Download N-Nitroso compounds in the gastrointestinal tract

Carcinogenesis vol.24 no.3 pp.595–603, 2003
N-Nitroso compounds in the gastrointestinal tract of rats and in the feces of mice
with induced colitis or fed hot dogs or beef
Sidney S.Mirvish1,2,4, James Haorah1, Lin Zhou1,
Melissa Hartman1, Chantey R.Morris1 and
Marge L.Clapper3
1Eppley
Institute for Research in Cancer and 2Departments of
Pharmaceutical Sciences and of Biochemistry and Molecular Biology,
University of Nebraska Medical Center, Omaha, NE 68198, USA and
3Division of Population Studies, Fox Chase Cancer Center, Philadelphia,
PA 19111, USA
whom correspondence should be addressed
Email: [email protected]
Because colonic N-nitroso compounds (NOC) may be a
cause of colon cancer, we determined total NOC levels by
Walters’ method in the gastrointestinal tract and feces of
rodents: (i) feces of C57BL mice fed chow and semipurified diets contained 3.2 ⍨ 0.4 and 0.46 ⍨ 0.06 NOC/
g, respectively (P < 0.01, mean ⍨ SD). (ii) NOC levels
for gastrointestinal contents of three groups of Sprague–
Dawley rats fed chow diet were 0.9 ⍨ 0.05 (diet), 0.2 ⍨ 0
(stomach), 0.3–0.4 (small intestine), 0.7–1.6 (cecum and
colon) and 2.6 ⍨ 0.6 (feces) nmol/g. NOC precursor
(NOCP) levels (measured as NOC after mild nitrosation)
for two rat groups fed chow diet showed a 16-fold increase
from stomach to proximal small intestine (mean, 6.2 µmol/
g), and a 1.7-fold increase from distal colon to feces (mean,
11.6 µmol/g). (iii) Eight Min and five C57BL/6J mice
received 4% dextran sulfate sodium in drinking water on
days 1–4 to induce acute colitis. This increased fecal NOC
levels 1.9-fold on day 5 in both strains (P ≤ 0.04), probably
due to NO synthase-derived nitrosating agents in the colon.
(iv) Following studies on humans fed beef [Hughes et al.
(2001) Carcinogenesis, 22, 199], Swiss mice received semipurified diets mixed with 18% of beef plus pork hot dogs
or sautéed beef for 7 days. On day 7, individual 24-h fecal
NOC outputs were determined. In three hot dog and two
beef groups with 5 mice/group, mean fecal NOC output/
day was 3.7–5.0 (hot dog) and 2.0–2.9 (beef) times that for
control groups fed semi-purified diet alone (P < 0.002 for
each of combined groups). These groups showed little
change in fecal NOCP output. (v) Initial purification of
rat fecal NOCP by adsorption–desorption and HPLC is
described. Results should help evaluate the view that
colonic NOC causes colon cancer associated with colitis
and ingestion of red and nitrite-preserved meat.
Introduction
N-Nitroso compounds (NOC) include nitrosamines and nitrosamides. They are produced by the reaction of nitrite and
nitrogen oxides with secondary amines and N-alkylamides, i.e.
with ‘NOC precursors’ (NOCP). We report here studies on
Abbreviations: DSS, dextran sulfate sodium; Exp., Experiment; GIT, gastrointestinal tract; iNOS, inducible nitric oxide synthase; NO, nitric oxide; NOC,
N-nitroso compounds; NOCP, NOC precursors; SP, semi-purified.
© All rights reserved. Oxford University Press 2003
595
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4To
NOC and NOCP in the gastrointestinal tract (GIT) and feces
of rats and mice. A basic question was to determine whether
NOC in the GIT and feces arise mainly from unabsorbed
dietary NOC or by in vivo nitrosation of NOCP in the GIT.
Carcinogenic NOC may be involved in the etiology of several
types of cancer, including those of the stomach and esophagus
(1). In 1996 Bingham et al. (2,3) proposed that NOC in colonic
contents are a cause of colon cancer. In earlier studies involving
the determination of total NOC in human feces by Walters’
method (4), mean total NOC levels were 8.4 nmol/g of human
feces compared with 1.0–1.5 nmol/g of fasting gastric juice
(5), and feeding nitrate raised mean fecal NOC levels from
109 (with a low nitrate diet) to 7.0 nmol NOC/g (6). In the
only related animal study, the colonic contents of conventional
and germ-free rats fed a semi-purified (SP) diet showed mean
values of 0 and 0.9 µmol NOC/kg, respectively, and colonic
NOC levels in conventional but not germ-free rats rose (to a
mean of 6.6 µmol/kg) when 500 mg nitrate/liter drinking water
was fed (7).
In 2001 we described improvements and simplifications of
the Walters’ method for determining total NOC and described
a method for measuring NOCP by nitrosation of aqueous food
extracts under mild conditions (110 mM nitrite, pH 1.5–2.0,
incubation for 1 h at 37°C), addition of sulfamic acid to destroy
excess nitrite and analysis for NOC (8). Nitrosation under
these conditions gave poor NOC yields when the NOCP were
simple secondary amines and N-alkylureas, but high (艌45%)
NOC yields when the NOCP was a rapidly nitrosated amine
such as morpholine, or was N-1-fructosyl valine. We examined
hot dogs (franks, frankfurters, wieners and sausages) because
they are a widely consumed nitrite-preserved meat product,
with US sales of ⬎800 million pounds in 2001 (9). We purified
the NOCP in hot dogs by adsorption–desorption and HPLC
(10). After the purified fractions were nitrosated and treated
with sulfamic acid, they were directly mutagenic for Salmonella
typhimurium TA-100, with up to a 4-fold increase in mutagenicity [(10) and unpublished studies]. In part, because some
N-nitrosoglycosyl amines are direct mutagens (11,12), we
suggested that the NOCP in hot dogs are N-glycosyl amino
acids (Figure 1) and N-glycosyl peptides (10).
Red meat (beef, pork and mutton) and, especially, processed
red meat (most of which is probably preserved with nitrite)
are probable risk factors for colon cancer (14). A recent metaanalysis of 13 studies concluded that the risk of colorectal
cancer was increased 12–17% by daily ingestion of 100 g red
meat and was increased by a mean of 49% on daily ingestion
of 25 g processed meat (15). Another recent review reached
a similar conclusion (16), but a third review disputed this
conclusion and emphasized that meat is a nutritionally important component of most Western diets (17). Fecal NOC excretion
in humans increased 3.7-fold when 420 g/day of beef was
consumed, did not decline even after this diet was consumed
for 40 days, showed a dose–response relationship for the
amount of beef consumed and was not affected when chicken
S.S.Mirvish et al.
Fig. 1. Structures of two types of N-glycosyl amino acids: (A) N-1-deoxy-1glycosyl amino acids; and (B) N-2-deoxy-2-fructosyl amino acids. Glucosyl
derivatives can rearrange to fructosyl derivatives by the Amadori reaction
(13).
Materials and methods
Animals, diets and general procedures
We used male Sprague–Dawley rats and male mice of various strains, with
both species aged 6–9 weeks, except in Experiment (Exp.) 4. The animals
were obtained from the National Institutes of Health unless mentioned
otherwise. To prevent coprophagy (eating the feces), in most tests the animals
were kept in cages fitted with floors of stainless steel wire grids mounted
2.5 cm from the bottom of the cages, with absorbent paper under the grids to
soak up spilt water and urine. Feces excreted over 24 h (except in Exp. 1)
were collected with tweezers after removing most of the spilt diet with a
kitchen strainer.
The diets were supplied by Harlan Teklad (Madison, WI) except in Exp. 4.
The chow diet was pelleted Sterilizable Rodent Diet W-8656, which contained
24.5% (by weight) of protein, 4.4% fat, 3.8% fiber, 7.4% ash, 47% nitrogen-
596
Analysis for NOC and NOCP
Samples were stored at –15°C for up to 3 weeks before analysis. For all
analyses, up to 1 g of diet, GIT contents or feces were weighed, dried
overnight at room temperature and ⬍1 Torr (this brought the samples to
constant weight) and weighed again. Mouse fecal pellets were then freed of
adhering diet by gentle rubbing with tissue paper. We analyzed several fecal
pellets of rats and mice or individual rat fecal pellets in Exp. 1: 24-h fecal
collections from mice in Exps 2, 4 and 5; and 1 g of diet, homogenized
sections of the GIT contents or several fecal pellets totaling ~1 g from
rats in Exp. 3. The samples were soaked for 30 min in a measured volume
(6–10 ml) of distilled water, vortexed, blended for 2 min in a motor-driven
Potter-Elvejhem homogenizer and centrifuged for 10 min at 10 500 g to give
‘supernatant A’. To determine NOC (8), 850 µl of supernatant A was mixed
with 50 µl of 2 N HCl and 100 µl of a freshly prepared saturated solution of
sulfamic acid in water to destroy any nitrite present. After the mixture was
kept for 15 min at room temperature and up to 4 h on ice, 100–200 µl samples
were injected into a modified Walters’ system for determining total NOC, in
which NOC are decomposed to NO by an HBr/HCl/HOAc/EtOAc mixture
refluxing under reduced pressure at 28°C (8,33,34). The NO is swept in a
stream of argon through seven wash bottles to dry and remove acid from the
gas stream, and is then passed into a Thermal Energy Analyzer to determine
the NO. N-Nitrosoproline (0.1 nmol) was injected as a standard once per hour.
To determine NOCP (8), samples of supernatant A were nitrosated by treatment
for 1 h at 37°C with 110 mM NaNO2 adjusted with HCl to pH 1.5–2.0, treated
with excess sulfamic acid, diluted 100 times with distilled water and analyzed
for NOC as just described. Analyses for NOC and NOCP are based on
duplicate and single analyses, respectively, of each extract. Duplicate analyses
generally agreed within 10%.
Exp. 1: initial analyses of rat and mouse feces
Rats and Swiss mice were fed chow or TD-98061 SP diets for 1 week.
Samples composed of several fecal pellets were then collected and the undried
samples were analyzed for NOC. In other tests, dried individual fecal pellets
were taken from 24-h fecal collections from individual rats and were analyzed
for NOC.
Exp. 2: fecal NOC in mice transferred from chow to SP diet
Four cages of five mice, two with IL10(–/–) and two with their wild-type
C57BL/6J mice (both from Jackson Laboratories, Bar Harbor, Maine) were
maintained on chow diet for 7 days. Then, starting on ‘day 1’, 24-h fecal
samples were collected daily for 6 days and analyzed for NOC. The chow
diet was continued until 10 a.m. on day 2, when the diet was switched to the
TD-98061 SP diet.
Exp. 3: NOC and NOCP in GIT contents and feces of rats
Rats were fed tap water and pelleted chow diet or powdered TD-98061 SP
diet for at least 1 week and were then killed with CO2. The contents of
different GIT sections were collected, combined for all rats on the same diet,
weighed and well mixed. The total collection (if ⬍1 g) or 1 g samples thereof
were analyzed for NOC or NOCP. The lengths of the GIT sections designated
as proximal, middle and distal small intestine and proximal and distal colon
were ~70, 70, 15, 8 and 8 cm, respectively. Each test involved two to three
rats fed chow diet and the same number fed SP diet. In two of these tests,
daily dietary intake and fecal output were determined for 2–4 days before death.
Exp. 4: fecal NOC in mice with acute colitis
Female Min and wild-type C57BL/6J mice from breeding colonies at Fox
Chase Cancer Center were treated there when they were 60–70 days old.
They were fed Purina Rodent chow 5013 (PMI-Nutrition, Brentwood, MO).
A 4% solution in distilled water of DSS (molecular weight 36 000–44 000
Da, ICN, Costa Mesa, CA) was administered as drinking water on days 1–4.
Body weight, stool consistency and results of a fecal occult blood test were
recorded daily. During days 5 and 7, feces were collected for 24 h while the
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or fish were eaten (2,3,18). These findings suggest that a cause
of colon cancer may be NOCs that occur in the colon and are
associated with the consumption of red meat.
Carcinogenic heterocyclic amines in charred meat may help
explain the apparent relationship between meat consumption
and colon cancer (19), and colon cancer was correlated
with certain polymorphic forms of N-acetyl transferase and
cytochrome P4501A2 that are involved in the activation
of these carcinogens (20). Carcinogenic polycyclic aromatic
hydrocarbons also occur in charred meat and can reach the
colon (21,22). Accordingly, NOC, heterocyclic amines and
polycyclic aromatic hydrocarbons might all be causes of
colon cancer associated with meat consumption. However, the
increased risk for processed meat suggests a special role for
NOC in this case.
Patients with inflammatory bowel disease (ulcerative colitis
and Crohn’s disease) show raised incidences of colon cancer
(23) and increased levels of inducible NO synthase (iNOS)
(24). During inflammatory bowel disease, colonic iNOS may
produce excess NO, which is oxidized to nitrogen oxides and
nitrite, which in turn could react with NOCP in colonic
contents to produce NOC. These NOC could initiate colon
cancer. Acute and chronic colitis were induced in mice by
treating them with 5% dextran sulfate sodium (DSS) in the
drinking water (25). Recently, acute colitis was induced by
treating mice for 4 days with 4% DSS (26). We applied the
latter treatment to Min (multiple intestinal neoplasia) and their
wild-type C57BL/6J mice, because Min mice spontaneously
develop adenomas of the small intestine and, to some extent,
the colon (27), and these tumors could be induced by NOC
formed in the intestines.
Rats were used here to study NOC and NOCP levels in the
GIT because it was easier to obtain useful amounts of GIT
contents from these animals than from mice. Mice were used
to study the effects of colitis and of feeding hot dogs and beef
on fecal NOC and NOCP because the most useful models of
colitis have been developed in mice and the small bulk of
their feces made it easy to dry and analyze 24-h samples.
Some of our results were presented at national meetings
(28–30).
free extract and added vitamins and minerals. Pelleted TD-98061 semi-purified
(SP) diet was based on the AIN-76A diet (31), with replacement of sucrose
by maltodextrin to mimic human diets more closely and still be able to pellet
the mixture. TD-01407 SP diet was powdered and was a modification of the
AIN-93G diet (32). The TD-98061 and TD-01407 diets contained, respectively,
in g/kg diet, 200 and 214 casein, 378 and 417 corn starch, 150 and 161
maltodextrin, 120 and 0 sucrose, 0 and 61 dextrose.H2O, 50 and 0 corn oil,
0 and 24 soybean oil, 50 and 61 cellulose, 35 and 0 AIN-76 mineral mixture,
0 and 43 AIN-93G-MX mineral mixture, 10 and 0 AIN-76A vitamin mixture,
0 and 12 AIN-93-VX vitamin mixture, 3 and 0 DL-methionine, 2 and 0
CaCO3, 2 and 3 choline bitartrate, and 0 and 0.017 t-butylhydroxyquinone.
Except in Exp. 4, the animals received tap water for drinking; this contained
~3 mg nitrate (as NO3–)/l according to our local water company. In chemical
experiments, suspensions were mixed with a Vortex-Genie mixer (Fisher
Scientific, Pittsburgh, PA).
N-Nitroso compounds in rodent feces
Table I. Composition of meat and control diets fed to mice in Exp. 5
Statistics
The Wilcoxon rank order test was used to determine the significance of
differences between groups.
Composition (g/kg diet)a
Control for
hot dog diet
Beef
diet
Control for
beef diet
820
0
0
180
917
26
57
0
820
0
0
180
909
51
40
0
Protein and fat in diets
Protein in SP diet
153
Protein in added casein
0
Protein in hot dog or beef 22
Total protein
175
Fat in SP diet
21
Fat in added soy oil
0
Fat in hot dog or beef
47
Total fat
68
171
20
0
191
22
57
0
79
153
0
52
205
20
0
36
56
169
44
0
213
22
40
0
62
Components of diet
SP diet TD-10407
Casein
Soy oil
Hot dog or beef
aBased
on the wet (undried) weights of each component.
mice were kept in metabolic cages and fasted. The feces was frozen, mailed
on dry ice to Omaha and analyzed for NOC.
Exp. 5: fecal NOC and NOCP in mice fed hot dogs and beef
The TD-01407 SP diet was designed so that addition of hot dog or beef at
18% by weight of the final diet brought the fat and protein contents to 5.6–
7.9 and 17.5–21.3% by weight, respectively (Table I). According to their
labels, the hot dogs were manufactured from beef and pork, and contained
26 g fat, 12 g protein and 1.7 g sugar/100 g (based on the reported composition
of a 57 g hot dog). The hot dogs, which are cooked during manufacture, were
mixed in the diet without further cooking. The beef was labeled as containing
20 g fat/100 g, normally contains 29 g protein/100 g (35) and was sautéed
(browned in a pan without added fat). The hot dog or beef (180 g) was
blended for several minutes in a Black and Decker Quick ‘N Easy Plus food
processor (Hampstead, MD) and 820 g of the SP diet was gradually blended
in. For the control diets (Table I), soy oil and casein were blended with the
SP diet. In each test, two cages of five Swiss mice were weighed. One cage
received the hot dog or beef diet and the other cage received the corresponding
control diet. The diets were supplied for 6 days in glazed clay pots (4.4 cm
high, diameter 6 cm at bottom and 3.5 cm at top). On day 7 the mice were
placed singly in cages each supplied with one pot of the same diet as before.
Feces were collected for 24 h, the mice were reweighed (there was little
change in weight over 7 days) and the feces analyzed. In each test, one sample
of each diet was analyzed for NOC and NOCP. Food consumption was
measured in one of the hot-dog tests.
Exp. 6: partial purification of NOCP in rat feces
Solutions were concentrated in a rotary evaporator at ⬍25 Torr and ⬍40°C
(solutions in organic solvents) or ⬍50°C (aqueous solutions). Feces were
collected from four rats maintained on chow diet. Dried feces (45 g) were
ground with 300 ml water in a Waring blender for 15 min. The mixture was
kept for 4 h at room temperature with occasional blending, centrifuged for 30
min at 6600 g and 4°C, filtered through Whatman no. 1 paper and evaporated
to 150 ml. The concentrate was mixed with 200 ml silica gel (Merck, grade
60, 70–230 mesh, Aldrich, Milwaukee, WI). A mixture of the resulting wet
solid and 300 ml acetonitrile (MeCN) was stirred occasionally for 30 min.
The supernatant (425 ml) was decanted. The sediment was extracted similarly
with 300 ml methanol (MeOH). The MeCN and MeOH extracts from the
silica gel were each evaporated to dryness and dissolved in 5 ml water. The
aqueous solutions were stirred for 5 min with 25 ml of the acidic form of a
cation exchange resin (50W-8X, Bio-Rad, Hercules, CA) at pH 2. The resin
was stirred (i) for 5 min with 300 ml water, which remained at pH 2 and was
decanted and discarded, (ii) with 2 N NH4OH until the pH reached 9.0 and
(iii) with 300 ml water adjusted to pH 9 with NH4OH. The pH 9 solution
derived from the MeOH extract was evaporated and dissolved in 5 ml water.
Of this solution, 50 µl was diluted with water to 2 ml. Of the diluted solution,
250 µl was injected onto a Rexchrom 25⫻1 cm amino HPLC column (Regis,
Morton Grove, IL), which was eluted with MeCN–water 7:3 at 3 ml/min.
The eluate was monitored at 260 nm. Ten 2-ml fractions were collected
and analyzed.
Results
Stability of NOC and NOCP in mouse feces
Feces were routinely collected for 24 h and dried under vacuum
overnight, both at room temperature. Some samples were
stored for up to 3 weeks at –15°C before analysis. To examine
the stability of fecal NOC and NOCP under these conditions,
two cages of mice were fed the hot dog diet for 6 days. Feces
were collected from each cage (batches 1 and 2) and dried by
our standard method, and ground to a powder with a mortar
and pestle. Samples (200–300 mg) of each of the two batches
were stored for 3 weeks at –70°C (control samples 1 and 2)
or at –15°C (samples 3 and 4), or were left for 24 h at room
temperature and then stored at –70°C (samples 5–8). We
prepared samples 1, 3, 5 and 6 from batch 1 and the remaining
samples from batch 2. Samples 1 and 2 showed 8.0 and
12.7 nmol NOC, and 4600 and 4700 nmol NOCP/g feces,
respectively. Sample 3 showed 98% for NOC, and samples 3
and 4 showed 86 and 120% for NOCP of the mean values for
the corresponding control samples. Samples 5–8 showed 76
⫾ 7% for NOC and 118 ⫾ 18% for NOCP of the mean results
for samples 1 and 2 (mean ⫾ SD). Some of this variability
may have been due to non-homogeneity of the ground feces,
a problem that did not arise in the main experiments where
entire 24-h fecal collections were analyzed without grinding.
These results indicate that NOC and NOCP were stable for 3
weeks at –15°C and that NOC, but not NOCP, decomposed
by ~25% when mouse feces were kept for 24 h and evaporated,
both at room temperature. This is far less than the severalfold changes observed in most of the reported experiments.
It remained possible that a fraction of the fecal NOC
or NOCP decomposed extensively during the initial 24-h
collection, even though storage for a second day at room
temperature caused only limited decomposition of NOC. Such
a finding would be analogous to the report of an unstable
NOC fraction in human gastric juice (36). Four batches of
feces were collected at room temperature during only 6–8 h,
stored overnight at –15°C, vacuum dried for 36 h at 0°C to
constant weight and ground as before. From each batch, one
90–150 mg sample (control samples 9–12) was stored for 3
weeks at –70°C and another similar sample (samples 13–16)
was stored at room temperature for 24 h and then for 20 days
at –70°C. Samples 9–12 showed 12 ⫾ 7 nmol NOC and 3600
⫾ 1000 nmol NOCP/g. The results for samples 13–16 were
118 ⫾ 12% for NOC and 82 ⫾ 29% for NOCP of the mean
levels for samples 9–12, and hence do not indicate extensive
instability of NOC or NOCP in freshly collected mouse feces
stored for 24 h at room temperature.
Exp. 1: initial analyses of rat and mouse feces
In tests on four rats maintained on chow diet, we found 0.44
⫾ 0.06 nmol NOC/g feces and 430 ⫾ 140 nmol NOCP/g
feces. The feces of C57BL mice (four mice/diet) fed chow
and SP diet contained 3.2 ⫾ 0.4 and 0.46 ⫾ 0.06 nmol NOC/
g feces, respectively. We determined NOC in three individual
fecal pellets from each of nine rats maintained on chow diet.
The difference between NOC concentration in each pellet and
the mean value for the three pellets was 42 ⫾ 28% of the
mean values. This large variation demonstrates the need to
analyze 24-h samples wherever possible.
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Hot dog
diet
S.S.Mirvish et al.
598
0.43 ⫾ 0.06 nmol NOC/g and 4900 ⫾ 700 nmol NOCP/g. In
the present test, the DSS-treated mice showed gross signs of
acute colitis, i.e. loss of body weight, blood in the stools and
diarrhea. Two of the eight DSS-treated Min mice died on day
8. Mean body weights on days 1, 5 and 7 were, respectively,
16, 16 and 13 g for the DSS-treated Min mice, and 18, 18
and 16 g for the DSS-treated C57BL/6J mice. Fecal blood
was detected in all DSS-treated mice on both days 5 and 7.
Stool consistency on days 5–7 was soft in almost all the DSStreated mice. Feces from days 5 and 7 were analyzed for NOC.
The results (Table III) are presented as fecal NOC concentration
rather than output per day because the mice were fasted
overnight (to facilitate urine collection for another purpose),
which reduced fecal output, and the diarrhea may have prevented collection of all the feces. On day 5, the DSS-treated
mice of both strains showed significant (P 艋 0.04; mean, 1.9fold) increases in fecal NOC concentration compared with
untreated mice of the same strains. On day 7, the DSS-treated
mice of both strains showed a consistent but smaller mean
increase of 1.6-fold in fecal NOC level, with P 艋 0.07.
Exp. 5: fecal NOC and NOCP in mice fed hot dogs and beef
The beef and hot dog diets (Table I) were eaten freely from
day 1 of each test, but it took 1–2 days for the mice to eat
freely of the control diets. NOC concentrations in the hot dog
and sautéed beef used to prepare these diets were 0.4–0.8
nmol/g, consistent with the NOC content of the complete diets.
Mean body weights in each test were 26–30 g. The results
(Table IV) are quite striking. Fecal NOC excretion (in nmol/
day) showed mean values that were 3.7, 5.0 and 3.8 times
higher for the three tests of hot dog diet, and 2.0 and 2.9 times
higher for the two tests of beef diet, as compared with control
groups in the same test. The differences in fecal NOC excretion
(nmol/mouse/day) between meat and control groups of the
same test were significant, with P ⬍ 0.002 for each of the
three hot dog tests, 0.01 and 0.06 for each of the two beef
tests, ⬍0.0001 for the combined hot dog tests, and 0.0013 for
the combined beef tests (the results for the combined tests
were obtained after expressing the result for each treated and
control mouse as a percentage of the mean control value in
the individual test). Because the amount of feces excreted in
24 h did not differ much between the groups (Table IV), fecal
NOC concentration paralleled fecal NOC output/day. The
differences between fecal NOC concentration in the treated
and control groups showed P values of 0.012 for each of the
three hot dog groups, 0.06 and 0.02 for the two beef groups,
and ⬍0.002 for the combined hot dog and the combined
beef groups.
Mean fecal NOC output was 33–41% (for the groups fed
the hot dog and beef diets) and 15–69% (for the control
groups) of mean dietary NOC intake (both intake and output
were expressed as nmol/mouse/day) (Table IV). The estimation
of dietary NOC intake assumes that the mice ate 5 g diet/day,
which is standard for this species (39) and is similar to
consumptions of 4.5–5.5 g/day observed in one of our tests.
Whereas the entire 24-h fecal collection was homogenized and
analyzed, only single food samples were analyzed in most of
these tests. This and the low NOC levels in the control diets
probably explain the wide variations in fecal NOC output
expressed as percentages of dietary NOC. In one test each of
the hot dog and beef diets, fecal NOCP was determined in
addition to NOC (Table IV). Mean NOCP outputs in the
treated and control groups were 1100–2000 nmol/day, ~1000
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Exp. 2: fecal NOC levels in mice transferred from chow to
SP diet
IL10(–/–) and their wild-type C57BL/6J mice were examined
here because IL10(–/–) mice can spontaneously develop colitis
(37). We determined how rapidly the fecal NOC level would
change after these mice were transferred from chow to SP
diet. Similar to our findings in rats, SP diet gave feces with
only ~25% of the NOC level obtained with chow diet (Table II).
It took 3 days after the diets were switched (from days 2 to
5) for fecal levels to drop to the value for SP diet. No
differences were seen between the two mouse strains, perhaps
because colitis does not always occur in IL10(–/–) mice on
a C57BL/6J background (37). [IL10(–/–) mice on a C3H
background have just been reported to develop colitis more
consistently (38).] Hence we did not use IL10(–/–) mice for
studying the effect of colitis on fecal NOC excretion (see
Exp. 4).
Exp. 3: NOC and NOCP in GIT contents and feces of rats
NOC and NOCP were determined in the diet, feces and
contents of individual GIT sections of rats fed chow and SP
diets (Figure 2). NOC and NOCP concentrations are presented
per gram wet weight (weight before drying) rather than per
gram dry weight because the GIT mucosa is exposed to the
undried contents. In two tests, mean dietary intake per rat per
day was 25 and 28 g for chow diet and 23 and 26 g for SP
diet, and mean fecal output per rat per day was 13 and 21 g
for chow diet and 4.7 and 5.2 g for SP diet. The wet weights
of all GIT sections were higher for chow than for SP diet
(Figure 2A). The dry/wet weight ratio (Figure 2B) remained
~0.23 in the upper GIT, but increased sharply in the colon and
feces. In three tests each on rats fed chow and SP diets, NOC
concentration was somewhat lower in the stomach than in the
diet and then increased steadily towards the distal GIT, with
especially large increases for chow diet just beyond the distal
small intestine, and for both diets in the colon and feces
(Figure 2C). NOC levels were higher with chow than with SP
diet, with chow/SP ratios for nmol NOC per gram wet weight
of 6.3 for the diet, 1.5–3.3 for the GIT contents and 3.8 for feces.
In two tests each on rats fed chow and SP diets, NOCP
levels per gram wet weight were similar in the stomach and
diet, rose by a surprising mean of 16-fold for chow diet and
2.6-fold for SP diet in the proximal small intestine, fell in the
cecum (especially for SP diet) and (for chow diet) rose in the
distal colon and feces, with a mean 1.7-fold increase from
distal colon to feces (Figure 2D). The two tests gave similar
results. The difference between the NOCP levels in the stomach
and proximal small intestine was significant (P ⫽ 0.03) when
the results for the chow and SP diets were combined. Mean
NOCP concentrations for rats on chow diet were four (in the
distal colon) and 23 times (in the feces) higher than those for
rats on SP diet. The differences between the results for the
two diets were significant for the combined distal colon and
feces, with P ⫽ 0.005 for NOC and 0.03 for NOCP.
Exp. 4: fecal NOC in mice with acute colitis
Min and their wild-type C57BL/6J mice were treated on days
1–4 with DSS to induce acute colitis (26). Chow diet was fed
here because previous studies by one of us (M.L.C.) showed
that DSS was highly toxic in mice fed an AIN-76A SP diet.
Thus, only half of a group of Swiss-Webster mice that were
given this diet survived DSS treatment for 7 days, whereas all
mice of this strain that were fed chow diet survived the DSS
treatment. The samples of the chow diet used here contained
N-Nitroso compounds in rodent feces
Downloaded from http://carcin.oxfordjournals.org/ at oxford university press on November 23, 2016
Fig. 2. Analysis of diet, GIT sections and feces for NOC and NOCP in rats fed chow and SP diets. (A) Wet weights; (B) dry weights as percentages of the
wet weights; (C) NOC concentration as nmol NOC/g wet weight; (D) NOCP concentration as µmol NOCP/g wet weight. Results are given for the different
GIT sections and (except in A) for the diet and feces. Note that NOC are expressed in nmol/g and NOCP in µmol/g. These figures show the mean and SD for
three tests each on rats fed chow and SP diet. The ‘GIT sections’ are: 1, diet; 2, stomach; 3–5, proximal, middle and distal small intestine, respectively; 6,
cecum; 7, appendix; 8, proximal colon; 9, distal colon; and 10, feces. T-bars show standard of deviations.
599
S.S.Mirvish et al.
Table II. Fecal NOC in male mice transferred from chow to SP dieta
Day
1
2
3
4
5
6
NOC (nmol/g feces)
IL-10(–/–) mice
C57BL mice
2.3,
2.2,
1.3,
0.6,
0.5,
0.5,
2.3,
2.2,
1.1,
0.6,
0.5,
0.5,
2.5
2.3
1.3
0.8
0.5
0.5
2.5
2.4
1.3
0.7
0.5
0.7
Fig. 4. HPLC on an amino column of the meOH eluate from purified rat
fecal NOCP. This shows the last stage in the purification of rat fecal NOCP
outlined in Figure 3. Diagonals, nmol NOCP/fraction. Cross-hatches, area
(integration units) for UV absorption of each fraction at 260 nm.
Table III. NOC in feces of mice treated with 4% DSS in drinking water on
days 1–4
Mice
No. of
mice
DSS
aMice
were switched to SP diet on 10 am of day 2.
Results are shown for two cages of each mouse strain.
times the output of NOC, with no significant differences
between the hot dog or beef groups and the control groups.
To check whether the single diet analyses used in Table IV
were representative of the total diet, we prepared fresh batches
of the hot dog diet and its control diet, and analyzed three
samples of each batch by duplicate analysis for NOC and
single analysis for NOCP. The results were 0.75, 0.81 and
1.05 nmol NOC/g and 390, 600 and 680 nmol NOCP/g for
the hot dog diet; and 0.43, 0.44 and 0.61 nmol NOC/g and
1320, 1330 and 1530 nmol NOCP/g for the control diet,
indicating that the single diet analyses used in Table IV were
somewhat inaccurate.
Variability of duplicate analyses
All NOC results were based on duplicate analyses of each
extract. The NOC results for the hot dog and its control diet
(tests 1–3 of Table IV) are used here to illustrate the agreement
between these duplicate analyses. The average difference
between the individual and mean values was 23 ⫾ 15% of the
mean values for the three hot dog groups and 37 ⫾ 14% of
the mean values for the three control groups. These percentages
are small relative to the 3.7-fold difference between the treated
and control groups, and hence do not affect the significance
of the comparison between these groups. The other NOC
analyses showed similar variations between the duplicate
analyses.
600
C57BL/6J
C57BL/6J
Min
Min
7
5
8
8
–
⫹
–
⫹
NOC (nmol/g feces,
mean ⫾ SD)
Significance
(P ⬍)
Day 5
Day 5
Day 7
–
0.04
–
0.01
–
0.07
–
0.05
20
38
22
41
⫾
⫾
⫾
⫾
17
18
9
16
Day 7
18
29
27
42
⫾
⫾
⫾
⫾
9
12
5
14
Exp. 6: partial purification of NOCP in rat feces
NOCP were studied here rather than NOC because they are
about 1000 times more abundant in rat feces than the NOC
(see Exp. 1) and identification of the NOC would probably be
easy once the NOCP were identified. NOCP from the feces of
rats fed chow diet were purified as summarized in Figure 3,
which also gives the NOCP yield at each stage of the
purification. The method involved batchwise adsorption on
silica gel, desorption with MeCN and then MeOH (these
contained 44 and 56%, respectively, of the total eluted NOCP),
followed by batchwise adsorption onto cation exchange resin
in its H⫹ form and elution from the resin at pH 9. Cation
exchange resin was used because, if the NOCP are secondary
amines, which is probable, they should be adsorbed on the
resin in their protonated form (RNH2R⬘⫹) and eluted from the
resin in their basic form (RNHR⬘). In the final step, HPLC on
an amino column of the pH-9 eluate from the MeOH fraction
(Figure 4) showed a principal NOCP peak in fractions 2–3
and three minor NOCP peaks, and UV absorption at 260 nm
that coincided with two of the NOCP peaks.
Downloaded from http://carcin.oxfordjournals.org/ at oxford university press on November 23, 2016
Fig. 3. Flow sheet for purification of NOCP in rat feces. The parentheses
show the amount of NOCP in each fraction.
N-Nitroso compounds in rodent feces
Table IV. Dietary intake and fecal output of NOC and NOCP in mice fed hot dog and beef dietsa
1
2
3
4
5
3
5
Test diet
Parameter measured
Hot dog
NOC
Hot dog
NOC
Hot dog
NOC
Beef
NOC
Beef
NOC
Hot dog
NOCP
Beef
NOCP
Mice fed control diets
Dietary NOC or NOCP level (nmol/g)
Dietary NOC or NOCP intake (nmol/mouse/day)b
Weight of feces (mg/mouse/day)
Fecal NOC or NOCP level (nmol/g)
Fecal NOC or NOCP output (nmol/mouse/day)c
Fecal output/dietary NOC or NOCP intake (%)d
0.35
0.32
1.77
1.62
260 ⫾ 110
310 ⫾ 60
0.98 ⫾ 0.33 0.79 ⫾ 0.13
0.27 ⫾ 0.19 0.24 ⫾ 0.07
15
15
0.23
0.18
1.15
0.92
420 ⫾ 80
280 ⫾ 80
2.00 ⫾ 1.18 1.29 ⫾ 0.86
0.79 ⫾ 0.43 0.39 ⫾ 0.29
69
43
0.39
1400
113
1.94
7000
565
380 ⫾ 100
420 ⫾ 80
380 ⫾ 100
1.38 ⫾ 0.85 2800 ⫾ 1600 4560 ⫾ 1260
0.45 ⫾ 0.19 1160 ⫾ 670 1800 ⫾ 850
23
17
320
Mice fed meat diets
Dietary NOC or NOCP level (nmol/g)
Dietary NOC or NOCP intake (nmol/mouse/day)b
Weight of feces (mg/mouse/day)
Fecal NOC or NOCP level(nmol/g)
Fecal NOC or NOCP output (nmol/mouse/day)c
Fecal output/dietary NOC or NOCP intake (%)d
Meat/control ratio for fecal NOC or NOCP output/daye
0.80
0.50
4.02
2.70
240 ⫾ 30
290 ⫾ 60
5.58 ⫾ 1.70 3.23 ⫾ 1.26
1.35 ⫾ 0.47 0.89 ⫾ 0.30
34
33
5.0
3.7
1.12
7.3
390
7.34
2.99
41
3.8
0.79
560
650
3.94
2800
3250
360 ⫾ 50
390 ⫾ 80
360 ⫾ 50
3.68 ⫾ 1.12 4800 ⫾ 2600 5100 ⫾ 1600
1.31 ⫾ 0.37 2000 ⫾ 1500 1900 ⫾ 700
33
71
530
2.9
0.71
1.05
aAll measurements are based on dry weights of the fecal and dietary samples.
bAssumes that mice ate 5 g diet/day.
cCalculated from fecal NOC concentrations and weight of feces for each individual
dMean daily fecal NOC output as percentage
eCalculated from the mean values.
⫾
⫾
⫾
⫾
0.45
2.9
2.26
80
330 ⫾ 50
2.97 2.38 ⫾ 0.65
1.74 0.77 ⫾ 0.18
34
2.0
mouse.
of mean daily NOC intake.
Discussion
Our studies used both chow and SP diets because chow diets
are better surrogates for human diets and are more suitable
for inducing colitis with DSS (Exp. 4), whereas SP diets are
more defined and showed lower NOC levels (Figure 2C) and
smaller variations in NOC content of the diet and feces than
did chow diet. Our results were mostly consistent within
each experiment but varied extensively between experiments,
especially when chow diet was fed. Thus, with chow diet the
mean fecal NOC levels in nmol NOC/g were 0.44 for rats in
Exp. 1, 2.4 for mice in Exp. 2 (Table II), 3.1 for rats in Exp.
3 (Figure 2C) and 18–20 for mice in Exp. 4 (Table III),
where the source of the diet differed from that for the other
experiments. For animals fed one of the SP diets, mean fecal
results in nmol NOC/g were 0.46 for mice in Exp. 1, 0.5–0.7
for mice in Exp. 2 (Table II), 0.69 for rats in Exp. 3 (Figure
2C) and 0.8–2.0 for mice in Exp. 5 (Table IV). The mean
fecal NOC levels for rats and mice fed chow diet are in the
same range as the 8.4–9.5 nmol NOC/g reported for the feces
of humans fed control diets (6,18). The low fecal NOC level
with SP diet may explain why Massey et al. (7) did not detect
NOC in the colons of rats fed SP diet. NOCP levels also
varied, with mean values for nmol NOCP/g feces of 430 for
rats fed chow diet in Exp. 1, 11 600 and 510 for rats fed chow
and SP diet, respectively, in Exp. 3 (Figure 2D), and 1160–
1800 for mice fed SP diet in Exp. 5 (Table IV).
Tap water containing ~3 mg nitrate (as NO3–) per liter was
fed in all tests except Exp. 4, where distilled water was given.
This nitrate was probably insufficient to produce significant
amounts of NOC in vivo, e.g. lung adenomas were induced in
strain A mice by feeding piperazine (a readily nitrosated
amine) together with as little as 250 mg NaNO2 per liter, but
no lung tumors were induced by feeding piperazine with 12.3
g NaNO3/liter drinking water (40), suggesting that nitrate is
not reduced extensively to nitrite in mice and rats, unlike the
situation in humans (41).
The results in Exp. 3 (Figure 2) extend the sections of the
GIT known to contain NOC from the previously studied
stomach (33) and colon (7) to the entire GIT, and suggest that
at least some fecal NOC arise from dietary NOC that pass
unabsorbed through the GIT. Most likely, NOC concentration
increases on descending the GIT because the GIT mucosa
absorbs water and digested food components, but not some of
the NOC. In support of this view, percent dry weight increased
2.7-fold and NOC level increased by a similar 3.3-fold on
proceeding from the proximal colon to the feces in rats fed
chow diet (Figure 2B and C). For SP diet, the corresponding
increases were 1.37- and 1.92-fold. Mean NOC level/g wet
weight in the distal colon was 82 and 92% of that in the feces
for rats fed chow and SP diet, respectively (Figure 2C). Hence,
at least in rats, fecal NOC level is a good indicator of NOC
level in the distal colon. If NOC are a cause of colon cancer,
the low NOC level in the small intestine relative to that in the
colon helps explain why the incidence of small intestine cancer
in the USA is only 5% of that for colorectal cancer (42).
The large NOCP peak in the proximal small intestine
(duodenum) with both chow and SP diets (Figure 2D) suggests
that NOCP is secreted into or produced in this section of the
GIT, and is absorbed or degraded in the distal small intestine.
The source of the duodenal NOCP could be bile salts secreted
in the bile or N-glycosyl amino acids (Figure 1) produced by
reactions between monosaccharides and amino acids under the
neutral or slightly basic conditions of the duodenum (43).
NOCP concentrations from the cecum to the feces were far
higher for chow than for SP diet, e.g. 23-fold higher for feces
(Figure 2D). From the proximal colon to the feces, percent
dry matter increased 2.72-fold with chow diet and 1.37-fold
with SP diet (Figure 2B), but NOCP level increased only 2.03fold with chow diet and fell 62% with SP diet (Figure 2D),
indicating some loss of NOCP in the colon and/or feces.
In Exp. 2 the 3 days required for mouse fecal NOC to drop
after the switch from chow to SP diet (Table II) probably
reflects transit time through the GIT. The finding that IL10(–/–)
mice on a C57BL/6J background did not show raised NOC
601
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Test no.
S.S.Mirvish et al.
602
tion of the fecal NOCP were less hydrophilic glycosyl amino
acids. UV absorption at 260 nm of the HPLC eluate fractions
was followed because it could be used to identify fractions
from different HPLC runs when they are combined. In Figure
4, the coincidence of the major NOCP peak (fraction 2) with
a major peak of UV absorption probably does not mean that
this NOCP was UV-absorbing as the NOCP was probably a
minor component of this fraction.
In conclusion, our results support the view that colonic
NOC are a cause of colitis-associated colon cancer and, as
proposed by Bingham et al. (2), of sporadic colon cancer
linked with consumption of red and processed meat. It remains
to establish in rodent models and in humans whether fecal
NOC represent unabsorbed dietary NOC, as suggested by Exp.
5 on the meat diets and by the finding in Exp. 3 that NOC
occur throughout the GIT; or whether some fecal NOC arise
by in vivo nitrosation, as suggested by Exp. 4 on colitis and
by the Hughes experiment (18). To help establish whether
NOC are a significant factor for the etiology of colon cancer,
it is urgent to identify the NOC in feces and to establish
whether they are absorbed from the GIT and are genotoxic
and carcinogenic in the colon.
Acknowledgements
We thank Elizabeth Lyden and Lynda Hock (Department of Preventive and
Societal Medicine, University of Nebraska Medical Center) for the statistical
analyses, Bryan Lee and Dr Wen-Chi Chang for technical assistance in
inducing colitis in mice, the Instrument Shop at Fox Chase Cancer Center for
constructing metabolic cages, Dr Ron Rose (Harlan-Teklad, Madison, WI) for
advice in formulating the diets, and Lynda Mirvish for technical and editorial
advice. This study was supported by grants RO1-CA-71483 and CA-06927
and core grant P30-CA-36727 from the National Cancer Institute, grant 94B28
from the American Institute for Cancer Research and support from the
Commonwealth of Pennsylvania.
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hot dog diets, and fecal NOC represented the fraction (15–
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absorbed from or degraded in the GIT. The concentration of
NOC in the feces was far higher than that in the diet, e.g. in
test 1 of the hot dog diet, fecal NOC concentrations were 5.58
and 0.98 nmol/g in the hot dog and control groups, respectively,
compared with dietary NOC levels of 0.80 and 0.38 nmol/g,
respectively, in the same groups (Table IV). This difference
was more than counterbalanced by the small amount of feces,
e.g. in test 1, fecal output was 210 mg/day whereas diet
consumption was assumed to be 5 g/day. The mean of 2.4–
7.3 nmol NOC/g feces for the mice fed beef and hot dogs
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NOC/g feces (Table II).
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beef diets. However, we reported (8) that fresh meat, including
beef, contains ~0.5 µmol NOC/kg and that heating hot dog
extracts at 100°C slightly lowered their NOC content (8). On
this basis, the diet with 420 g beef/day fed by Hughes et al.
contained 0.21 µmol NOC/day, far less than the average
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personal communication]. Hence, in the test on humans, ⬎90%
of the excreted NOC may have been produced in vivo.
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partial purification (Figures 3 and 4) suggests that these NOCP
are similar to those in hot dogs, as they were purified by
similar methods. The purified fractions were not weighed, so
that activity (µmol NOCP/g) could not be determined. However, purification of the hot dog NOCP showed a 19-fold
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