Carcinogenesis vol.20 no.11 pp.2167–2170, 1999
Mutagenesis induced by oral carcinogens in lacZ mouse
(Muta™Mouse) tongue and other oral tissues
Marcia d.M.von Pressentin1, Wieslawa Kosinska1 and
Joseph B.Guttenplan1,2,3
1Division
of Basic Sciences/Biochemistry, New York University, Dental
Center, 345 East 24th Street and 2Department of Environmental Medicine,
New York University, Medical Center, New York, NY 10100, USA
3To
whom correspondence should be addressed
Email: joseph.guttenplan@nyu.edu
Animal models for carcinogenesis of the oral cavity are
limited, although this disease is often fatal or disfiguring
and its incidence in the USA is ~30 000 cases/year. Shortterm whole-animal models for this disease should prove
valuable in the investigation of factors affecting oral carcinogenesis. In this study we observed that a group of oral
carcinogens are clearly mutagenic in the lacZ transgenic
mouse oral cavity. The carcinogens 4-nitroquinoline-Noxide (4-NQO), benzo[a]pyrene (B[a]P), N-nitroso-Nmethylurea (NMU), 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK), nitrosonornicotine (NNN) and 7,12dimethylbenzanthracene (DMBA) were all mutagenic in a
mixture of pooled oral tissues (gingival, buccal, pharyngeal
and sublingual) and in the tongue. All agents except DMBA
(which was swabbed in the oral cavity) and B[a]P (by
gavage) were given in drinking water for 2–4 weeks followed
by a 2 week expression period before killing. With one
exception, groups of 4–5 female mice were treated. The
doses and mutant fractions (MF) in DNA isolated from
pooled oral tissues (in mutants/105 p.f.u. K SD) were:
4-NQO (20–80 µg/ml, over 4 weeks) 78 K 16; B[a]P (five
doses of 125 mg/ml) 33.2 K 10.9; NMU (20–80 µg/ml over
4 weeks) 7.8 K 2.6; NNK (0.1 mg/ml, weeks 1–2, 0.2 mg/
ml, weeks 3–4) 9.1 K 3.0; NNN (same dose as NNK) 9.2
K 1.6 and DMBA (0.5 mg/ml in corn oil, 3 weeks) 7.1 K
2.7. The corresponding value for untreated controls was
3.2 K 1.8. Values for induced mutagenesis in tongue from
the same animals were similar except for 4-NQO which
was about twice as potent in tongue. Mutagenesis by several
compounds was compared in other organs. B[a]P was
assayed in lung and kidney and was about twice as
mutagenic in oral tissues as in lung, but several times less
mutagenic in kidney. Lung, but not kidney is a target
organ for B[a]P-induced carcinogenesis in the mouse. NNK
was somewhat more mutagenic in lung (MF of 15.0 K 5.5)
than in oral tissues, corresponding with previous reports
on carcinogenesis by NNK. Mutagenesis induced by NNN
was also assayed in esophagus, a target organ in rodents,
and was similar to that in oral tissue. In all cases the MF
in untreated control group was about 3–4. These results
suggest that: (i) the oral cavity has a significant capacity
for metabolic activation of carcinogens; (ii) DNA damage
in the oral cavity can be converted to mutations; and (iii)
Abbreviations: B[a]P, benzo[a]pyrene; DMBA, 7,12-dimethylbenzanthracene; MF, mutant fraction; MNU; N-nitroso-N-methylurea; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, nitrosonornicotine; 4-NQO, 4nitroquinoline-N-oxide.
© Oxford University Press
there is significant target organ specificity. The results also
tend to support the concept that the anatomical components
of the upper aerodigestive tract, in general, behave similarly
with respect to genotoxicity. As carcinogenesis is believed
to involve mutagenesis, this study demonstrates the utility
of the lacZ mouse for investigations involving initiation of
carcinogenesis of the oral cavity.
Introduction
Cancer of the oral cavity represents a significant public health
problem, with ~30 000 new cases and 8000 deaths per year
(1). In addition it often results in significant disfiguration and
other complications in the oral cavity and face. There are two
whole-animal models for this disease, 7,12-dimethylbenzanthracene (DMBA)-induced carcinogenesis in the hamster
cheek pouch (2) and 4-nitroquinoline-N-oxide (4-NQO)induced carcinogenesis in the rat oral cavity (3). The latter
model is anatomically closer to the human situation (3), more
convenient and probably more reproducible as the carcinogen
is administered in the drinking water, as opposed to painting
in the former. Experiments in cultured cells are of course
much more time efficient, but they require major extrapolations
to in vivo situations and generally there is no accurate method
for performing such extrapolations, as carcinogen-activating
and -detoxifying enzymes, the concentrations of carcinogens
and their metabolites, and a host of other conditions are much
different in culture than in animals. Since carcinogenesis assays
involving the oral cavity require significant time and resource
investment, a shorter term in vivo assay would offer considerable utility in establishing conditions for carcinogenesis assays
and possibly for mechanistic studies.
The lacZ and related lacI mouse in vivo mutagenesis assays
have been developed as shorter-term models for the initiation
steps of carcinogenesis (4,5). In addition the assay is sensitive
to increases in cellular proliferation at certain time intervals
after administration of the genotoxin (6,7), and therefore the
effects of certain tumor promoters are detectable (6,7). This
system should then be applicable to the initiation stages of
carcinogenesis, including the effects of cellular proliferation
on initiation. In an initial study we reported that benzo[a]pyrene
(B[a]P), a carcinogen known to produce tumors in the mouse
tongue, among other organs (8) was mutagenic in lacZ mouse
tongue and a number of other organs (9). Here we extend that
work and report that a number of known oral carcinogens,
including several present in tobacco products, are mutagenic
in tongue and other pooled areas of the oral cavity following
oral administration.
Materials and methods
Animals
Female mice (Muta™Mouse, 6 weeks old) were purchased from Covance
Research Products (Denver, PA) and acclimated for 1 week before the start
of treatments. Mice were housed in polycarbonate micro-isolation cages, with
2167
M.d.M.von Pressentin, W.Kosinska and J.B.Guttenplan
Shepard Paper Products dry bedding. The housing quarters were maintained
on a 12 h light/dark cycle, 21°C, 50% relative humidity. The mice were
maintained on an AIN-76 diet (ICN Biomedicals, Costa Mesa, CA) and were
allowed food and water ad libitum.
Chemicals
B[a]P, 4-NQO, N-nitroso-N-methylurea (MNU), DMBA, protease K and
RNase A were purchased from Sigma (St Louis, MO) and nitrosonornicotine
(NNN) and 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) were
purchased from Chemsyn Science Laboratories (Lenexa, KS).
Dosing
DMBA was swabbed (0.5 mg/ml solution in corn oil) for 2 min/mouse for 3
weeks (weekdays only). The animals were killed after another 2 weeks.
4-NQO and MNU were given at a concentration of 20 µg/ml for 2 weeks,
followed by 40 and 80 µg/ml for 1 week each in the drinking water. The
mice were killed 2 weeks later.
B[a]P was administered by gavage in corn oil (0.25 ml of 10 mg/ml every
other day for the first 5 days and then twice more every third day, for a total
of five doses. As the mice weighed ~20 g at the time of administration, the
individual doses administered were 125 mg/kg.
NNK and NNK were given at a concentration of 0.1 mg/ml for 2 weeks
followed by 0.2 mg/ml in drinking water for 2 weeks followed by a 2 week
expression period.
Drinking water containing 4-NQO, NNK and NNN was changed twice
weekly. Drinking water containing NMU was changed every other day.
DNA isolation
A portion of the tissue was gently homogenized by hand in a ground glass
homogenizer using three volumes of 10 mM Tris–HCl (pH 8.0), 10 mM
EDTA, 150 mM NaCl per part tissue weight (w/v). To this mix, 1% SDS and
1 mg/ml protease K were added. The mixture was incubated overnight at
37°C or 2–3 h at 50°C, and then for 30 min at 37°C with 0.1 mg/ml RNase
A. After this, one-third volume of 6 M ammonium acetate (pH 7.4) was
added, the mixture was gently mixed and then centrifuged at 14 000 r.p.m. in
an Eppendorf microfuge. The supernatant was carefully removed, leaving a
small volume behind, so as not to transfer any of the precipitate. An equal
volume of isopropyl alcohol was added to the supernatant at room temperature
to precipitate DNA. The supernatant was removed, the DNA was washed
once with 70% ethanol, suspended in 10 mM Tris–HCl (pH 8.0), 1 mM
EDTA, and left at room temperature overnight to dissolve. The samples
designated, ‘pooled oral tissues’ consist of a mixture of gingival, buccal,
pharyngeal and sublingual tissue.
Mutagenesis assay
Phage packaging was carried out using a Transpack packaging mix (Stratagene,
La Jolla, CA) and the positive selection (galE–) mutation assay was performed
according to published techniques (10). The necessary bacterial strain, Escherichia coli C lac– galE–, was obtained from Ingeny (Leiden, The Netherlands).
Results
All of the compounds tested were mutagenic in tongue and
other pooled oral tissues under the conditions employed (Tables
I and II). 4-NQO was the most potent, producing very high
levels of mutagenesis (78 and 144 mutants/105 p.f.u., or ~18
and 353 background in oral tissue and tongue, respectively)
after 4 weeks in drinking water at 20–80 µg/ml. B[a]P also
showed considerable mutagenic activity, resulting in mutant
fractions of 33 and 26 mutants/105 p.f.u. in oral tissue and
tongue, respectively, after five doses of 125 mg/kg. NNK and
NNN were administered in drinking water for 2 weeks at 0.1
mg/ml followed by 2 weeks at 0.2 mg/ml. Both were mutagenic
in tongue and oral tissue, resulting in mutant frequencies 2–
3-fold above spontaneous levels. NNN was somewhat more
potent in tongue than NNK, and was similarly mutagenic in
other pooled oral tissue (Tables I and II). The total dose was
estimated as 16.8 mg (588 mg/kg). DMBA was administered
using an oral painting protocol similar to that previously used
to initiate tumors in the hamster cheek pouch model (2). It
resulted in about a doubling in the mutant frequency. We
estimate that 0.1 ml per administration was applied, for a total
dose of 7.5 mg (262 mg/kg). However, the exact amount
2168
Table I. Mutant frequencies in pooled oral tissue and selected other tissues
of LacZ mice treated with oral carcinogens
Carcinogen
Tissue
4-NQO
DMBA
NNK
Pooled oral
Pooled oral
Pooled oral
Lung
Pooled oral
Esophagus
Pooled oral
Lungc
Kidneyc
Pooled oral
Pooled oral
Lung
Esophagus
Kidney
NNN
B[a]P
NMU
Control
No. mice/group MF (mutants/105 p.f.u.)a
tissueb 4
tissue 5
tissue 5
5
tissue 5
5
tissue 5
5
5
tissue 2
tissue 6
5
5
5
77.8 6 33**
7.1 6 2.7**
9.1 6 3.0**
15.0 6 5.5**
9.2 6 1.6**
9.8 6 1.8**
33.2 6 10.9**
57.9 6 33.0**
11.6 6 6.4**
7.8 6 2.6*
3.2 6 1.8
3.1 6 1.6
3.7 6 1.9
2.9 6 2.0
The range of p.f.u. (3103) and mutants are, respectively, for each group:
4-NQO (oral tissue) 24–841, 18–1054; DMBA (oral tissue), 266–947,
20–48; NNK (oral tissue), 303–427, 21–59; (lung) 147–341, 15–71; NNN
(oral tissue) 57–322, 4–31; (esophagus) 177–685, 21–56; B[a]P (oral tissue)
208–333, 56–129; (lung) 143–320, 59–272; (kidney) 61–661, 4–113; NMU
(oral tissue) 340–353, 20–34; control (oral tissue) 401–829, 4–40; (lung)
294–593, 6–22; (esophagus) 201–611, 5–11; (kidney) 540–1004, 10–34.
aGroup mean 6 SD.
bPooled oral tissue: gingival, buccal, pharyngeal and sublingual.
cTaken from Kosinska and Guttenplan (9).
**P , 0.01; *P , 0.05 in Student’s t-test versus control group.
Table II. Mutant frequencies in tongue of LacZ mice treated with oral
carcinogens
Carcinogen
Tissue
No. mice/group
MF (mutants/105 p.f.u.)a
4-NQO
DMBA
NNK
NNN
B[a]P
NMU
Control
Tongue
Tongue
Tongue
Tongue
Tongue
Tongue
Tongue
4
3b
5
5
5
2
6
144 6 73**
7.9 6 4.6
5.6 6 1.2*
7.5 6 1.7**
25.7 6 6.1**
9.7 6 2.5*
3.8 6 1.4
The range of p.f.u. (3103) and mutants are, respectively, for each group:
4-NQO, 12–185, 18–235; DMBA, 373–788, 28–47; NNK, 538–936, 22–63;
NNN, 244–906, 17–93; B[a]P, 66–404, 16–132; NMU, 342–834, 39–66;
control, 62–727, 2–23.
aGroup mean 6 SD.
bTwo samples did not package.
**P , 0.01, *P , 0.05 in Student’s t-test versus control group.
applied to the oral cavity cannot be determined because some
of the DMBA probably remained on the cotton swab, and
some may have been immediately swallowed. In a pilot
experiment, MNU was administered to two mice in drinking
water under the same conditions and concentrations as 4-NQO,
but was much less mutagenic, resulting in an ~2-fold increase
mutagenesis.
To compare mutagenesis in the oral cavity with that in other
target and non-target organs the MFs induced by several of
the carcinogens were determined in certain other organs. 4NQO, which is not carcinogenic in the rat liver (in contrast to
tongue) after administration in drinking water (3) was not
mutagenic in liver. B[a]P, which is a lung carcinogen in certain
strains of mice (11,12) was about twice as potent in lung than
in oral cavity tissues (Tables I and II); it was much less
mutagenic in the kidney which is a non-target organ (8,11–
13). NNK, a potent lung carcinogen (14,15), was about twice
Mutagenesis in lacZ mouse oral tissue
as potent a mutagen in lung than in the pooled oral tissues
and somewhat more potent than in tongue. NNN, an esophageal
carcinogen in hamsters and rats (15), was similarly potent in
esophagus, tongue and pooled oral tissues (Tables I and II).
The standard deviations of the means of the individual groups
were sometimes relatively high and did not seem to be
tissue specific.
Discussion
All of the known oral carcinogens tested here were mutagenic
in the oral cavity of lacZ mice. These agents exhibited
mutagenic activity in tongue and other pooled oral tissues
when administered by several different routes. By far the most
potent of the group was 4-NQO. 4-NQO is carcinogenic in
the rat tongue and to a lesser extent in other areas of the oral
cavity when administered orally (3,16). The higher mutagenic
activity of 4-NQO in tongue than other oral tissues is consistent
with this report. The mutagenic activity in tongue and lack of
mutagenic activity of 4-NQO in liver is strikingly consistent
with the carcinogenic effects of 4-NQO in the rat (3). The
inital concentration of 4-NQO used here for 2 weeks was the
same as that used in an 8 week administration in carcinogenesis
experiments (3). Here, however, in each of the subsequent 2
weeks the dose was doubled. Thus, in mutagenesis and
carcinogenesis protocols the exposure, calculated as concentration versus duration of exposure, was similar. B[a]P was
significantly mutagenic in tongue and oral tissue. In contrast
to 4-NQO, B[a]P was slightly more mutagenic in oral tissue
than tongue. When administered in feed in a 2 year feeding
study of carcinogenicity at 100 p.p.m., the tongue and forestomach were major target organs (8). The authors estimate a daily
consumption of B[a]P of ~0.4 mg/day compared with 2.5 mg/
day for 5 days here. Thus, the total dose was much higher in
the long-term feeding study, but the daily dose and the route
of administration were different. The target organs for B[a]Pinduced carcinogenesis in mice are dependent on the administration protocol and the strain, (8,11–13 and references therein).
For B[a]P then, the mutagenic activity observed in lacZ mouse
tongue is consistent with the results of carcinogenicity studies,
but here mutagenesis was also observed in other areas of the
oral cavity, and thus far there are no reports of B[a]P-induced
carcinogenesis at these sites. Also, B[a]P was not reported to
be carcinogenic in any other area of the oral cavity of male
lacZ mice under similar conditions (13). It is, of course,
possible that life-shortening by other tumors in certain studies
precluded the development or detection of oral tumors. It
is, however, noteworthy that B[a]P was relatively weakly
mutagenic in the kidney, consistent with carcinogenicity data.
It appears then that at least for B[a]P, the lacZ mutagenesis
assay is sensitive to initiation, but this is not sufficient to
predict carcinogenesis. In an initial study B[a]P was mutagenic
in tongue and other oral tissues when administered as a
suspension in drinking water followed by a single administration by gavage (9). It was assumed that the oral cavity was
primarily exposed via the drink, but in the current study
gavage administration alone was very effective at inducing
mutagenesis in oral tissues. Here, exposure of oral tissue could
be physical, via contact with B[a]P solution during gavage,
by regurgitation or reflux, and/or via systemic circulation.
B[a]P is metabolically activated by cytochrome P450 1A1 and
perhaps less efficiently by other forms of cytochrome P450
(17). Previous studies have reported that it can be metabolized
to DNA binding products in cultured rodent and human oral
tissue cells (18), indicating cytochrome P450 is constitutively
expressed in oral tissue. Cytochrome P450 1A1 is also highly
inducible by polycyclic aromatic hydrocarbons and related
compounds (17) and in the current study B[a]P was administered over a 12 day time period. Thus, it seems possible that
some induction of cytochrome P450 1A1 occurred during the
treatment period, and this would result in enhanced genotoxicity
of B[a]P.
NNK and NNN were both mutagenic when given in drink.
A long-term lower dose administration of a mixture of NNN
and NNK has been reported to induce tumors in the rat oral
cavity when administered by swabbing or via a test canal (14)
although which of these agents was most active is uncertain.
The total dose on a weight/kg basis was similar to that
administered here. NNK is activated to genotoxic products by
a cytochrome P450-dependent pathway (17), and NNK has
been reported to be metabolized to genotoxic products in
cultured human oral tissue (18). Therefore, it is reasonable to
assume it is metabolized by a cytochrome P450-dependent
pathway in oral tissues. NNK is reported to be a potent lung
carcinogen in rodents, but it also targets other areas of the
upper aerodigestive tract, while NNN is carcinogenic in
esophagus, nasal cavity, trachea and lung (15). Most carcinogenesis studies using NNK did not involve oral administration
in drink and there is a possibility that NNK might also be
carcinogenic in the oral cavity if given in drink. Also, the A/
J mouse, often used in studies reporting murine lung tumors
(15,19,20), is particularly sensitive to lung carcinogenesis (19)
and, thus, may be a less appropriate model for certain other
organs. Both NNK and NNN are usually present together in
tobacco products, but NNN is present in significantly higher
concentrations (15). Thus, if both are similarly potent initiators,
and other factors were equal, NNN would contribute more to
carcinogenesis in the oral cavity than NNK.
Both NNN and NNK were more mutagenic in the pooled
oral tissue than tongue. This was in contrast to results with
4-NQO. As NNN and NNK are presumably activated by
cytochrome P450 isozymes in lacZ mouse oral tissue and it
seems possible that the sites of greatest mutagenic activity are
those with the highest levels of the specific forms of P450
that activate NNN and NNK. 4-NQO, in contrast to NNK and
NNN, can be activated by several reductases (21,22). The
relatively higher mutagenic activity of 4-NQO in tongue rather
than other oral tissue may result from a different distribution
of these reductases (22) than that of P450. Rat tongue contains
significant levels of reductases, and levels in other areas of
the oral cavity are lower (22). Additionally, if both oral cavity
and liver can metabolize 4-NQO to a genotoxin, the high
mutagenic activity in oral cavity relative to liver may reflect
dilution of the 4-NQO in body water before absorption by
the liver.
DMBA was also mutagenic via oral swabbing of a 0.5%
solution in corn oil over a 3 week period. This concentration
has been used to induce tumors in the hamster cheek pouch
(2). Although the contact time of the DMBA with the oral
cavity may be relatively short, DMBA is such a potent
genotoxin that even this exposure was sufficient for mutagenesis. As with B[a]P, it seems likely that either constitutive
and/or induced cytochrome P450 activities contribute to the
metabolic activation of DMBA.
A pilot experiment on two mice was performed where
MNU was administered under the same conditions as 4-NQO.
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M.d.M.von Pressentin, W.Kosinska and J.B.Guttenplan
Although NMU is a carcinogen in rodents in a number of
organs including the oral cavity (20), and a mutagen in certain
organs of lac mice when administered i.p. (4,5) it was a
relatively weak mutagen in the oral cavity. MNU is relatively
unstable at neutral pH (20) and, in contrast to 4-NQO (which
is activated intracellularly) it is likely that a significant fraction
of the MNU decomposes extracellularly. It might be thought
to rapidly decompose in drinking water, but after a trace
amount decomposed the water became slightly acidic due to
decomposition products, and UV analysis indicated relatively
little decomposition at the time of water change.
In conclusion, several oral carcinogens were all mutagenic
in lacZ mouse tongue and other pooled oral tissues. 4-NQO,
which is the most potent of the carcinogens for the oral cavity,
was the most potent mutagen. Although conditions of previous
carcinogenicity assays and the present mutagenicity assays are
not directly comparable (because carcinogenicity assays are
usually carried out for longer time periods), when the total
applied dose was compared in the two assays, they were
similarly effective in detecting significant increases over background. It is noteworthy that the increases in mutant frequency
were observable with relatively small numbers of mice per
group and occasional large standard deviations of the means.
Usually, the large deviations resulted from results with a single
outlier per group. Possibly these outliers may result from
‘jackpot’ mutations, or the fact that lacZ mice are not inbred.
It should also be noted that mutagenesis has been observed in
several organs of the lacZ mouse which have not been reported
to be target organs for carcinogenesis (9,13). Thus, mutagenesis
in lacZ mouse oral tissue appears to be a useful and appropriate
short-term in vivo model for the initiation of carcinogenesis
in the oral cavity.
Acknowledgements
The authors thank Kurosh Haghighi for carrying out some of the some of the
preliminary experiments in this study. This work was supported in part by a
grant from the Smokeless Tobacco Research Council, # 0727, and the
American Institute for Cancer Research, #95B-104.
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Received April 6, 1999; revised July 15, 1999; accepted July 16, 1999