University of Minnesota Cancer Center (P.U., S.S.H.) and College of
Pharmacy (C.L.Z.), Minneapolis, Minnesota
 |
Introduction |
NNN1
is a tobacco-specific nitrosamine formed by nitrosation of the tobacco
alkaloids nornicotine and nicotine (Hu et al., 1974
; Hecht et al.,
1978). It is the most prevalent strongly carcinogenic nitrosamine in
smokeless tobacco products, such as chewing tobacco and snuff, and
occurs in substantial quantities in cigarette smoke (Hoffmann et al.,
1994). NNN causes tumors of the esophagus and nasal mucosa in rats,
respiratory tract in hamsters, and lung in mice (Hecht, 1998). When NNN
was administered to rats together with NNK, oral cavity tumors resulted
(Hecht et al., 1986). NNN is likely to play an important role as a
cause of esophageal cancer in smokers and oral cavity tumors in people
who use smokeless tobacco products (Hoffmann and Hecht, 1990).
Metabolic activation of NNN is a prerequisite for its mutagenicity, DNA
binding, and most likely its carcinogenicity (Chen et al., 1978; Hecht,
1998). The metabolism of NNN in rodents is summarized in Fig.
1(Hecht, 1998).
Pyridine-N-oxidation produces NNN-N-oxide,
whereas denitrosation and oxidation yield norcotinine. These two
pathways result in detoxification of NNN. The major hydroxylation
reactions occur 
to the N-nitroso group, yielding 2'-hydroxyNNN and 5'-hydroxyNNN. 2'-HydroxyNNN undergoes spontaneous ring opening, producing a diazohydroxide, which reacts with
H2O yielding a keto alcohol. This keto alcohol is
then metabolically oxidized to a keto acid or reduced to a diol.
Alternatively, 2'-hydroxyNNN can lose nitrous acid, yielding
myosmine. 5'-Hydroxylation of NNN gives 5'-hydroxyNNN, which ring opens
to a diazohydroxide that reacts with H2O
producing a lactol. This lactol is metabolically converted to a hydroxy
acid, either directly or through a lactone. Studies to date indicate
that 2'-hydroxylation of NNN is its major metabolic activation pathway,
yielding target tissue DNA adducts that release the keto alcohol upon
hydrolysis (Hecht, 1998). Minor hydroxylation pathways also occur at
the 3'- and 4'- positions of NNN, yielding the stable metabolites
3'-hydroxyNNN and 4'-hydroxyNNN.
We are particularly interested in identifying a urinary metabolite of
NNN that could be used as a biomarker of its uptake in humans exposed
to tobacco products. This represents a considerable challenge because
studies in rodents have shown that NNN itself is extensively
metabolized and its known major urinary metabolites such as the hydroxy
acid and keto acid are also metabolites of nicotine. Since nicotine
levels in tobacco products are as much as 10,000 times greater than
those of NNN, the hydroxy acid and keto acid cannot be used as specific
biomarkers of NNN uptake (Hecht et al., 1999; Trushin and Hecht, 1999).
Metabolites of NNN that retain unique structural features related to
NNN, such as NNN-N-oxide, 3'-hydroxyNNN, and 4'-hydroxyNNN,
are relatively minor urinary metabolites in rodents. It is possible
that the qualitative or quantitative aspects of NNN metabolism could
differ in rodents and primates. We have observed such differences in the metabolism of the related nitrosamine NNK (Hecht et al.,
1993). The results of those studies ultimately led to the development of an assay for uptake of NNK in humans by measurement of its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide in urine (Carmella et al., 1993; Hecht, 1998). Therefore, in this study, we examined the metabolism of NNN in the patas monkey.
There has been only one previous investigation of NNN metabolism in a
nonhuman primate (Castonguay et al., 1985). The tissue distribution of
NNN and its metabolites was explored in the marmoset monkey
(Callithrix jacchus) by whole body autoradiography and HPLC
analysis of tissues. The metabolites illustrated in Fig. 1 were
detected in several tissues and in urine, but limited structural and
quantitative data were available.
| |
Materials and Methods |
Instrumentation.
1H NMR data were obtained on either a 600 or 800 MHz instrument (Varian Medical Systems, Inc., Palo Alto, CA). MS
were run on Finnigan TSQ 7000 and LCQ-Deca instruments (Thermo Finnigan MAT/Thermoquest, San Jose, CA), and a Finnigan MAT 90/95
instrument (Thermo Finnigan MAT GmbH, Bremen, Germany). GC-MS was
carried out with a model 6890 gas chromatograph equipped with an
autosampler and model 5973 mass selective detector (Agilent
Technologies, Wilmington, DE). HPLC was performed with a Waters
Associates system equipped with a model 440 UV detector and a 
-RAM
radioflow detector (IN/US Systems, Tampa, FL).
Chemicals.
Racemic [5-3H]NNN (1.9 Ci/mmol, tritium at the
5-position of the pyridine ring) was purchased from ChemSyn
Laboratories (Lenexa, KS). NNN and its metabolites were
synthesized (McKennis et al., 1964; Hu et al., 1974; Chen et al., 1978;
Hecht et al., 1980; Upadhyaya et al., 2001).
trans-3'-Hydroxycotinine and norcotinine were purchased from
Toronto Research Chemicals (Toronto, ON, Canada). cis-3'-Hydroxycotinine was a gift from Dr. Shantu Amin,
American Health Foundation (Valhalla, NY).
Norcotinine-1N-oxide and 3'-methoxycotinine were prepared as
described below. All other chemicals were purchased from Aldrich
Chemical Co., (Milwaukee, WI).
Norcotinine-1N-Oxide.
Norcotinine (0.005 mmol) was dissolved in
CH2Cl2 (5 ml), and
m-chloroperbenzoic acid (0.09 mmol) was added. The reaction
mixture was stirred overnight at room temperature. The solvent was
removed at reduced pressure, and the residue was purified using HPLC
system 1 (retention time 16 min) and 2 (retention time 20 min) to yield norcotinine-1N-oxide. 1H NMR
(D2O) 
8.19 (s, 1H, pyridyl 2H), 8.12 (d, 1H,
J = 6.4 Hz, pyridyl 6H), 7.61 (d, 1H, J = 8.0 Hz, pyridyl 4H), 7.48 (dd, 1H, J = 6.4, 8.0 Hz,
pyridyl 5H), 4.82 (t, 1H, J = 6.4 Hz, 5'H), 2.56 (m,
1H, 3'H), 2.38 (m, 1H, 4'H) and 1.87 (m, 1H, 3'H). CI-MS
(NH3) m/z (relative
intensity) 357 (2M + 1, 70), 196 (M + NH4, 75), 179 (M + 1,100), 163 (85).
3'-Methoxycotinine.
Trans- or cis-3'-hydroxycotinine (0.01 mmol) was
dissolved in 0.5 ml of DMSO. NaH (0.02 mmol) was added, and the
reaction mixture was stirred for 5 min. CH3I
(0.02 mmol) was added, and the reaction mixture was stirred for 10 min.
The reaction was quenched by addition of 0.5 ml
H2O. The mixture was purified by HPLC using a
4.6 × 200 mm Luna C18 column (Phenomenex,
Torrence, CA) with elution by 100% 10 mM ammonium acetate, pH 6.5, to
100% methanol in 40 min at a flow rate of 1 ml per min, retention time 22 min. This gave 0.003 mmol (30%) trans- or
cis-3'-methoxycotinine: 1H NMR
(CDCl3) trans-3'-methoxycotinine; 8.60 (d, 1H, J = 4.8 Hz, pyridyl 6H), 8.48 (d, 1H,
J = 2.4 Hz, pyridyl 2H), 7.46 (d, 1H, J = 8.0 Hz, pyridyl 4H), 7.34 (dd, 1H, J = 8, 4 Hz,
pyridyl 5H), 4.65 (dd, 1H, J = 4.8, 4.8 Hz, H3'), 4.11 (dd, 1H, J = 4.8, 6.4 Hz, H5'), 3.59 (s, 3H,
OCH3), 2.72 (s, 3H, NCH3),
2.47 (m, 1H, H4'), 2.2 (m, 1H, H4'). Electrospray ionization-MS
m/z (relative intensity) 207, M + 1 (100); MS/MS
of m/z 207; 175 (100), 149 (60), 118 (40), 80 (32). 1H NMR (CDCl3)
cis-3'-methoxycotinine, 8.60 (d, 1H, J = 4.8 Hz, pyridyl 6H), 8.54 (d, 1H, J = 1.6Hz, pyridyl
2H), 7.65 (ddd, 1H, J = 8.0,1.6,1.6 Hz, pyridyl 4H),
7.34 (dd, 1H, J = 8.0, 5.2 Hz, pyridyl 5H), 4.41 (dd,
1H, J = 6.4, 7.2 Hz, 3'H), 4.04 (dd, 1H, J = 6.4, 7.2 Hz, 5'H), 3.61 (s, 3H,
OCH3), 2.66 (s, 3H, NCH3), 2.8 (m, 1H, 4'H), 1.9 (m, 1H, 4'H).
3'-Hydroxynorcotinine, collected from monkey urine using HPLC system 2, was similarly derivatized.
Animal Experiments.
Male patas monkeys were housed according to the National Institutes of
Health Guide for the Care and Use of Laboratory Animals at Bioqual,
Inc. (Rockville, MD). Four experiments were carried out.
In experiment 1, monkey R-234 (4.8 kg) was injected i.v.
with ~302 µCi (4 µg/kg) [5-3H]NNN in
1.8-ml sterile phosphate-buffered saline (PBS). In experiment 2, monkey
R-238 (4.1 kg) was injected i.v. with ~336 µCi (7.6 µg/kg) in 1.8 ml PBS. In experiment 3, monkey R-241 (3.0 kg) was injected i.v. with ~381 µCi (11.8 µg/kg) in 1.8 ml PBS.
For collection of metabolites, experiment 4 was carried out. Monkey
R-238 (4.1 kg) was injected i.v. with ~422 µCi (12.7 mg/kg) in 1.8 ml PBS.
Urine samples were collected at intervals of 0 to 2, 2 to 6, 6 to
24 h. Blood samples were collected at 2, 5, 10, 30, 60, 90, 120, 240, and 360 min after injection, then centrifuged at 2000 rpm for 30 min. Serum and urine samples were frozen at 20°C and shipped to the
University of Minnesota Cancer Center for analysis.
Analytical Methods.
Serum was purified by ultrafiltration using an Amicon Centrifree
micropartition system (Millipore Corporation, Bedford, MA) prior to
HPLC analysis. Urine samples were centrifuged at 6000 rpm and filtered
through a 0.45 µm × 3 mm Acrodisc (Gelman Sciences, Ann Arbor,
MI) prior to HPLC analysis. Some samples were subjected to enzyme
hydrolysis as follows. A 0.5-ml aliquot of urine was adjusted to pH 7.0 and treated with 20,000 units of -glucuronidase (type-1XA from
Escherichia coli; Sigma-Aldrich, St. Louis, MO). The
mixture was incubated at 37°C for 12 h and filtered through a
Centrifree-YM 30 filter (Millipore Corporation) prior to HPLC analysis.
Metabolites and synthetic standards were analyzed by reverse phase HPLC
using system 1, a 300 × 3.9 mm, Phenomenex C18 Bondclone 10-µ column (Phenomenex), with
elution by a linear gradient from 10 mM ammonium acetate, pH 6.5, to
30% methanol at 1 ml/min in 60 min or system 2, isocratic elution with
10 mM ammonium acetate containing 1% methanol. For collection of
urinary metabolites, a semipreparative 300 × 7.80 mm Phenomenex
C18 Bondclone 10-µ column was used, and the
HPLC conditions were the same as system 1 except the flow rate was 3 ml/min.
For GC-MS analysis, fractions corresponding to labeled urinary
metabolites were evaporated to dryness, redissolved in acetonitrile, and analyzed using a 30 m × 0.32 mm i.d., 0.25-µm film
thickness, DB-1701 column (J & W Scientific, Folsom, CA). The injection
port temperature was 250°C, and the injection mode was splitless. The oven temperature was 90°C for 5.0 min, then raised at 10°C per min
to 240°C, and held for 15 min. The carrier gas was He at a flow rate
of 1.3 ml/min.
Acetylation of 3'-Hydroxynorcotinine.
Urinary 3'-hydroxynorcotinine (5 µCi) was collected by HPLC. It was
dissolved in 1-ml ethyl acetate and to this was added 4-dimethylaminopyridine (4 mg) followed by acetic anhydride (0.1 ml).
The reaction mixture was stirred overnight at room temperature then
quenched by addition of 1 ml H2O. The product was
collected by HPLC system 1, retention time 59 min.
Pharmacokinetic Data Analysis.
The serum concentration versus time data for NNN and its metabolites in
experiments 1 to 3 were analyzed by noncompartmental analysis (Gibaldi
and Perrier, 1982). The slope of the terminal phase of the serum
concentration rate versus time curve was determined by fitting the data
to a monoexponential equation. The terminal rate constant, 
, was
determined from the slope. For both NNN and its metabolites, the area
under the serum concentration-time curves from time 0 to time
t [AUC (0-t)] was determined by the linear
trapezoidal rule up to the last measured concentration. The AUC
(t-
) was determined by dividing the last measured
concentration by the terminal rate constant . The AUC (0-) was
the sum of the two partial AUCs. The elimination half-life
(t1/2) was calculated as 0.693 divided
by . The total body clearance (CL) for NNN was calculated as
|
(1)
|
where D is the intravenous dose of NNN.
The steady-state volume of distribution of NNN was calculated as
|
(2)
|
where AUMC was the area under the first moment of the plasma
concentration-time curve.
The renal clearance (CLR) of NNN and metabolites
were calculated as
|
(3)
|
where Xu was the amount of drug collected in the urine up to
24 h. However, renal clearance could only be determined for those
metabolites for which there were both serum and urine data. The time at
which the maximal concentration occurred
(tmax) was determined by direct
observation of the concentration-time data.
| |
Results |
Identification of Metabolites.
Urine samples were analyzed by HPLC with radioflow detection. A typical
chromatogram of 24-h urinary metabolites is illustrated in Fig.
2. Peak 1 was identified as the hydroxy
acid by coelution with a standard. The assignment was confirmed by
converting the urinary hydroxy acid to its methyl ester then allowing
it to react with (S)-()-methylbenzyl isocyanate, as
described previously (Trushin and Hecht, 1999). The resulting carbamate
diastereomers coeluted with standards. Peak 4 was identified as the
keto acid by coelution with a standard and by reduction to the hydroxy
acid upon treatment with NaBH4.
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Fig. 2.
Chromatogram obtained upon HPLC analysis
(system 1) of urine from a patas monkey treated with
[5-3H]NNN.
Peaks number 1-6 are 1, hydroxy acid; 2, 3'-hydroxynorcotinine-O-glucuronide; 3, norcotinine-1N-oxide; 4, keto acid; 5, 3'-hydroxynorcotinine; 6, norcotinine; and 7, NNN. Structures of the
metabolites are shown in Fig. 6.
|
|
Peaks 2, 3, and 5 did not correspond in retention time to any known
metabolites of NNN. Treatment of the urine with -glucuronidase caused Peak 2 to disappear with a corresponding increase in Peak 5. The
same result was obtained when collected Peak 2 was treated with
-glucuronidase. Peak 2 was stable to basic conditions
1N NaOH,
80°C, 1h that cleave pyridine-N-glucuronides. These
results indicated that Peak 2 was an O-glucuronide and that
Peak 5 was its aglycone. Electrospray ionization-MS analysis of
collected Peak 5 gave a molecular ion of m/z 178, with prominent fragments at m/z 135 (85%) and
m/z 106 (100%). CI-MS analysis
(NH3) gave a base peak of
m/z 179. Acetylation of Peak 5 followed by
atmospheric pressure chemical ionization-MS analysis produced a base
peak of m/z 263, corresponding to addition of 2 acetyl groups. MS/MS analysis of m/z 263 gave
daughter ions at m/z 221 and 179. These results
are also consistent with the presence of two acetyl groups. Therefore,
there were two protons in the metabolite that could be replaced by
acetyl groups. The results suggested that the unknown aglycone, Peak 5, was 3'-hydroxynorcotinine (Fig. 3). This
compound has not been reported in the literature, and we were unable to synthesize it. Therefore, we confirmed its identity as illustrated in
Fig. 3. Peak 5 was methylated with NaH and CH3I.
This produced a new HPLC peak with retention time identical to that of
both cis- and trans-3'-methoxycotinine (inset of
Fig. 3), which were prepared by methylation of the known compounds,
cis- and trans-3'-hydroxycotinine. Further
evidence was obtained by GC-MS analysis of the methylated metabolite
(Fig. 4). The two peaks coeluted with
standard cis- and trans-3'-methoxycotinine and
had MS essentially identical to those of standards. These results
demonstrate that Peak 5 is a mixture of cis- and
trans-3'-hydroxynorcotinine, with a cis-/trans- ratio of approximately 40:60. Peak 2 is therefore
3'-hydroxynorcotinine-O-glucuronide.
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Fig. 3.
Scheme for identification of
3'-hydroxynorcotinine by conversion to 3'-methoxycotinine and
3'-acetoxy-N'-acetylnorcotinine.
The urinary metabolite was methylated or acetylated to give the
structures illustrated. The methylated material was identified as a
mixture of cis- and
trans-3'-methoxycotinine by comparison to standards
prepared by methylation of cis- or
trans-3'-hydroxycotinine. Inset, HPLC analysis of
standard 3'-methoxycotinine and methylated 3'-hydroxynorcotinine from
monkey urine.
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Fig. 4.
GC-positive ion CI-MS analysis of material
obtained upon methylation of urinary 3'-hydroxynorcotinine.
A, separation of cis- and
trans-3'-methoxycotinine formed upon methylation of
urinary 3'-hydroxynorcotinine; B, MS of
trans-3'-methoxycotinine formed in this reaction; and C,
MS of cis-3'-methoxycotinine formed in this reaction.
Identical spectra were obtained from standards.
|
|
CI-MS analysis of Peak 3 gave a base peak of m/z
179, which could be M + 1 of norcotinine-1N-oxide. This
compound was synthesized by treatment of norcotinine with
m-chloroperbenzoic acid. The HPLC retention times of the
metabolite and standard were identical in HPLC systems 1 and 2 (Fig.
5A). The CI-MS of synthetic
norcotinine-1N-oxide and the metabolite were essentially
identical (Fig. 5B). GC-MS analysis of norcotinine-1N-oxide
and the metabolite further confirmed the identity of this metabolite.
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Fig. 5.
A, Chromatogram obtained upon HPLC analysis
(system 2) of standard norcotinine-1N-oxide and metabolite peak 3 from
patas monkey urine; and B, CI-MS (NH3) of
standard and urinary metabolite.
|
|
Peaks 6 and 7 were identified as norcotinine and NNN by comparison of
their HPLC retention times to those of standards. GC-MS analysis of
peak 6 collected from HPLC confirmed the assignment as norcotinine.
We also considered the possible formation of
pyridyl-N--D-glucopyranuronosyl-N'-nitrosonornicotinium
inner salt, which eluted at 7 min in HPLC system 1. There was no
indication of the presence of this metabolite in urine. Moreover,
treatment of the urine with -glucuronidase did not result in an
increase in the NNN peak.
Relative amounts of the metabolites in 24 h urine were (% of
radioactivity eluting from HPLC ± S.D.) hydroxy acid (43.8 ± 4.0), 3'-hydroxynorcotinine-O-glucuronide (5.4 ± 1.0), norcotinine-1N-oxide (16.5 ± 1.3), keto acid
(2.7 ± 0.66), 3'-hydroxynorcotinine (16.9 ± 2.0),
norcotinine (13.1 ± 2.7), and NNN
(0.63 ± 0.15), respectively. Metabolite structures
are illustrated in Fig. 6.
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Fig. 6.
Scheme showing possible pathways of
metabolite formation upon administration of NNN to patas monkeys.
|
|
Analysis of pharmacokinetic parameters (Fig. 7, Tables
1 and 2) demonstrated that NNN was
rapidly eliminated from the body in an
apparent monoexponential manner with an elimination half-life of
26.8 ± 12.7 min. Its mean total body clearance was 86.2 ± 24.5 ml/min, and its volume of distribution at steady state was
0.58 ± 0.32 L/kg. Less than 1% of NNN was excreted unchanged in
the urine, with a renal clearance of 0.27 ± 0.24 ml/min. The two
major metabolites in serum were the hydroxy acid and norcotinine, as indicated by the metabolite/NNN AUC ratios.
NNN was rapidly metabolized to the hydroxy acid, with its maximal serum
concentration (tmax) occurring at 30 min. The hydroxy acid had an apparent elimination half-life of
42.1 ± 16.3 min, which was not significantly different from that
of NNN, indicating a formation rate-limitation on the elimination of
hydroxy acid (Table 2). The formation of norcotinine was somewhat
delayed with a tmax of 80 ± 35 min. It was very slowly eliminated, once formed, with an elimination
half-life of 198.7 ± 128 min. The slow formation of its
sequential metabolite, norcotinine-1-N-oxide, was consistent
with the kinetics of norcotinine. The
tmax for the
norcotinine-1-N-oxide was 90 ± 30 min and its
half-life was 162.4 ± 54.4 min. There was no significant
difference in either tmax or
elimination half-life of norcotinine and
norcotinine-1-N-oxide, indicating that the
N-oxide elimination was formation-rate limited.
3'-Hydroxynorcotinine was formed relatively quickly after the dose of
NNN with a tmax of 40 ± 17 min
but was in significantly lower concentrations than the two primary
serum metabolites, the hydroxy acid and norcotinine. Its
half-life was 174 ± 26.8 min, which was significantly longer than
that of NNN, but not significantly different from that of norcotinine.
The half-life data are consistent with 3'-hydroxynorcotinine being
either formed directly from NNN or formed from norcotinine. The
tmax of 3'-hydroxynorcotinine appears
to be shorter than that of norcotinine, a finding that would not
support its formation from norcotinine. However, the difference in the
tmax values did not reach statistical significance.
| |
Discussion |
The results of this study demonstrate considerable differences
between NNN metabolism in rodents and patas monkeys. In rodents, the
major urinary metabolites of NNN are the keto acid and hydroxy acid,
resulting from 2'-hydroxylation and 5'-hydroxylation, as illustrated in
Fig. 1. Relatively small amounts of norcotinine and
NNN-N-oxide are observed, whereas the other metabolites of Fig. 1 are barely detectable in rodent urine (Hecht, 1998). A common
feature of rodent and monkey metabolism of NNN is the relatively large
amount of the hydroxy acid in urine. However,
norcotinine-1N-oxide and 3'-hydroxynorcotinine are found in
patas monkey urine but not in rodent urine. In addition, the amount of
norcotinine in patas monkey urine is considerably greater than that
observed in rodent urine. Finally, there is less of the keto acid in
patas monkey urine than in rodent urine.
A scheme rationalizing the formation of NNN metabolites in the patas
monkey is presented in Fig. 6. The hydroxy acid results from
5'-hydroxylation of NNN, as in the rat. It may also be formed by
reduction of keto acid. The keto acid most likely is formed by
2'-hydroxylation, but it could also be produced from norcotinine (see
below). In the patas monkey, 5'-hydroxylation of NNN appears to exceed
2'-hydroxylation, which is consistent with results obtained in human
liver microsomes (Staretz et al., 1997). Rodent 2'-hydroxylation appears to be more extensive than that observed here (Hecht et al.,
1981).
The metabolic conversion of NNN to norcotinine, which may play a
central role in NNN metabolism in the patas monkey, is poorly understood. Our earlier studies in rodents suggest that 5'-hydroxyNNN and nornicotine are not precursors to norcotinine (McIntee and Hecht,
2000). Two other potential pathways to norcotinine are shown in Fig. 6.
In one, loss of HNO from NNN would produce isomyosmine, which
can be converted to norcotinine by epoxidation followed by spontaneous
rearrangement of the epoxide (National Institutes of Health shift).
Little is known about the chemical properties of isomyosmine, nor has
it been identified as a metabolite in any system to date. Nevertheless,
it is possible that this compound could be an intermediate in the
formation of both norcotinine and 3'-hydroxynorcotinine, as illustrated
in Fig. 6. A second possible intermediate in the formation of both
norcotinine and 3'-hydroxynorcotinine is -oximinonornicotine (Fig.
6). An analogous -oximoamine has been invoked as an intermediate in
the formation of metabolites of N-nitrosohexamethyleneimine
in the rat (Grandjean, 1976). -Oximinoamines are known compounds,
being produced in the photolysis of nitrosamines (Chow, 1973), but they
have not been isolated as nitrosamine metabolites. Thus, norcotinine
and 3'-hydroxynorcotinine could both be formed via isomyosmine or -oximinonornicotine.
Further metabolism of norcotinine leads to
norcotinine-1-N-oxide, and possibly to 3'-hydroxynorcotinine
and 3'-hydroxynorcotinine-O-glucuronide. Since
3'-hydroxycotinine is a major metabolite of cotinine, in humans and
rhesus monkeys, it is not unreasonable to propose that 3'-hydroxynorcotinine is a metabolite of norcotinine (Dagne and Castagnoli, 1972; Gorrod and Schepers, 1999). However, we were unable
to detect 3'-hydroxynorcotinine as a metabolite of norcotinine in vitro
with patas monkey liver microsomes (data not shown). Pharmacokinetic
considerations also suggest that 3'-hydroxynorcotinine is not a
metabolite of norcotinine. Moreover, norcotinine is excreted in urine
mainly unchanged after administration to rats; the only metabolite
detected was the keto acid (Hecht et al., 1981). Rat liver microsomes
convert norcotinine to 4-oxo-4-(3-pyridyl)butyramide and nicotinamide
(Eldirdiri et al., 1997). As mentioned above, 3'-hydroxynorcotinine
could be produced from isomyosmine or -oximinonornicotine, as well
as from norcotinine. The origin of 3'-hydroxynorcotinine requires
further study.
A major goal of this study was to identify a metabolite that could be
used as a biomarker of NNN uptake in humans. Such a metabolite should
not be formed from nicotine or other constituents, which are present in
tobacco products at levels considerably greater than those of NNN.
3'-Hydroxynorcotinine may qualify, but extensive further evaluation,
beyond the scope of the present study, would be necessary.
3'-Hydroxynorcotinine has not, to our knowledge, been reported as a
metabolite of nicotine, nornicotine, cotinine, or norcotinine in any
system, and it has not been reported as a constituent of tobacco
products. However, we cannot exclude the possibility that it simply has
not been found because no standard was available. There are several
experiments that should be carried out to further evaluate the
potential utility of 3'-hydroxynorcotinine as a biomarker of NNN
uptake. First, norcotinine and nicotine should be administered to patas
monkeys to determine whether 3'-hydroxynorcotinine is a urinary
metabolite. Second, the urine of nonsmokers who are using nicotine
replacement products should be analyzed for 3'-hydroxynorcotinine. Third, the urine of smokers should be analyzed for
3'-hydroxynorcotinine. If the results of the first two experiments were
negative, demonstrating that 3'-hydroxynorcotinine is not a metabolite
of norcotinine or nicotine, but 3'-hydroxynorcotinine were found in
appropriate quantities in the urine of smokers, its source likely would
be NNN. This possibility could perhaps be further evaluated by analysis of the urine of individuals using low nitrosamine tobacco products. These proposed experiments are justifiable because previous studies have clearly shown that aspects of the metabolism of the related tobacco-specific nitrosamine NNK are similar in patas monkeys and humans.
In summary, the results of this study demonstrate considerable
differences between rodent and patas monkey metabolism of NNN. In
particular, norcotinine, 3'-hydroxynorcotinine,
3'-hydroxynorcotinine-O-glucuronide, and
norcotinine-1-N-oxide are all quantitatively significant
metabolites of NNN in patas monkey urine whereas the pathways leading
to these compounds in rodents are relatively minor or nonexistent.
This study was supported by Grants CA-44377 and CA-81301 from
the National Cancer Institute
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Abstract
Introduction
), hydroxy acid (
), norcotinine (
),
3'-hydroxynorcotinine (X), norcotinine-1N-oxide (
).