American Journal of Molecular Biology, 2011, 1, 70-78
doi:10.4236/ajmb.2011.12009 Published Online July 2011 (http://www.SciRP.org/journal/ajmb/
AJMB
).
Published Online July 2011 in SciRes. http://www.scirp.org/journal/AJMB
N–nitrosodiethylamine cytochrome P450 induction and
cytotoxicity evaluation in primary cultures of rat hepatocytes
Claudia Alessandra Fortes Aiub1,2, Gabriele Gadermaier3, Fátima Ferreira3, Israel Felzenszwalb4,
Peter Eckl1, Luis Felipe Ribeiro Pinto2
1Department of Cell Biology, Division of Genetics, Salzburg University, Salzburg, Austria;
2Department of Biochemistry, Rio de Janeiro State University, Rio de Janeiro, Brazil;
3Department of Molecular Biology, Salzburg University, Salzburg, Austria;
4Department of Biophysics and Biometry, Rio de Janeiro State University, Rio de Janeiro, Brazil.
Received 28 March 2011; revised 27 May 2011; accepted 18 June 2011.
ABSTRACT
The primary routes of potential human exposure to
N-nitrosodiethylamine (NDEA) are ingestion, inhala-
tion, and dermal contact. Air, diet and smoking con-
tribute to potential huma n exposure at levels of a few
µg of NDEA/day. Potential exposure depends on the
ability of the nitrosamines to migrate from the prod-
uct into the body. The first step in the metabolic deg-
radation of NDEA by cytochrome oxidase (CYPs)
enzymes is the introduction of a hydroxyl group and
in human esophage and liver CYP2A3 and CYP2E1
participate on this metabolism. Measuring cytotoxic-
ity in female rat primary hepatocytes cultures, were
used to understand the CYP induction and metaboli-
zation correlated with low NDEA concentrations. We
observed that NDEA at different concentrations in
the absence of CYPs inducers, was able to induce
CYP2B1, CYP2B2, CYP2E1, CYP3A1 and CYP4A3.
A positive NDEA synergistic effect on the levels of
mRNA, was observed in the presence of pyrazole
(300 µM) for CYP2B1 and CYP2B2 and for pregne-
nolone 16-α carbonitrile (0.15 µM) for CYP2E1.
Negative NDEA synergistic effects were observed for
ethanol (0.3%) for CYP3A1, pyrazol (300 µM) for
CYP2A1 and CYP2E1, and phenobarbital (1 mM)
for CYP2A1. These facts are extremally important
once that these metabolites can be directly related to
the primary DNA lesions. We consider that studies to
elucidate the biological factors that determine the
shape of the dose-response curve are crucial for low-
dose extrapol ations of risk.
Keywords: N-Nitrosodiethylamine; Cytochrome P450;
Cytotoxicity; Primary Culture; Hepatocyte; Real-Time
PCR.
1. INTRODUCTION
Cytochrome oxidase (CYPs) form a superfamily of haem-
thiolate proteins present in prokaryotes and throughout
the eukaryotes. CYPs act as mono-oxygenases, with
functions ranging from the synthesis and degradation of
endogenous steroid hormones, vitamins and fatty acid
derivatives (“endobiotics”) to the metabolism of foreign
compounds such as drugs, environmental pollutants, and
carcinogens (“xenobiotics”) [1]. The majority of the
CYP isoforms are found in the liver, however other ex-
tra-hepatic sites include the center nervous system, gas-
trointestinal tract, kidney, lungs, and adrenal glands [2].
CYP gene expression is regulated by several factors,
as gender, microsomal enzyme inducers, age, diet, and
hormones. Differences in the amounts and intrinsic ca-
pacities of CYP forms to metabolize a particular drug or
chemical may influence profoundly drug-drug interac-
tions, drug or carcinogen activation and detoxification
contributing to the development of cancer, Parkinson’s
disease, and adrenal hyperplasia [3].
N-nitrosodiethylamine (NDEA) is activated by cyto-
chrome P450 enzymes, resulting in ethylation of N and
O atoms of most bases from DNA. The N7, and O6 po-
sitions of guanine are preferable ethylated, and a lower
level of ethylation is also observed at the O4 position of
thymine [4]. O6-ethylguanine and O4-ethylthymine, if
not repaired, will lead to mutation and tumour formation
in superior animals [5]. NDEA is able to produce tu-
mours in many species of animals and in a variety of
organs, requiring metabolic activation through P450-
catalyzed α-hydroxylation, generating unstable metabo-
lites that will alkylate the DNA at the site of activation,
in order to induce tumors [6]. Till the present, there is no
evidence that NDEA is a CYP inducer or if CYP’s in-

C. A. F. Aiub et al. / American Journal of Molecular Biology 1 (2011) 70-78 71
duction can increase or decrease others previously in-
duced CYPs.
The presence of NDEA in foodstuffs has been a sub-
ject of several reviews [7-11]. Druckrey and Preussmann
(1962) postulated that NDEA formation could arise in
tobacco smoke via the interaction of nitrogen oxides and
tobacco amines [12]. Additionaly, mechanisms for the in
vivo formation of NDEA may involve chemical and en-
zymatic nitrosation especially dependent on the presence
of both nitrate and nitroreductases [13-16].
In Wistar rats, a commonly used experimental model
for oesophageal carcinogenesis, NDEA induces tumours
at low doses. When higher doses are given for a shorter
period, tumours originate mainly in the liver [5]. Rea-
sons for such interorgan differences are currently un-
known but it has been hypothesized that this could be
due to different enzymes being responsible for the me-
tabolic activation of NDEA [17,18].
The differences in the amounts and intrinsic capacities
of CYP forms to metabolize a particular chemical may
influence: drug-drug interactions, drug or carcinogen
activation and detoxification. This knowledge has con-
tributed to an increase in the scientific literature on
CYP-dependent drug metabolism. Predicting the ability
of a drug, like NDEA, to modulate CYP expression at an
early stage may permit the identification of alternative
noninducing chemical structures. For the pharmaceutical
industry the potential induction of various CYPs by
drugs candidates, is important once they can lead to tox-
icity or reduce drug-drug interactions efficacy.
During the last few years, there has been a great in-
terest in developing rapid and simple tests to identify the
effects of exposure to environmental agents that can in-
duce DNA damage. Therefore, in the present study using
primary female rat hepatocytes we 1) Analyzed the ef-
fects of six microsomal enzyme inducers on CYPs ex-
pression, and 2) Investigated NDEA participation on
CYPs mRNA regulation. The data will provide essential
information to whether classes of microsomal enzyme
affect rat CYPs mRNA levels at low NDEA doses sug-
gesting potential roles of specific ligand-activated tran-
scription factor pathways.
2. MATERIAL AND METHODS
2.1. Animal Model
This study was conducted in compliance NRC (National
Research Council and the “Guide for the Care and Use
of Laboratory Animals”. Female albine Fischer 344 rats
(F-344/DuCrl) from Charles River Laboratories (Ger-
many) between 6 - 8 weeks old were used for the ex-
periments. Light/dark regime was 12/12 h, and standart
pelleted rat feed and drinking water were supplied ad
libitum.
2.2. Hepatocyte Isolation and Culture
Hepatocytes were prepared according to the two-step
collagenase perfusion method [19] with modifications
[20]. Fischer 344 female rats were anesthetized with
sodium pentobarbital (200 mg/kg) and following hepatic
portal vein cannulation, livers were perfused with 200
mL of solution A (NaCl 142 mM, KCl 6.7 mM, HEPES
10 mM) pH 7.4, during 12 - 15 min at 15 mL/min. Liv-
ers were subsequently perfused with 200 mL of solution
C (9:1 of solution A and CaCl2 5.7mM), pH 7.4, con-
taining 0.5 mg/mL collagenase (Collagenase Sigma IV,
125 CDU/mg, CAS 9001-12-1) for 20 min at 10 mL/min.
Perfused livers were excised and dispersed in 50 mL of
solution A and shaken in a closed, sterile container at
37˚C for 10 min. Hepatocyte preparation was filtered
through 180 µm nylon filter and centrifuged at 500 rpm,
for 10 min, at 4˚C. The wash was repeated and the cells
resuspended in MEM eagle Ca++ 1.8 mM (Gibco), sup-
plemented with NaHCO3 (26.2 mM), pyruvate 1mM,
aspartic acid 0.2 mM and L-serine 0.2 mM. Hepatocytes
were counted by hemacytometry, and (2 - 5) × 105
cells/mL were added to 60 mm, and (2 - 5) × 106
cells/mL were added to 90 mm collagen-coated dishes.
Hepatocytes were allowed to attach for 2 hours. Viability,
measured by Trypan blue staining, was approximately
85% - 90% and only preparations with viabilities greater
than 80% were used in experiments. After attachment,
the medium was removed and replaced with fresh MEM
eagle Ca2+ 1.8 mM (Gibco).
2.3. Incubation of Cells
To investigate the effects of drugs on CYP induction,
primary rat hepatocytes were incubated after the first
medium changed, for 14 - 16 h with the CYP inducers
drug. Ethanol (ETOH), Pyrazole (PYR), and Phenobar-
bital (PB) were prepared in NaCl 0.9% solution and
added directly to cultures to give a final concentration of
0.3%, 300 µM and 1 mM. 3-Methylcholanthrene (3-MC),
Streptozotocin (STR) and Pregnenolone 16-

carboni-
trile (PNC) were dissolved in ethanol 30% and added to
culture medium to give a final concentration of 2 µM, 25
µM and 0.15 µM, respectively.
After 14 - 16 h in the presence or in the absence (W/O
group) of CYP inducers, rat hepatocytes were incubated
during 3 h, at NDEA final concentrations ranging from
0.21 µg/mL to 105 µg/mL. All experiments were per-
formed in triplicate, using five isogenic animals.
2.4. Cytotoxicity
The studies were performed in triplicate as described by
Eckl and Riegler (1987) with some modifications [20].
For determination of the number of apoptotic and ne-
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crotic cells, MEM Eagle 0.4 mM was replaced by cold
fixative methanol-glacial acetic acid (3:1) for 15 min on
the petri dishes, rinsed with distilled water for 2 min and
air dried. The fixed cells were stained with DAPI (0.2
pg/ml) dissolved in McIlvaine buffer (Citric acid 0.1M,
Na2HPO4 0.2M, pH 7.0) for 40 min, washed with McIl-
vaine buffer for 2 min, briefly rinsed with distilled water
and mounted in glycerol. 1000 cells/petri dish (3000
cells per animal/group concentration) were analyzed
under the fluorescence microscope (Reichert Univar).
The cell death processes are presented as the percentage
of cells in 3000 total cells per animal/group concentra-
tion) analized. Apoptotic cells were characterized by the
occurrence of nuclear condensation, DNA fragmentation,
and perinuclear apoptotic bodies, using a fluorescence
microscopy (×100 or higher). Necroses were character-
ized by clumping, increased vacuolation and swelling on
the cell membrane.
2.5. RNA Isolation
Total RNA was isolated with TRIzol (Invitrogen, Ger-
many) and treated with DNase I (0.5 Units) (Invitrogen),
according to the manufacturer’s instructions, and disol-
ved in DEPC water. The concentration of the isolated
total RNA was spectrophotometricaly determined at
A260nm and diluted in DEPC water at 2 µg/µL. Aliquots
of RNA were analyzed by agarose/formadehyde gel
electrophoresis to check RNA integrity and stored at
–20˚C until further use.
Development of primers for quantitative Real Time
PCR Coding sequences for the genes listed in Tab le 1,
were obtained from GenBank (http://www.ncbi.nml.nih.
gov/GenBank). Target regions within the coding se-
quences were determined through nucleotide sequence
alignment comparisons of target within multiple member
gene families using Vector NTI (Informax, Inc., Be-
thesda, MD). A subfamily-specific region for each CYP
was selected as the site of hybridization for either the 5’
or 3’ CYP PCR primer, and then complementary PCR
oligos were screened on the basis of 1) Similar melting
temperatures, 2) Similar oligo length and, 3) The produ-
tion of a PCR amplicon with greater than 50% GC con-
tent. All primers were submitted to the National Center
for Biotechnological Information for nucleotide com-
parison using the basic logarithmic alignment search tool
(BLASTn) to ensure specificity.
2.6. cDNA Synthesis
First-strand cDNA synthesis was performed on the re-
maining DNase-treated total RNA which was reverse-
transcribed using Oligo primers (0.3 ng/µL) and Super-
scriptTM III bulk mix (Invitrogen), according to the
manufacter’s instructions. In addition, duplicate no tem-
plate control samples were run in identitical conditions.
Table 1. Primer sequences used for Real-Time Quantitative
RT-PCR.
Gene/locationTm ˚CPrimerSequence Amplicon bp
67.5F AGTCCCTGCCCTT
TGTACACACCGC
18S RNA
(V01270) 69.5R
ACCATC-
CAATCGGTAG-
TAGCGACGGG
152
68.1F
TCAATCCTCACTG
GCCAC-
TATGCTGGACA
CYP2A1
(J04187)
69.5R
CAGAGGGACAC-
CAAGAGCAT-
GACGCTC
90
68 F
CAGCCAGGTGTTT
GAGTTCTTCTCTG
GG
CYP2B1
(AJ320166)
66.4R CCCTGTGCTTCTCC
ACAATATGGCCA
122
66.1F
ACATGTGAACA-
GAGATTTCAT-
GAGTACA-
CATCTCAT CYP2B2
(J00720)
68.3R
TGTAGACATAG-
CACTGAGAC-
CATATACA-
GAGTCCAT
148
66.8F
CTCCTCGTCATATC
CATCTGGAAGAA-
GATCT
CYP2E1
(J02627)
63.7R
TGGTGAAA-
GACTTGGGGA-
TATCCTTCAA
127
CYP3A1
63.9F
GTTTATGGAAATTC
GATGTGGAGTGCC
AT 153
(M10161) 64.4R CCCGCCGGTTTGT
GAAGACAGAAA
2.7. Real Time qRT-PCR Analysis
Quantitative RT-PCR reactions were performed using
the reagent mix and protocol contained in the BioRad
iCycler iQ Real-Time Detection System (BioRad). Re-
actions were run in duplicate, in a volume of 10 µL,
containing 5 µL of the cDNA diluted 1:20 in DEPC wa-
ter, 4.4 µL of the iQ SYBR Green Supermix and 0.6 µL
of the primers mix (forward and reverse primers) at a 3
pmol/µL. The cycle was programmed to 95˚C, 3 min; 40
× (95˚C 1 min, 65˚C 1 min and 72˚C 1 min); 95˚C 1
min; 55˚C 1 min. For the melting curve analysis, it was
used a gradient from 55˚C to 95˚C. Real-time PCR data
were collected and analyzed on a Rotor-Gene 3000
(Corbett Research). The products were checked by aga-
rose gel electrophoresis and by sequencing.
The endpoint used in the real-time PCR quantification,
CT value, is defined as the PCR cycle number that
crosses an arbitrarily placed signal threshold. Average
CT values from duplicate PCR reactions were normal-
ized to average CT values for housekeeping gene from
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Tabl e 2. NDEA cytotoxicity followed administration of classi-
cal CYP inducers ETOH (0.3%), PYR (300 µM) and PB (1
mM).
Inducer Assay condi-
tions % Survival % Necrosis % Apoptosis
NaCl 0.9% 98.7 ± 0.3 1.1 ± 0.4 0.2 ± 0.1
0.21 µg/mL 98.2 ± 0.4 1.6 ± 0.3 0.2 ± 0.1
2.1 µg/mL 97.4 ± 0.5*2.4 ± 0.5* 0.2 ± 0.1
21 µg/mL 97.2 ± 0.7*2.4 ± 0.6* 0.4 ± 0.2
W/O
105 µg/mL 97.2 ± 1.0 2.5 ± 1.0 0.4 ± 0.1*
NaCl 0.9% 98.1 ± 0.6 1.4 ± 0.4 0.6 ±0.2
0.21 µg/mL 97.7 ± 0.6 1.6 ± 0.4 0.7 ± 0.1†
2.1 µg/mL 97.5 ± 0.6 1.5 ± 0.3 1.0 ± 0.3†
21 µg/mL 96.6 ± 1.4 2.5 ± 0.7 1.2 ± 0.4†
ETOH
105 µg/mL 95.7 ± 1.1 2.6 ± 0.8 1.7 ± 0.2*††
NaCl 0.9% 98.7 ± 0.5 0.9 ± 0.1 0.4 ± 0.4
0.21 µg/mL 98.1 ± 0.2 1.2 ± 0.2 0.8 ± 0.0††
2.1 µg/mL 97.0 ± 0.7 2.1 ± 0.4 0.9 ± 0.3†
21 µg/mL 95.1 ± 1.0*3.0 ± 0.2** 2.0 ± 0.8†
PYR
105 µg/mL 95.9 ± 0.4*3.0 ± 0.1** 1.1 ± 0.2†
NaCl 0.9% 98.0 ± 0.2 1.3 ± 0.1 0.8 ± 0.1††
0.21 µg/mL 97.9 ± 0.5 1.5 ± 0.3 0.7 ± 0.2†
PB 2.1 µg/mL 93.8 ± 0.3**†† 4.8 ± 0.5*† 1.4 ± 0.2††
21 µg/mL 93.4 ± 0.4**†† 4.7 ± 0.0**† 2.0 ± 0.4*††
105 µg/mL 92.9 ± 0.4**† 3.6 ± 0.3* 3.6 ± 0.1**††
Mean ± SEM of 3 female Fischer 344 rat livers. Were analysed 3000 cells
per animal/group concentration. Statistical analysis, in order to compare the
NDEA cytotoxic effect related with the control (NaCl 0.9%) shown signifi-
cant differences (*p < 0.05 and **p < 0.01) into the same inducer group.
Comparative statistical analysis were made between W/O group and the
CYP inducers group (†p < 0.05 and ††p < 0.01) at same NDEA concentra-
tion.
the same cDNA preparations (Δ CT). The ratio of ex-
pression of each CYP gene induced vs. expression of
each CYP gene spontaneously induced was calculated as
ΔΔCT. The fold induction of each CYP gene is calcu-
lated as 2–(ΔΔCT) as recommended by Perkin-Elmer. Val-
ues were reported as an average of triplicate analyses.
The amount of each gene target in different groups
was normalized to an endogenous control (18S ribo-
somal mRNA).
3. RESULTS
The incubation of the hepatocytes with either each in-
ducer or with different concentrations of NDEA alone
did not result in cell viability lower than 90%, as shown
in Tables 2 and 3.
However, a discrete cytotoxicity was observed when
PB, STR and PNC were incubated with hepatocytes in
the presence of NDEA at 2.1 - 105 µg/mL, 21 µg/mL,
and 0.21 µg/mL, respectively. Most of the cytotoxicity
was a result of the increase of the necroses rate. ETOH,
PYR and PB increased the rate of apoptoses compared to
the control group either with or without different con-
centrations of NDEA (Tables 2 and 3).
The effect of different classical CYP inducers and of
different concentrations of NDEA, either alone or to-
gether, on the expression of CYP mRNAs in rat hepato-
cytes is shown in Figures 1-5. Figure 1 shows that STR
Table 3. NDEA cytotoxicity followed administration of classi-
cal CYP inducers ETOH (0.3%), 3-MC (2 µM), STR (25 µM)
and PNC (0.15 µM)
Inducer Assay condi-
tions % Survival % Necrosis % Apoptosis
NaCl 0.9%98.1 ± 0.6 1.4 ± 0.4 0.6 ± 0.2
0.21 µg/mL97.7 ± 0.6 1.6 ± 0.4 0.7 ± 0.1
2.1 µg/mL97.5 ± 0.6 1.5 ± 0.3 1.0 ± 0.3
21 µg/mL 96.6 ± 1.4 2.5 ± 0.7 1.2 ± 0.4
ETH
105 µg/mL95.7 ± 1.1 2.6 ± 0.8 1.7 ± 0.2*
NaCl 0.9%95.4 ± 0.5 3.8 ± 0.6† 0.9 ± 0.1†
0.21 µg/mL93.6 ± 0.7 5.7 ± 0.9† 0.7 ± 0.2†
2.1 µg/mL95.6 ± 0.3 3.3 ± 0.2† 1.2 ± 0.0*
21 µg/mL 95.9 ± 0.1 2.8 ± 0.3 1.4 ± 0.1*
3-MC
105 µg/mL95.9 ± 0.6 3.1 ± 0.6 1.1 ± 0.0
NaCl 0.9%98.8 ± 0.4 0.9 ± 0.2 0.3 ± 0.2
0.21 µg/mL98.2 ± 0.0 1.1 ± 0.1 0.8 ± 0.1
2.1 µg/mL97.1 ± 0.6 2.1 ± 0.4 0.8 ± 0.2
21 µg/mL 93.7 ± 0.9*† 3.9 ± 1.0 2.5 ± 0.1**
STR
105 µg/mL96.2 ± 0.0** 2.8 ± 0.1** 1.0 ± 0.1*
NaCl 0.9%95.0 ± 0.1 4.3 ± 0.2† 0.8 ± 0.1†
0.21 µg/mL92.9 ± 0.3* 6.5 ± 0.2*†† 0.6 ± 0.1††
PNC 2.1 µg/mL95.2 ± 0.8 3.7 ± 0.9 1.1 ± 0.1
21 µg/mL 95.8 ± 0.1 3.0 ± 0.1* 1.3 ± 0.0**
105 µg/mL95.7 ± 0.5 3.1 ± 0.7 1.2 ± 0.2
Mean ± SEM of 3 female Fischer 344 rat livers. Were analysed 3000 cells
per animal/group concentration.Statistical analysis, in order to compare the
NDEA cytotoxic effect related with the control (NaCl 0.9%) shown signifi-
cant differences (*p < 0.05 and **p < 0.01) into the same inducer group.
Comparative statistical analysis were made between ETH group and the
CYP inducers group (†p < 0.05 and ††p < 0.01) at same NDEA concentra-
tion.
Figure 1. Data has been reported as fold induction of the
house keeping gene (18S), for mRNA levels of CYP2A1,
under different NDEA concentrations and CYP inducers,
measured by real-time RT-PCR.* represents the significant
difference (p < 0.05) between the NDEA treatment and the
control NaCl 0.9%, with the same inducer treatment. Ψ
represents the significant difference (p < 0.05) at same
NDEA concentration (or solvent) between the inducer con-
trol group (W/0 for ETOH, PYR and PB treatment, and
ETOH for 3-MC, STR and PNC treatment) and the inducer.
and PNC caused an induction (7- and 5-fold, respec-
tively) of CYP2A1 expression, when compared to the
solvent treated hepatocytes (W/o group with NaCl 0.9%).
NDEA produced a statistically significant decrease of
the CYP2A1 induction produced by STR (NDEA con-
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C. A. F. Aiub et al. / American Journal of Molecular Biology 1 (2011) 70-78 74
Figure 2. Data has been reported as fold induction of the
house keeping gene (18S), for mRNA levels of CYP2B1,
under different NDEA concentrations and CYP inducers,
measured by real-time RT-PCR.* represents the signifi-
cant difference (p < 0.05) between the NDEA treatment
and the control NaCl 0.9%, with the same inducer treat-
ment. Ψ represents the significant difference (p < 0.05) at
same NDEA concentration (or solvent) between the in-
ducer control group (W/0 for ETOH, PYR and PB treat-
ment, and ETOH for 3-MC treatment) and the inducer.
STR and PNC were not performed.
centrations of 2.1 and 105 µg/mL) and by PNC (NDEA
concentrations from 2.1 to 105 µg/mL). NDEA also re-
duced CYP2A1 mRNA levels in the presence of ETOH
(NDEA at 105 µg/mL), PYR (NDEA at all doses tested),
and PB (NDEA at 2.1 µg/mL).
Figure 2 shows that PYR and PB caused an induction
of (3.6- and 8.1-fold, respectively), whereas 3-MC re-
duced (3.2-fold) CYP2B1 expression. Surprisingly,
NDEA (0.21 µg/mL) produced a 2.5-fold induction of
CYP2B1. The two highest concentrations of NDEA
tested (21 and 105 µg/mL) increased the induction pro-
duced by PYR on CYP2B1 levels (1.6 and 2-fold, re-
spectively). When NDEA (at 2.1 µg/mL) was present
with 3-MC, it abolished the decrease on CYP2B1 ex-
pression produced by 3-MC. CYP2B2 mRNA expres-
sion was also induced by PYR and PB (10- and 2.3-fold,
respectively), and decreased by 3-MC (3.4-fold), as
shown in Figure 3. NDEA caused an increase of the
CYP2B2 mRNA levels (from 2.8- to 1.5-fold at concen-
trations ranging from 0.21 to 21 µg/mL). NDEA in-
creased the induction produced by PYR on CYP2B2
expression (1.4 to 2.1-fold for NDEA concentrations
ranging from 2.1 to 105 µg/mL), and, at 21 µg/mL, it
partially decreased the reduction produced by 3-MC.
CYP2E1 expression was decreased after PYR (2.0-
fold), STR (1.8-fold) and PNC (1.6-fold) treatments, as
shown in Figure 4. Similarly to CYP2B1 and CYP2B2,
CYP2E1 was also induced by NDEA, with an inverse
relationship between the NDEA dose tested and the fold-
induction observed (from 2.6- to 1.6-fold at NDEA con-
centrations ranging from 0.21 to 21 µg/mL). However,
Figure 3. Data has been reported as fold induction of the house
keeping gene (18S), for mRNA levels of CYP2B2, under dif-
ferent NDEA concentrations and CYP inducers, measured by
real-time RT-PCR. * represents the significant difference (p <
0.05) between the NDEA treatment and the control NaCl 0.9%,
with the same inducer treatment. Ψ represents the significant
difference (p < 0.05) at same NDEA concentration (or solvent)
between the inducer control group (W/0 for ETOH, PYR and
PB treatment, and ETOH for 3-MC treatment) and the inducer.
STR and PNC were not performed.”
Figure 4. Data has been reported as fold induction of the
house keeping gene (18S), for mRNA levels of CYP2E1,
under different NDEA concentrations and CYP inducers,
measured by real-time RT-PCR. *Represents the significant
difference (p < 0.05) between the NDEA treatment and the
control NaCl 0.9%, with the same inducer treatment. Ψ
Represents the significant difference (p < 0.05) at same
NDEA concentration (or solvent) between the inducer con-
trol group (W/0 for ETOH, PYR and PB treatment, and
ETOH for 3-MC, STR and PNC treatment) and the inducer.
different from CYP2B1 and CYP2B2, CYP2E1 expres-
sion was reduced when PB and PYR were incubated
with hepatocytes in the presence of NDEA. NDEA abol-
ished the decrease in CYP2E1 expression produced by
either STR or PNC.
CYP3A1 expression was induced by PNC (47-fold),
and decreased by ETOH (2.6-fold). NDEA (2.1 mg/ml)
also induced CYP3A1 mRNA. However, NDEA de-
creased the induction produced by PNC (4.8- to 2.7-fold,
for NDEA concentrations ranging from 2.1 to 105
µg/mL) and it increased the reduction produced by
ETOH on CYP3A1 expression (1.8-fold at 105 µg/mL).
There was also a reduction of CYP3A1 when STR was
incubated with the three highest doses of NDEA tested
(from 5.5- to 3.1-fold for NDEA concentrations ranging
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C. A. F. Aiub et al. / American Journal of Molecular Biology 1 (2011) 70-78 75
Figure 5. Data has been reported as fold induction of the
from 2.1 to 105 µg/mL) (Figure 5)
EA was considered a weak muta-
w that the administration of
ce
ta
ce at very
lo
house keeping gene (18S), for mRNA levels of CYP3A1,
under different NDEA concentrations and CYP inducers,
measured by real-time RT-PCR. *Represents the significant
difference (p < 0.05) between the NDEA treatment and the
control NaCl 0.9%, with the same inducer treatment. Ψ
Represents the significant difference (p < 0.05) at same
NDEA concentration (or solvent) between the inducer con-
trol group (W/0 for ETOH treatment, and ETOH for 3-MC,
STR and PNC treatment) and the inducer. PYR and PB were
not performed.”
4. DISCUSSION
Until last decades, ND
gen in classical genotoxicity assays [21-23]. Aiub, et al.
[4,24-25], standardized some genotoxicity assay (Ames
and SOS chromotest) conditions in order to show the
real genotoxicity potential of NDEA. Using more sensi-
tive strains for the Ames test, positive results were de-
tected for NDEA at doses between 1.01 ng/mL and 50.64
ng/mL, when 4% metabolic activation mixture (S9 mix)
was present [16]. Although short-term in vitro tests for
genotoxicity, play an important role in the initial screen-
ing of drugs in order to check their potential to induce
mutagenic/carcinogenic effects, it is very important to
include assays that use mammalian cells, such as pri-
mary cultures of rat hepatocytes, when these systems
maintain metabolic competence for the bioactivation of
xenobiotics by CYP enzymes. The genes encoding these
enzymes are either expressed constitutively or are in-
duced by various chemicals [26]. Hepatocytes, as the
cells that express higher CYP levels, are the most suit-
able model to investigate CYP induction and their rela-
tion with drug metabolism.
In the present work we sho
rtain chemicals results in an up or down regulation of
the transcription of different forms of CYP genes.
Human and rodent CYP2E1, 2A and 3A play impor-
nt roles in the metabolic activation of carcinogenic N-
nitrosamines [27-29]. From the present data we can
suggest the necessity of CYP2A1, 2B1, 2B2, 2E1 and
3A1 induction in NDEA-treated hepatocyte rats and also
recommend a combined treatment for preparation of the
S9 fraction in short-term genotoxicity assays.
The N-nitroso compound was able to indu
w doses, CYP2A1 (0.21 µg/mL ), CYP2B2 (0.21 - 21
µg/mL ), CYP2E1 (0.21 - 105 µg/mL ) and CYP3A1
(2.1 µg/mL ), in the absence of inducers (W/O group).
Otherwise for some of the tested inducers, in the pres-
ence of NDEA, a dose-dependent inhibition of CYP2A1
mRNA (for PYR, STR and PNC treatment, Figure 1),
CYP2B2 mRNA (for ETOH, Figure 3), CYP2E1 mRNA
(ETOH, PYR, PB, STR and PNC, Figure 4), and
CYP3A1 mRNA (for ETOH and STR treatment, Figure
5) was observed. It is known that CYP enzymes exhibit
liver-specific expression driven by transcriptional factors
[30]. One possibility is that NDEA metabolites or NDEA
by itself, are able to interact negatively on the orphan
receptors (ear-2 and ear-3) or in concert with other tran-
scriptional factors (HNF-4 and ER

). Miyazaki et al.
(2005) [31] and Weymarn, et al. (205) [32] have also
shown that the pre-treatment of female A/J mice with 8-
methoxypsoralen inhibits the CYP2A family.
Although 3-MC and PB are known to induce
0
CYP2A1
pr
nducer, fold in-
du
ress hepatic levels of CYP2E1
pr
arkable capacity to activate low
m
otein [33], the effect of NDEA on CYP2A induction
by PB did not show significant effects.
Concerning NDEA’s effects as CYP i
ction of CYP2B1 and CYP2B2 present similar profile
curves although with different intensities. Both CYPs
were strongly induced by PYR (CYP2B1 at 21 - 105
µg/mL NDEA concentration; CYP2B2 at 2.1 - 105
µg/mL) suggesting a synergistic effect on mRNA CYP
expression signaling (Figures 2 and 3). An increase in
the expression of CYP2B1 and CYP2B2 mRNA levels
in the presence of PYR can lead to an increase in the
NDEA metabolites and consequently on the DNA le-
sions. The data herein, show for the first time, the induc-
tion of a CYP2B1 and CYP2B2 by NDEA and provide
basis for a mechanism by which NDEA could modulate
biological processes.
3-MC and PB supp
otein and metabolic activity in rats [34-36]. We ob-
served a decrease on the CYP2E1 mRNA levels in the
presence of NDEA for ETH, PYR and PB inducer treat-
ments. The cultures CYP pre-induced by 3-MC did not
present significantly changes compared with the control
group (ETOH).
CYP2E1 has a rem
olecular weight N-nitrosamines such as NDEA [28].
Mori, et al. (2001) observed that the inducer 4-methylpy-
razole markedly inhibit DMN activity and showed a
higher induction of CYP2B1/2, rather than CYP2E1 [37].
This is in agreement with our findings for CYP2B1,
CYP2B2 mRNA, while for CYP2E1 mRNA is inhibited,
suggesting that NDEA activates CYP2B gene expression
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C. A. F. Aiub et al. / American Journal of Molecular Biology 1 (2011) 70-78 76
at a pretranslational level.
As described by Honkakoski and Negishi (2000) sev-
er
the only cul-
tu
CYP2B
C
e CYPs levels of mRNA in culture, it h
be
has a multifunction
xchange Service
mans, L., Kamataki, T., Stegeman, J.J.,
al CYPs degrade ligands that activate the NR respon-
sible for the regulation of this specific CYP form, creat-
ing a direct feedback loop [38]. In this way, the observed
induction of CYP2B1 and CYP2B2 by NDEA and PYR,
can act inhibiting CYP2A1 and CYP2E1.
In vitro assays with rodents, generally
red cells in which CYP2B genes respond normally to
PB treatment, are primary hepatocytes [39,40]. The me-
chanism for this is based on the PB response unit, that
confers PB inducibility on heterologous promoters pre-
senting transcriptional enhancer properties [41]. Com-
paring the effects on mRNA levels on Figures 4 and 5,
for PB treatment and concerning the fact that PB en-
hances the mRNA levels of CYP2B1 > CYP2B2, inde-
pendently of NDEA concentrations, we can suggest that
NDEA or it metabolites, can enhance the mRNA levels
of CYP2B1 by others unknown mechanisms.
Comparing the mRNA levels of CYP2B1, 2,
YP2E1 and CYP3A1 from ETOH-treated hepatocytes
with the cells without inducer, we can observe a repres-
sion in their expressions (Figures 2-5). Ethanol has been
known as a substrate of CYP2E1 [42]. The constitutive
level of hepatic CYP2E1 is regulated transcriptionally
by liver-enriched transcription factors, especially by
HNF-1 [43].
Regarding thas
en shown that rat hepatocyte in the presence of 3-MC
(2 µM) and PB (1 mM) after second day in culture need
supplementation [44]. The cells were submitted to in-
ducer drugs after 6 hours platting and kept for 16 - 18
hours until NDEA added. Then, these drugs (inducers
and NDEA) remained for 3 hours in the medium.
5. CONCLUSIONS
We have demonstrated that NDEAal
action with wide spectrum induction of phase I enzymes
and alone or in combination with different inducers
would be a pertinent inducer for metabolic enzymes in in
vitro bioassays. This pattern provides a general outline
of the induction/repression effects of NDEA and the in-
ducers used, leading to further mechanistic studies.
6. ACKNOWLEDGEMENTS
The authors were supported by the Austrian E
(OEAD), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do
Rio de Janeiro (FAPERJ) and SR2/UERJ.
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