in HepG2 cells, we used
permeable, specific and potent caspase inhibitors,
Z-DEVD-FMK (caspase-3 inhibitor), Z-VEID-FMK (ca-
spase-6 inhibitor), Z-IETD-FMK (caspase-8 inhibitor)
and Z-LEHD-FMK (caspase-9 inhibitor). HepG2 cells
were treated with 50 mM NPYR (48 h) or 68 mM NDMA
(72 h) in the presence or absence of 100 μM of caspase
inhibitors. After the incubation, the percentage of apop-
totic cells was determined by TUNEL assay and flow
cytometry.
2.7. Statistical Analyses
The Student’s t-test was used for statistical comparison
and differences were considered significant at p ≤ 0.01.
Descriptive and graphical methods were used to charac-
terize the data. All tests were performed with the soft-
ware package Statgraphics Plus 5.0.
3. Results
3.1. Analysis of Morphological Changes Induced
by NPYR and NDMA
Initial studies were performed to investigate whether
NPYR and NDMA-induced apoptosis in human cancer
cells. Thus, cells were treated with 10-50 mM NPYR or
27-135 mM NDMA for different time periods (24-72 h),
and nuclear morphology was observed by fluorescence
microscopy using acridine orange. 24 hours treatment of
HL-60 cells at the highest doses of NPYR (50 mM) and
NDMA (135 mM) induced 58% and 39% of apoptosis,
respectively. In HepG2 cells, 24 h of treatment with
NPYR (50 mM) and NDMA (135 mM), induced above
62-46% of apoptosis, respectively.
3.2. TUNEL Assay
The TUNEL assay is a sensitive test to detect the DNA
strand breaks that are a hallmark of the late stages of
apoptosis [32]. TUNEL analysis showed that NPYR and
NDMA-induced apoptosis in HL-60 (Figure 1) and Hep-
G2 cells (Figure 2), in a concentration and time depend-
ent-manner. An increase in the number of apoptotic (41-

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines 317
(a)
(b)
Figure 1. Flow cytometric analysis using TUNEL assay of
HL-60 cells treated with different concentrations of NPYR
(a) and NDMA (b) for 24 h ( ) , 48 h ( ) and 72 h ( ).
Co, untreated cells; C1, cells treated with etoposide (5 μM).
Asterisks indicate significant difference from control *** p
≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05.
51%) HL-60 cells was apparent after 72 h incubation with
10-50 mM NPYR (Figure 1(a)). The short-time treat-
ments (24 and 48 h) with NDMA (27 and 68 mM), did
not induce a significant percentage of apoptotic cells
(4%), whereas 72 h induced about 43-48%, respectively
(Figure 1(b)). After treatment with 135 mM of NDMA,
an increase in the percentage of apoptotic cells was de-
tected from 48 to 72 h of incubation (41-49%, respec-
tively).
The presence of apoptotic cells was noted in HepG2
cells treated with NPYR for 48 h (24-52%) (Figure 2(a)).
When the cells were treated for 72 h, the highest number
of apoptosis was induced with 50 mM of NPYR (68%).
As shown in Figure 2(b), only a moderate number of
TUNEL positive cells were observed after treatment with
NDMA for 24 and 48 h. However, at 72 h, a remarkable
percentage of apoptotic cells occurred at all concentra-
tions of NDMA. More than 70% of HepG2 cells were
TUNEL positive after treatment with 135 mM of NDMA.
3.3. Western Blot
In view of the ability of NPYR and NDMA to induce
apoptosis by the TUNEL assay, it was considered of in-
(a)
(b)
Figure 2. Flow cytometric analysis using TUNEL assay of
HepG2 cells treated with different concentrations of NPYR
(a) and NDMA (b) for 24 h ( ) , 48 h ( ) and 72 h ( ).
Co, untreated cells; C1, cells treated with etoposide (100
μM). Asterisks indicate significant difference from control
*** p ≤ 0.001 and ** p ≤ 0.01.
terest to examine the role of caspases. PARP is a prefer-
ential substrate for caspase-3 and is cleaved by this pro-
tein into 85 and 24 KDa fragments during the apoptotic
mode of cell death [33]. Thus, protein extracts from
HL-60 and HepG2 cells untreated and treated with
NPYR, NDMA and etoposide, were electroblotted and
probed against a PARP polyclonal antibody that recog-
nizes the 116-kDa intact PARP as well as an 85-kDa
cleaved product. Quantification of PARP cleavage was
determining by densitometry of the intensity of full-
length protein signal visualized by polyclonal anti-PARP
antibody.
As shown in Figure 3, untreated HL-60 cells showed
only intact PARP at 116 KDa (Figures 3(a) and 3(b),
lane 1). In contrast, all the PARP present in the 5 μM
etoposide-treated cells had been cleaved into the 85 kDa
fragment (Figures 3(a) and 3(b), lane 2). No PARP
cleavage was detectable after incubation of cells with 10
mM NPYR (Figure 3(a), lanes 3 and 4). Treatment of
HL-60 cells with 30 mM NPYR induced the cleavage of
PARP from 116 to 85 kDa at 48 h (Figure 3(a), lanes 5
and 6). Some uncleaved PARP remained in 50 mM
NPYR treated HL-60 cells at 24 h, whereas a PARP
cleavage product of 85kDa was prominent at 48 h (Fig-
ure 3(a), lanes 7 and 8). As shown in Figure 3(b),

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines 318
(a)
(b)
Figure 3. Western blot of PARP cleavage in HL-60 cells
treated with NPYR (a) and NDMA (b). Lane 1 represents
untreated cells and lane 2 represents cells treated with eto-
poside. Lane 3 cells treated with (a) 10 mM or with (b) 27
mM for 24 h and lane 4 for 48 h. Lane 5 cells treated with (a)
30 mM or with (b) 68 mM for 24 h and lane 6 for 48 h. Lane
7 cells treated with (a) 50 mM or with (b) 135 mM for 24 h
and lane 8 for 48 h.
treatment of the cells with NDMA caused the proteolytic
cleavage of PARP with accumulation of an 85 KDa frag-
ment and the concomitant disappearance of the original
116 KDa PARP at all test doses.
Figure 4 shows the results obtained in HepG2 cells
treated with NPYR and NDMA. In HepG2 cells un-
treated and treated with etoposide (100 μM for 24 and 72
h), western blot revealed only a single band at 116 KDa
representing full-length enzyme (Figures 4(a) and 4(b),
lanes 1 and 2). Similarly, PARP cleavage could not be
detected after the treatment of HepG2 cells with different
doses of NPYR or NDMA for 24 to 72 h (Figures 4(a)
and 4(b), lanes 3 to 8).
3.4 Effects of NPYR and NDMA on the Caspase
Pathway in HepG2 Cells
Since the key effector molecules of the apoptotic process
belong to the caspase family, we evaluated the ability of
NPYR (50 mM, 48 h) and NDMA (68 mM, 72 h) to in-
duce apoptosis in HepG2 cells in the presence or absence
of different caspase inhibitors (100 μM).
(a)
(b)
Figure 4. Western blot of PARP cleavage in HepG2 cells
treated with NPYR (a) and NDMA (b). Lane 1 represents
untreated cells and lane 2 represents cells treated with eto-
poside. Lane 3 cells treated with (a) 10 mM or with (b) 27
mM for 24 h and lane 4 for 72 h. Lane 5 cells treated with (a)
30 mM or with (b) 68 mM for 24 h and lane 6 for 72 h. Lane
7 represents cells treated with (a) 50 mM or with (b) 135
mM for 24 h and lane 8 for 72 h.
As shown in Figure 5, the addition of Z-IETD-FMK
(caspase-8 inhibitor) significantly diminished NPYR-
and NDMA-induced apoptosis in a 76-64%, respectively.
TheZ-DEVD-FMK (caspase-3 inhibitor) reduced the
apoptotic effect of NPYR and NDMA in 79-58%, re-
spectively, and Z-VEID-FMK (caspase-6 inhibitor) in-
hibited a 63% both N-nitrosamines. The blockage of
apoptosis by Z-LEHD-FMK (caspase-9 inhibitor) caused
an inhibition of NPYR- and NDMA-induced apoptosis of
56 and 52%, respectively.
4. Discussion
Apoptosis induced by carcinogens seems to have an im-
portant role in cancer development [34]. Accordingly, the
mechanism and cell signalling pathways involved in food
carcinogens-induced cell death or cell survival and pro-
liferation have recently received much interest [26,35]. In
1978 the International Agency for Research on Cancer
(IARC) classified NPYR and NDMA as possibly and
probably carcinogenics to humans, respectively [36]. It is
widely accepted that N-Nitrosamines require metabolic

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines 319
(a)
(b)
Figure 5. Effect of specific caspase inhibitor on apoptosis
induced by (a) 50 mM NPYR (48 h) or (b) 68 mM NDMA
(72 h) in HepG2 cells, using TUNEL assay and flow cy-
tometry. C ( ), HepG2 cells treated with N-nitrosamines
and without caspase inhibitor. ( ) HepG2 cells treated
with N-nitrosamines and specific caspase-3, -6, -8 or -9 in-
hibitor. Asterisks indicate significant difference from con-
trol *** p ≤ 0.001 and ** p ≤ 0.01.
activation by cytochrome P-450 to become carcinogenic.
The activatedN-Nitrosamine attacks and covanlently
binds to DNA, forming detectable DNA adducts. Wang
et al. [37] showed that there was a significant positive
association between NPYR exposures and having de-
tectable several adducts in hepatic DNA of rats. More-
over, Cheng et al. [38] have just demonstrated that
NDMA damage calf thymus DNA through reactive me-
tabolites. DNA lesions are converted into double strand
breaks that act as a trigger for the up-regulation of p53,
which induces apoptosis by the cell death receptor path-
way [39].
In the present study, a variety of methods have been
employed to detect and quantify apoptosis, since many
studies suggest that the utilisation of two or more diffe-
rent techniques may be convenient to avoid determina-
tion errors [40,41]. Our results clearly demonstrated that
NPYR and NDMA-induced apoptosis in a concentration
and time dependent-manner as judged by the TUNEL
assay (Figures 1 and 2). Moreover, we observed that
HepG2 cells were more sensitive to the treatments than
HL-60 cells. At 72 h, 50 mM NPYR induced 51% of
apoptosis in HL-60 and 68% in HepG2 cells, whereas
135 mM NDMA caused 49% and 78% of apoptosis in
HL-60 and HepG2 cells, respectively. It has been re-
ported that cell lines differ substantially in their sensitiv-
ity towards various classes of apoptotic chemicals [42].
Thus, Duc and Leong-Morgenthaller [43] found that the
heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,
5-b]pyridine (PhIP) induced different apoptotic response
in two human lymphoblastoid cell lines (TK6 and MT1).
A five to six fold increase and less than a two fold in-
crease in the fraction of apoptotic cells were observed in
TK6 and MT1, respectively. In addition, in our previous
studies [44,45] HepG2 cells were more resistant to the
apoptosis induction by N-nitrosodibutylamine (NDBA)
and N-nitrosopiperidine (NPIP) than HL-60 cell line.
N-nitrosamines are metabolised by enzymes of the mixed-
function cytochrome P-450-dependent monooxidase sys-
tem. Cyclic N-nitrosamines such as NPYR and NPIP are
primarily activated by CYP2A6. On the other hand, short
chain N-nitrosamines such as NDMA is activated by
CYP2E1 whereas CYP1A1 is involved in the metabolism
of the longer chain N-nitrosamines such as NDBA [46].
Thus, a possible explanation of the variation in the per-
centage of apoptotic cells induced by N-nitrosamines
could be attributed to the differences in the levels of en-
zymatic activities in both cell lines.
Both N-nitrosamines induce apoptosis in HL-60 and
HepG2 cells, even though NPYR was most effective than
NDMA at lower concentration. Numerous studies have
reported that genotoxicity of NDMA in human hepatoma
cell lines was observed only at high concentrations [47,
48]. Furthermore, we have demonstrated that NPYR ex-
erted greater oxidative DNA damage in HepG2 cells than
NDMA by using the Comet assay [49]. The lowest con-
centration of NDMA required to cause a significant in-
crease in DNA damage was approximately 5-fold higher
than that of the corresponding NPYR. In comparison
with our previous studies [44,45] NDBA was the most
effective N-nitrosamine to induce apoptosis in both cell
lines by the TUNEL assay. Thus, after 24 h incubation
with NDBA at 3.5 mM, the percentage of apoptotic
HepG2 cells reached 95%, whereas it was necessary to
use doses of 45 mM NPIP (86%), 50 mM NPYR (68%)
and 135 mM NDMA (77%) and longer incubations times
(72 h) to obtain a high percentage of apoptotic HepG2
cells. Similar findings have been obtained in the leuke-
mia HL-60 cell line. At 72 h, 2.5 mM NDBA induced
69% of apoptotic HL-60 cells, whereas it was necessary
doses of 20 mM NPIP (75%), 50 mM NPYR (51%) and

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines 320
135 mM NDMA (49%) to obtain a high percentage of
apoptotic cells. The fact that the percentage of apoptotic
cells varied with the type of N-nitrosamine suggests that
the apoptotic effect depended on the chemical structure
of N-nitrosamine.
Activated caspase-3 cleaves PARP, a 116 kDa enzyme,
generating a fragment of 85 kDa [50]. Our results
showed that NPYR and NDMA caused PARP cleavage
in HL-60 cells, in a concentration and time dependent
manner (Figure 3). In contrast, no PARP cleavage (re-
flecting caspase-3 activation) was detected in HepG2
cells (Figure 4). These results are in agreement with Di-
bartolomeis and Moné [51] who assumed that the PARP
cleavage was based on the disappearance of the 116 kDa
fragment in Jurkat cells treated with with 500 μM eto-
poside.
To determine whether the caspases were involved in
NPYR and NDMA-induced apoptosis in HepG2 cells,
we also analysed the effects of the specific inhibitors of
caspase activity. The two major apoptotic pathways de-
scribed in eukaryotic cells are extrinsic (triggered by
death receptors) and intrinsic mediated by mitochondrial
events [52]. The apical proteases in the extrinsic and in-
trinsic pathways are caspase-8 and caspase-9, respec-
tively. Activated caspase-8 and -9 further initiates the
activation of caspase cascade leading to biochemical and
morphological changes associated with apoptosis [53].
Caspase-3 and -6 are well-known downstream effector
caspases which can be proteolytically activated by cas-
pase-8 or -9 via different signalling pathways [54].
Our results confirmed that NPYR and NDMA-induced
cell death in HepG2 cells was due to caspase-dependent
apoptosis (Figure 5). Caspase-8 seems to be the central
caspase in the NPYR and NDMA-induced apoptosis be-
cause blocking of its activity had the highest percentage
of reduction of apoptosis (76-64%, respectively). Inhibi-
tion of caspase-3 and -6 activities partially inhibited the
NPYR and NDMA-induced apoptosis which suggested
that caspase-8 was upstream of caspase-3 and -6. How-
ever, may be other mechanism for caspase-3 and -6 acti-
vations, apart from the cascade mediated through cas-
pase-8 activation [55]. Caspase-9 activity was slightly
reduced by the inhibitor, suggesting the involvement of
intrinsic pathway, but might not be the major way to in-
duce apoptosis in HepG2 cells. It is now recognized that
some chemicals induce apoptosis via the mitochon-
dria-dependent pathway in which caspase-9 is initially
activated [56] and also activate caspase-8 in the absence
of the death receptor signalling [57]. Our results are con-
sistent with the report in which heterocyclic amine, 3-
Amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1)-
induced apoptosis, mainly operates the caspase-8-depen-
dent pathway, and there is also a caspase-9-dependent
side pathway [21]. Moreover, we found that both the
intrinsic and extrinsic pathways were similarly involved
in the NPIP and NDBA-induced apoptosis in HepG2
cells [45].
Taken together, the results reported in this work
demostrate that NPYR and NDMA induce apoptosis in
HepG2 and HL-60 cell lines via caspase dependent path-
way. Further studies are needed to determine the mo-
lecular mechanism of NPYR and NDMA induce apop-
tosis.
5. Acknowledgements
This work has been supported by Grant ALI2002-01033
from the Ministerio de Ciencia y Tecnología (Spain) and
by Grant 910177 from the Comunidad de Madrid and the
Universidad Complutense (UCM). N. Arranz is recipient
of Fellowship from the Ministerio de Ciencia y Tec-
nología, Spain.
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