Toxicology 215 (2005) 245–253

Acute and subacute effects of tobacco alkaloids, tobacco-specific

nitrosamines and phenethyl isothiocyanate on
N
-nitrosonornicotine metabolism in rats
Stefan Tyroller ∗ , Wolfgang Zwickenpflug, Charlotte Thalheim, Elmar Richter
Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians University, Goethestrasse 33, D-80336 Munich, Germany
Received 6 July 2005; received in revised form 14 July 2005; accepted 14 July 2005
Available online 22 August 2005

Abstract
N
-Nitrosonornicotine (NNN) was the first tobacco-specific nitrosamine (TSNA) identified as carcinogen in tobacco smoke, but no
data exist on in vivo interactions between NNN and other tobacco alkaloids, TSNA or phenethyl isothiocyanate (PEITC) which have
been demonstrated in various studies on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Acute effects on NNN metabolism
were tested in male Fischer F344 rats injected s.c. with 30 nmol/kg body weight (bw) [5-3 H]NNN either alone or simultaneously with
15 ␮mol/kg bw nicotine, nornicotine, anatabine, or anabasine, 150 ␮mol/kg bw cotinine, 3 ␮mol/kg bw myosmine, or 300 nmol/kg
bw of either N
-nitrosoanatabine or N
-nitrosoanabasine. Another group of rats was fed a diet supplemented with PEITC at 1 ␮mol/g
diet starting 24 h before NNN treatment. Within 24 h more than 80% and about 10% of the radioactivity was excreted with urine and
feces, respectively. Urinary metabolites were separated by reversed-phase radio-HPLC and identified by co-chromatography with
UV standards. In two sets of experiments with control rats treated with NNN only, 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy
acid, 44.4/44.8%), 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 32.4/31.5%), NNN-N-oxide (5.0/3.8%), 4-(3-pyridyl)butane-1,4-
diol (diol, 1.1/1.0%) and norcotinine (2.3/1.0%) were consistently detected besides unmetabolised NNN (4.7/3.3%). Co-treatment
with nicotine, cotinine, nornicotine and PEITC shifted the contribution of the two major metabolites significantly in favor of
hydroxy acid (108–113% of control) as compared to keto acid (86–90% of control). The same treatments also increased norcotinine
(135–170% of control). These changes are consistent with a decreased metabolic activation of NNN. In subacute studies rats received
NNN in drinking water for 4 weeks at a daily dose of 30 nmol/kg bw with or without nornicotine at 15 ␮mol/kg bw or myosmine
at 3 ␮mol/kg bw. On the last day of the experiment all rats received [5-3 H]NNN at 30 nmol/kg bw with a contaminated apple
bite followed by collection of urine and feces for 18 h. Most of the radioactivity, 87–96% of the dose, was recovered in urine and
only minor amounts have been excreted in feces or persisted in blood. In urine of the NNN-control group keto acid (32.2%) and
unmetabolised NNN (3.9%) were present in identical amounts as in the acute experiment whereas hydroxy acid (41.4% of total
radioactivity in urine, 93% of acute NNN control) was reduced in expense of the minor NNN metabolites. Co-administration of
nornicotine resulted in a small but significant rise of keto acid (107% of control) and a significant decrease in NNN-N-oxide (76%
of control). After co-treatment with myosmine the increase of keto acid (104% of control) was even less but still significant whereas
NNN-N-oxide and diol were significantly reduced to 72% and 79% of control, respectively.
Our experiments with rats indicate significant mutual effects of some of the major tobacco alkaloids and most relevant TSNA.
Further studies on the impact on smokers and the inhibitory effects of isothiocyanates are needed for a final risk assessment.
© 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Rat; Metabolism; NNN; Tobacco-specific nitrosamines; Tobacco alkaloids; Phenethyl isothiocyanate

∗ Corresponding author. Tel.: +49 89 2180 75744; fax: +49 89 2180 75701.
E-mail address: stty@freenet.de (S. Tyroller).

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2005.07.011
246 S. Tyroller et al. / Toxicology 215 (2005) 245–253
1. Introduction
N
-Nitrosonornicotine (NNN) was the first tobacco-
specific nitrosamine (TSNA) for which tumorigenicity
(Boyland et al., 1964) and the occurrence in tobacco
smoke (Hoffmann et al., 1974) has been proven. In vivo
metabolism of NNN has been studied in rodents (Chen
et al., 1978; Hecht et al., 1980, 1981; Hecht and Young,
1982; Hoffmann et al., 1981; Trushin and Hecht, 1999;
McIntee and Hecht, 2000), miniature pigs (Domellöf
et al., 1987) and marmoset monkeys (Castonguay
et al., 1985). Main metabolic pathways are 2
- and
5
-hydroxylation leading to 4-hydroxy-1-(3-pyridyl)-
1-butanone (HPB), 4-(3-pyridyl)butane-1,4-diol (diol),
5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactol), and 2-
(3-pyridyl)butyrolactone (lactone) which are further
metabolized to the major urinary NNN metabolites 4-
(3-pyridyl)-4-oxobutanoic acid (keto acid) and 4-(3-
pyridyl)-4-hydroxybutanoic acid (hydroxy acid), respec-
tively (Fig. 1). Other minor metabolites are NNN-N-
Fig. 1. NNN metabolism pathways in rodents (modified from Hecht, 1998).
oxide resulting from N-oxidation, nornicotine after den-
itrosation, 3
- and 4
-hydroxy-NNN from hydroxylation
of the pyrrolidine ring, and norcotinine, whose forma-
tion is not yet clearly understood (Fig. 1) (Hecht, 1998).
Recently, additional urinary metabolites, norcotinine-1-
N-oxide, 3
-hydroxynorcotinine and its O-glucuronide
have been detected in patas monkeys (Upadhyaya et al.,
2002).
According to current knowledge only 2
-hydro-
xylation but not 5
-hydroxylation leads to DNA adduct
formation via its reactive intermediate, 4-(3-pyridyl)-
4-oxobutane-1-diazohydroxide. Therefore, this pathway
leading finally to HPB and keto acid is regarded as
the major route of NNN metabolic activation (Hecht,
1998; Hecht and Tricker, 1999). Using racemic NNN,
hydroxy acid, the end product of 5
-hydroxylation is the
major urinary metabolite in rats, whereas with (S)-NNN,
the predominant isomer occurring in tobacco products,
2
-hydroxylation to keto acid is predominating (McIntee
and Hecht, 2000).
 S. Tyroller et al. / Toxicology 215 (2005) 245–253
Research has been focused mainly on the TSNA 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
after detection of its biological activity as systemic
lung carcinogen in rodents (Hecht, 1998). NNK and
NNN have been recently classified as “carcinogenic to
humans” by the IARC (IARC, 2004). Studies on interac-
tions between NNN and different tobacco constituents,
ethanol or dietary chemopreventive compounds have
been performed in the course of investigations on car-
cinogenicity, mutagenicity and in vitro metabolism using
rather high concentrations which are not expected to
occur in tobacco smokers (Hecht, 1998). Reduced car-
cinogenicity of a combination of NNN and NNK in the
oral cavity and the lungs of F344 rats has been detected
after co-administration of a snuff extract (Hecht et al.,
1986). Whereas, inhibition of NNK activation by other
smoke constituents has been proven in vivo (Brown et al.,
1999, 2001; Kutzer et al., 1995; Richter and Tricker,
1994, 2002; Tricker et al., 2001) and in vitro (Charest
et al., 1989; Murphy and Heiblum, 1990; Schuller
et al., 1991; Castonguay and Rossignol, 1992; Hong
et al., 1992; Schulze et al., 1998) only a few data exist for
in vitro inhibition of NNN activation. NNN metabolism
is strongly inhibited in oral tissues cultures from rats
by co-incubation of nicotine or phenethyl isothiocyanate
(PEITC) or pretreatment of rats with a diet contain-
ing 3 ␮mol PEITC/g diet (Murphy and Heiblum, 1990;
Murphy et al., 1991) and by co-incubation with nico-
tine, cotinine and PEITC in precision-cut lung and liver
slices from hamsters (Marchand et al., 2000; Richter
et al., 2000) and rats (Lassnack et al., 2004).
No data on effects of tobacco alkaloids, other TSNA
or PEITC on in vivo NNN metabolism have been yet pub-
lished. Therefore, NNN metabolism was studied in F344
rats after single doses of NNN either alone or in com-
bination with nicotine, cotinine, PEITC, nornicotine,
anatabine, anabasine, myosmine, N
-nitrosoanatabine or
N
-nitrosoanabasine. In addition, the interaction of NNN
metabolism with nornicotine or myosmine was tested
after co-administration with NNN in the drinking water
for 28 days.
2. Materials and methods
2.1. Materials
[5-3 H]NNN with a specific activity of 27 Ci/mmol
and a radiochemical purity of 99.9% was purchased from
Hartmann Analytic (Braunschweig, Germany). Unla-
beled NNN, anabasine, anatabine, N
-nitrosoanabasine
and N
-nitrosoanatabine were obtained from Toronto
Research Chemicals Inc. (Toronto, Canada). (−)-
247
Nornicotine, (−)-cotinine, [−]-nicotine di[+]tartrate salt
and ␤-PEITC were purchased from Sigma (Deisenhofen,
Germany). Myosmine was synthesized in our labora-
tory (Brandänge and Lindblom, 1976). All other chem-
icals and solvents were of analytical grade and obtained
from Merck (Darmstadt, Germany). A mixture of NNN
metabolite standards for HPLC was kindly donated by
Dhimant H. Desai and Shantu Amin (Penn State Univer-
sity Medical Center, Hershey, PA, USA).
2.2. Animals
Male F344 rats (200–250 g body weight) were pur-
chased from Harlan-Winkelmann (Borchen, Germany).
They were housed in groups of three rats each in stain-
less steel cages with a 12-h light/dark cycle at 20 ± 2 ◦ C
with relative humidity of 50 ± 10%, and allowed ad libi-
tum access to water and ssniff R laboratory animal diet
(ssniff, Soest, Germany). The rats were allowed to accli-
matize to these conditions for at least 7 days before use.
The animal experiments were officially approved by the
Government of Upper Bavaria (AZ 211-2531-79/99).
2.3. NNN administration
2.3.1. Acute experiments
For collection of urine and feces, animals were trans-
ferred individually in stainless steel metabolism cages
with water and ssniff R/M-H diet (ssniff, Soest, Ger-
many) ad libitum 24 h prior to treatment. All com-
pounds except PEITC were dissolved in sterile physio-
logical saline at concentrations to give a total volume of
100 ␮l/100 g body weight for s.c. injection. All animals
were administered 30 nmol [5-3 H]NNN/kg (5.3 ␮g/kg;
2 ␮Ci/rat). The experiments have been divided into
two studies. In the first study, eight rats/group
were given either NNN only or NNN + 15 ␮mol
nicotine/kg (2.43 mg/kg), NNN + 150 ␮mol cotinine/kg
(26.4 mg/kg) or NNN + PEITC which was given to the
rats with a diet supplemented with 1 ␮mol PEITC/g diet
starting 24 h before NNN injection, and continued until
the end of the study. Urine and feces were collected in
24 h time intervals for 2 days.
In the second study, animals were arranged to
seven groups of four rats each given NNN only or
NNN + 15 ␮mol nornicotine/kg (2.22 mg/kg), NNN +
15 ␮mol anatabine/kg (2.40 mg/kg), NNN + 15 ␮mol
anabasine/kg (2.43 mg/kg), NNN + 3 ␮mol myos-
mine/kg (0.44 mg/kg), NNN + 300 nmol N
-nitrosoana-
tabine/kg (56.8 ␮g/kg), NNN + 300 nmol N
-nitro-
soanabasine/kg (57.4 ␮g/kg), respectively. Urine was
collected in intervals from 0 to 12 h, 12 to 24 h, 24 to 48 h,
248 S. Tyroller et al. / Toxicology 215 (2005) 245–253

24 to 48 h and 48 to 72 h. Feces were pooled for 72 h. In
both studies urine of the first collection period was kept
on ice and stored at −20 ◦ C until HPLC analysis.
2.3.2. Subacute experiment
Three groups of 12 rats each were treated with either
NNN only or with combinations of NNN and nornico-
tine or NNN and myosmine in the drinking water for
28 days. Aqueous solutions were prepared to ensure
daily uptake of approximately 30 nmol NNN/kg, 3 ␮mol
myosmine/kg and 15 ␮mol nornicotine/kg. At 18 h prior
to the end of the experiments the rats were separated
in metabolism cages and each rat received 30 nmol [5-
3 H]NNN/kg (5.3 ␮g/kg; 2 ␮Ci/rat) by feeding contami-
nated apple bites. Urine and feces were collected for 18 h.
The rats were killed by cervical dislocation in order to
collect blood and tissue samples for adduct analyses. All
samples were stored until analysis at −20 ◦ C.
2.4. Excretion of radioactivity
Aliquots of 50 ␮l from all urine samples were mixed
with 10 ml of Ultima Gold XR scintillator (Packard,
Frankfurt/Main, Germany) for determination of total
radioactivity in a Tri Carb 2500 TR Liquid Scintil-
lation Analyzer (Packard, Frankfurt/Main, Germany).
Feces were mixed with 2.5 ml methanol/g wet weight
and homogenized with an Ultra-Turrax (Bachofer, Reut-
lingen, Germany). To 0.1 g of the homogenates 0.2 ml
hydrogen peroxide and 0.2 ml perchloric acid were
added and incubated at 80 ◦ C for 30 min. After cool-
ing, 10 ml liquid scintillator was added to the samples
which were measured after overnight storage in the dark.
Aliquots of blood samples (100 ␮l) were incubated at
room temperature for 1 h with 1 ml soluene. After addi-
tion of 1 ml of a H2 O2 /2-propanol (2:3) mixture the
samples were incubated for another hour at 60 ◦ C. The
cooled samples were mixed with 10 ml of Ultima Gold
XR scintillator and stored in the dark prior analysis.
2.5. Metabolite assay
After centrifugation at 10,000 rpm for 5 min, super-
natants of urine samples were filtered through a 0.22 ␮m
Millipore Ultrafree filter (Millipore, Königstein, Ger-
many). Aliquots of 700 ␮l filtrate were analyzed by
reversed-phase HPLC (Dionex, Idstein, Germany) with
on-line radioflow detection (Ramona 2000, Raytest,
Straubenhardt, Germany) after mixing with Quickszint
Flow 302 (Zinsser, Frankfurt, Germany) at a flow rate
of 2.0 ml/min. The column was operated at a flow rate
of 0.7 ml with a gradient using acetonitrile and 10 mM
phosphate buffer, pH 6.8. After an initial time of 3 min at
0% CH3 CN/100% buffer, CH3 CN was linearly increased
over 27 min to 22% and within 2 min to 60%. After
3 min, the eluent was returned to 0% CH3 CN/100%
buffer within 2 min. The column was reconditioned for
at least 10 min. Radioactive metabolites were identified
by co-chromatography with unlabeled reference com-
pounds using UV detection at 232 nm and 254 nm.
2.6. Statistical analysis
Reported values represent mean ± standard deviation
(S.D.). Differences between the groups were tested for
statistical significance by the two-sided t-test for inde-
pendent samples using WINSTAT for MICROSOFT
EXCEL (R. Fitch Software, Staufen, Germany).

Table 1
Effect of nicotine, cotinine and PEITC on excretion of NNN metabolites in 24 h urine
Metabolite Percent of radioactivity in 24 h urine (percent of control)a

NNN only NNN + nicotine NNN + cotinine NNN + PEITC

Hydroxy acid 44.4 ± 2.9 48.0 ± 2.5(108)* 50.1 ± 2.9(113)** 48.1 ± 1.6 (108)**
Keto acid 32.4 ± 1.6 28.0 ± 1.9 (86)*** 29.3 ± 1.8 (90)** 28.0 ± 1.7 (86)***
NNN-N-oxide 5.0 ± 1.9 5.8 ± 1.6 (116) 3.5 ± 0.5 (70) 6.4 ± 0.8 (128)
Diol 1.1 ± 0.4 1.4 ± 0.4 (127) 1.0 ± 0.2 (91) 0.8 ± 0.3 (73)
Norcotinine 2.3 ± 0.7 3.4 ± 1.1 (148)* 3.3 ± 0.5 (143)** 3.1 ± 0.7 (135)*
NNN 4.7 ± 1.4 4.5 ± 2.3 (96) 2.4 ± 0.5 (51)*** 3.2 ± 0.5 (68)*

Male F344 rats were administered a single s.c. dose of 30 nmol [5-3 H]NNN/kg either alone or together with s.c. co-administration of 15 ␮mol
nicotine/kg or 150 ␮mol cotinine/kg. PEITC was given to the rats with a diet supplemented with 1 ␮mol PEITC/g diet starting 24 h before NNN
injection, and continued until the end of the study. Urinary metabolites of NNN were analyzed in urine collected for 24 h as indicated in the text.
a Mean ± S.D. of eight animals.
* P < 0.05 compared with control animals given NNN only.
** P < 0.01 compared with control animals given NNN only.
*** P < 0.001 compared with control animals given NNN only.
 S. Tyroller et al. / Toxicology 215 (2005) 245–253 249

3. Results Male F344 rats were administered a single s.c. dose of 30 nmol [5-3 H]NNN/kg either alone or together with s.c. co-administration of 15 ␮mol nornicotine/kg, 15 ␮mol anatabine/kg, 15 ␮mol
0.1 (150)*
0.3 (102)
0.9 (102)

0.1 (120)
0.4 (92)

0.3 (94)
NNN + NAB

3.1. Acute experiments

±
±
±
±
±
±
45.8
32.2
3.5
1.5
1.2
3.0

Excretion of NNN-derived radioactivity in urine and

feces was nearly complete within 48 h in both stud-
ies and was not affected by any co-treatment. In the
anabasine/kg, 3 ␮mol myosmine/kg, 0.3 ␮mol NAT/kg or 0.3 ␮mol NAT/kg. Urinary metabolites of NNN were analyzed in urine collected for 12 h as indicated in the text.

various groups the mean percentage of radioactivity in

0.2 (103)
0.9 (103)

0.2 (130)
0.2 (130)
0.1 (89)

0.3 (81)

urine accounted for 84.5–90.6% and 2.0–6.4% of totally

NNN + NAT

recovered radioactivity within the first and second 24 h

collection periods, respectively.
±
±
±
±
±
±
46.0
32.6
3.4
1.3
1.3
2.6

In both studies, five metabolites besides NNN were

detected with high reproducibility and little intraindivid-
Effect of nornicotine, anatabine, anabasine, myosmine, N
-nitrosoanatabine and N
-nitrosoanabasine on excretion of NNN metabolites in 12 h urine

ual variation in urine of control animals (Tables 1 and 2).

Hydroxy acid was the major metabolite accounting in
NNN + myosmine

0.8 (103)
0.4 (100)

0.1 (130)
0.1 (130)
0.2 (95)

0.2 (90)

study 1/2 for 44.4/44.8% of total radioactivity in urine

excreted within 24/12 h. Keto acid (32.4/31.5%) was
the second most important NNN metabolite followed
±
±
±
±
±
±
46.2
31.4
3.6
1.3
1.3
2.9

by NNN-N-oxide (5.0/3.8%), norcotinine (2.3/1.0%)

and diol (1.1/1.0%). Unmetabolised NNN accounted for
4.7% and 3.2%, respectively.
Co-administration of nicotine, cotinine or PEITC
NNN + anabasine

1.5 (100)
1.1 (101)
0.3 (100)
0.2 (140)
0.2 (140)
0.6 (106)

significantly reduced the contribution of keto acid to

totally excreted radioactivity in urine to 86.4 ± 5.9%
(P < 0.001), 90.4 ± 5.6% (P < 0.01) and 86.4 ± 5.2%
±
±
±
±
±
±
44.7
31.9
3.8
1.4
1.4
3.4

(P < 0.001) compared to control values, respectively

(Table 1). In contrast, levels of hydroxy acid were signif-
icantly increased in these three groups to 108.1 ± 5.6%
(P < 0.05), 112.8 ± 6.5% (P < 0.01) and 108.3 ± 3.6%
0.1 (140)*
0.2 (105)

0.1 (160)
0.7 (122)
NNN + anatabine

1.6 (99)
1.2 (98)

(P < 0.01) of control. Norcotinine formation was also

significantly increased by nicotine (147.8 ± 47.8%),
Percent of radioactivity in 12 h urine (percent of control)a

cotinine (143.4 ± 27.7%) and PEITC (134.8 ± 30.4%).

±
±
±
±
±
±
44.4
30.9
4.0
1.4
1.6
3.9

The excretion of unmetabolised NNN was significantly

lower after co-treatment with cotinine (51.1 ± 10.6%,
P < 0.001) or PEITC (68.1 ± 10.6%, P < 0.05). The
remaining minor NNN metabolites, diol and NNN-N-
0.1 (170)**

0.2 (134)**
NNN + nornicotine

* P < 0.05 compared with control animals given NNN only.

** P < 0.01 compared with control animals given NNN only.
(108)*

0.4 (118)*

0.3 (170)
0.5 (87)*

oxide, were not significantly changed by any treatment.

In the second study, only co-administration of nor-
1.0

nicotine had a significant influence on the major NNN

±
±
±
±
±
±

metabolites which was comparable to the nicotine effect

48.2
27.3
4.5
1.7
1.7
4.3

in the first study (Table 2). Nornicotine treatment

reduced the formation of keto acid to 86.7 ± 1.58%
(P < 0.05) and increased the metabolism to hydroxy
2.3
2.0
0.5
0.3
0.7
0.5

a Mean ± S.D. of four animals.

NNN only

acid to 107.6 ± 2.2% (P < 0.05) of control. In addition,

±
±
±
±
±
±

levels of NNN-N-oxide (118.4 ± 10.5%, P < 0.05), diol

44.8
31.5
3.8
1.0
1.0
3.2

(170.0 ± 10.0%, P < 0.01), norcotinine (170.0 ± 30.0%)

and unmetabolised NNN (134.4 ± 6.3%, P < 0.01) were
significantly higher compared to control animals. Co-
NNN-N-oxide
Hydroxy acid

administration of anatabine, anabasine, myosmine,

Norcotinine

N
-nitrosoanatabine and N
-nitrosoanabasine resulted

Metabolite

Keto acid
Table 2

in mostly unchanged levels of NNN metabolites

NNN
Diol

with a tendency to increased formation of diol and

250 S. Tyroller et al. / Toxicology 215 (2005) 245–253

Table 3
Subacute effect of nornicotine and myosmine on excretion of NNN metabolites in 18 h urine
Metabolite Percent of radioactivity in 18 h urine (percent of control)a

NNN only NNN + nornicotine NNN + myosmine

Hydroxy acid 41.5 ± 1.2 40.7 ± 3.5 (98) 41.3 ± 0.5 (99)
Keto acid 32.2 ± 0.8 34.5 ± 2.5 (107)* 33.5 ± 1.0 (104)**
NNN-N-oxide 6.0 ± 0.4 4.5 ± 0.6 (76)*** 4.3 ± 1.3 (72)***
Diol 2.2 ± 0.3 2.0 ± 0.6 (91) 1.7 ± 0.3 (79)**
Norcotinine 3.3 ± 0.2 2.9 ± 0.8 (88) 3.4 ± 0.9 (102)
HPB 0.9 ± 0.1 n.d. n.d.
NNN 3.9 ± 0.8 4.3 ± 1.0 (111) 4.3 ± 1.4 (111)

Male F344 rats were administered a single dose of 30 nmol [5-3 H]NNN/kg by feeding a contaminated apple bite after 28 days administration in
drinking water at concentrations corresponding to a daily uptake of either 30 nmol NNN/kg alone or together with co-administration of 15 ␮mol
nornicotine/kg or 3 ␮mol myosmine/kg. Urinary metabolites of NNN were analyzed in urine collected for 18 h as indicated in the text. (n.d.) not
detected.
a Mean ± S.D. of 12 animals.
* P < 0.05 compared with control animals given NNN only.
** P < 0.01 compared with control animals given NNN only.
*** P < 0.001 compared with control animals given NNN only.

norcotinine reaching significance for diol after co-
treatment with anatabine (140.0 ± 10%, P < 0.05) and
N
-nitrosoanabasine (150.0 ± 10%, P < 0.05).
3.2. Subacute experiment
Following four weeks of treatment with either
NNN only or with NNN + norcotinine or with
NNN + myosmine in the drinking water, the experiment
was terminated 18 h after oral administration of [5-
3 H]NNN by a contaminated apple bite to allow collection
of urine and feces as well as blood and selected tissues.
During this time between 92.3 ± 7.1% and 96.0 ± 2.5%
of total recovered radioactivity have been excreted with
urine. Only small amounts have been detected in feces
and blood ranging from 0.7 ± 0.7% to 3.2 ± 3.2% and
0.5 ± 0.1% to 1.7 ± 0.8%, respectively.
After four weeks pretreatment with NNN and oral
administration of [5-3 H]NNN the urinary metabolite pat-
tern (Table 3) was significantly different compared to the
acute experiments with rats given the same dose of [5-
3 H]NNN by s.c. injection (Tables 1 and 2). Whereas, the
percentage of keto acid (32.2% of total radioactivity in
urine) remained unchanged in the subacute experiment,
the contribution of hydroxy acid (41.5 ± 1.2%) was sig-
nificantly lower as compared to the acute experiments
(44.4 ± 2.9%, P < 0.001 and 44.8 ± 2.3%, P < 0.01).
The decrease in hydroxy acid was compensated by
an increase of minor NNN metabolites, NNN-N-oxide
(6.0%), norcotinine (3.3%) and diol (2.2%) but not of
unmetabolised NNN (3.9%). In addition, small amounts
of HPB (0.9%) have been identified in urine samples
of the NNN-pretreated group. HPB was not detected
after combined pretreatment with NNN and nornico-
tine or myosmine. Co-administration of nornicotine and
myosmine resulted in a small but significant increase of
keto acid to 106.9 ± 7.7% (P < 0.05) and 103.9 ± 3.2%
(P < 0.01) of control pretreated with NNN only, whereas
the amounts of hydroxy acid remained unchanged. For-
mation of NNN-N-oxide was significantly reduced by
subacute co-administration of nornicotine (76.1 ± 9.8%,
P < 0.001) and myosmine (71.6 ± 22.3%, P < 0.001).
Myosmine treatment also decreased the level of diol
(79.0 ± 15.9%, P < 0.01). All other changes of minor
NNN metabolites and unmetabolised NNN did not reach
significance.
4. Discussion
The interaction of different tobacco alkaloids and
TSNA with NNN was tested on the basis of expected
differences in uptake of these compounds by smok-
ers. Although nicotine is present in mainstream smoke
in about 5000-fold higher concentrations than NNN
(Roemer et al., 2004) its high acute toxicity allowed
only administration of a 500-fold higher dose which
did not cause any symptoms of acute poisoning in the
rats. Cotinine, a much less toxic major metabolite of
nicotine was given at a 5000-fold higher concentra-
tion than NNN. These dose ratios as well as the dose
of PEITC which was added to the diet at 1 ␮mol/g,
were the same as in our previous experiments on the
interaction with NNK metabolism in rats and ham-
sters (Richter and Tricker, 1994, 2002). Nornicotine,
 S. Tyroller et al. / Toxicology 215 (2005) 245–253
anatabine and anabasine are mostly present at 10- to
100-fold lower levels in cigarette tobacco than nicotine
(Chen and Moldoveanu, 2003) but are also less toxic than
nicotine (Werle and Schievelbein, 1961). Therefore, a
500-fold higher dose compared to NNN was chosen for
these alkaloids. Much lower concentrations are found for
myosmine which was given only at a 100-fold higher
molar dose than NNN. Although N
-nitrosoanatabine
and N
-nitrosoanabasine contribute not more than NNN
to total TSNA in mainstream smoke (Counts et al., 2005),
these nitrosamines were tested according to Murphy and
Heiblum (1990) at 10-fold higher doses as compared to
NNN.
Since NNN metabolism in rats is well documented
(Hecht et al., 1981; McIntee and Hecht, 2000), the
major urinary metabolites represented as signals in radio-
HPLC could be easily identified by co-chromatography
with reference compounds. Formation of norcotinine
and NNN-N-oxide is assigned to detoxification path-
ways in NNN metabolism. Hydroxylation of NNN
yields various reactive intermediates, but only 4-(3-
pyridyl)-4-oxobutane diazohydroxide formed after 2
-
hydroxylation has been identified to cause covalent
DNA binding which is thought to be a prerequisite of
its carcinogenic activity (Hecht, 1998, 2003). These
adducts release HPB upon alkaline or acidic hydroly-
sis. In vitro studies on the reaction of DNA with 4-
(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol, a
stable precursor to the product formed upon metabolic
methyl hydroxylation of NNAL, yielded adducts releas-
ing diol after hydrolysis (Upadhyaya et al., 2003). If
these diol-releasing adducts are also formed from NNN
5
-hydroxylation in vivo and if they are mutagenic
and carcinogenic, such as HPB-releasing adducts, this
pyridylhydroxybutylation pathway should be regarded
as an additional NNN activation step. However, at the
present standard of knowledge the amount of keto acid
and HPB formation represents the extent of NNN acti-
vation. Therefore, the significant decrease of keto acid
formation together with a significant increase of hydroxy
acid formation from NNN after acute co-administration
of nicotine, nornicotine, cotinine and PEITC could be
an indication of an impaired metabolic activation of
NNN. This is in line with the observation that NNN and
NNK were less tumorigenic when administered chroni-
cally to the oral cavity of rats together with snuff extract
than when administered alone (Hecht et al., 1986). This
effect could be mediated by nicotine which inhibits even
more efficiently both ␣-hydroxylation pathways of NNK
(Richter and Tricker, 1994). In the Ames test, nicotine
was also a much stronger inhibitor of the mutagenicity
of NNK than of NNN (Lee et al., 1996).
251
In our previous experiment with Syrian golden ham-
sters, acute co-administration of nicotine or PEITC
inhibited NNK metabolism to keto acid to a similar
extent, whereas cotinine was a more effective inhibitor
of both keto acid and hydroxy acid formation from
NNK (Richter and Tricker, 2002). Numerous studies
have demonstrated the chemopreventive effects of iso-
thiocyanates on NNK metabolism and tumorigenic-
ity (for review see Hecht, 1999, 2000; Chung, 2001)
but only few reports have dealt with effects on
NNN. Pretreatment of rats with PEITC strongly inhib-
ited in vitro metabolism of NNN in cultured tar-
get tissues of rats, esophagus (Chung et al., 1984;
Stoner et al., 1998) and oral cavity (Murphy et al., 1991).
In contrast to the specific inhibition of keto acid forma-
tion in our in vivo experiment, all metabolic pathways of
NNN were inhibited to a similar extent in these in vitro
studies. 3-Phenylpropyl isothiocyanate, the strongest
inhibitor of NNN metabolism in cultured esophagus
was also a highly significant inhibitor of NNN-induced
esophageal tumorigenesis in rats (Stoner et al., 1998).
All other tested compounds, anatabine, anabasine,
myosmine, N
-nitrosoanatabine and N
-nitrosoanabasine
had no appreciable effect on NNN metabolism after a
single dose co-treatment. Results with NAT are in accor-
dance with previous studies showing no effect on the
NNN metabolism in rat oral tissues when incubated with
10- and 100-fold higher concentrations of NAT (Murphy
and Heiblum, 1990).
The inhibitory effect of nornicotine could not be ver-
ified after subacute co-administration with NNN for 28
days. In contrast to the acute experiments where forma-
tion of keto acid was decreased, a small but significant
increase of keto acid was noted in the subacute exper-
iment. This was accompanied by a significant decrease
of the minor metabolite NNN-N-oxide whereas the uri-
nary excretion of the second major NNN metabolite,
hydroxy acid, was not changed at all. A similar but
even weaker effect, increase of keto acid, no change of
hydroxy acid and a decrease of NNN-N-oxide and diol,
was noticed after 28 days co-administration of NNN with
myosmine. There are no reports on similar studies with
these tobacco smoke constituents in the literature. With
NNK, the acute inhibitory effect of nicotine on NNK
metabolic activation (Richter and Tricker, 1994) was
also not reproduced in subchronic experiments (Kutzer
et al., 1995; Keyler et al., 2003). However, hemoglobin
adducts resulting from NNK-derived pyridyloxobuty-
lation were significantly reduced by nicotine or coti-
nine in one of these experiments (Kutzer et al., 1995).
Long-term co-administration of PEITC and NNK to
rats also resulted in significantly reduced hemoglobin
252 S. Tyroller et al. / Toxicology 215 (2005) 245–253
and lung DNA adducts releasing HPB (Hecht et al.,
1996; Boysen et al., 2003). Studies underway looking
for effects of nornicotine and myosmine on levels of
HPB-releasing DNA adducts from NNN will give fur-
ther insight into the impact of these compounds on NNN
tumorigenicity.
In conclusion, NNN metabolism was signifi-
cantly directed towards detoxification by acute co-
administration of nicotine, cotinine, nornicotine or
PEITC. The maximal 20% inhibition of NNN metabolic
activation has to be seen in context with a 25-fold varia-
tion of NNN delivery to mainstream smoke of commer-
cial cigarettes (Counts et al., 2005) and individual smok-
ing behavior. Our results contribute new information on
the interactions of tobacco alkaloids and TSNA but fur-
ther studies are needed to evaluate the NNN impact on
smokers.
Acknowledgement
Supported in part by a grant from the Institute for
Science and Health.
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