Carcinogenesis vol.18 no.5 pp.1045–1048, 1997
Relative mutagenicities of gaseous nitrogen oxides in the supF
gene of pSP189
David J.Kelman, Danae Christodoulou1, David A.Wink2,
Larry K.Keefer1, Aloka Srinivasan1 and Anthony Dipple3
Chemistry of Carcinogenesis Laboratory, ABL–Basic Research Program and
1Chemistry Section, NCI Laboratory of Comparative Carcinogenesis, NCI–
Frederick Cancer Research and Development Center, Frederick, MD 21702
and 2Radiation Biology Branch, NCI, National Institutes of Health,
Bethesda, MD 20892, USA
3To
whom correspondence should be addressed
Gaseous nitric oxide (NO), an environmental pollutant
found in cigarette smoke and diesel exhaust, has been
shown to generate mutations in aerobic in vitro assays. The
objective of this study was to identify which oxides of
nitrogen, formed in the gaseous phase from NO, possess
mutagenic activity. Samples of the plasmid pSP189, in 1 M
HEPES buffer, pH 7.4, were exposed to preparations of
nitrogen dioxide (NO2), dinitrogen trioxide (N2O3) or an
air control. The gas mixtures were formed in a gas-tight
syringe and were then introduced into 1 l flasks. The
plasmid solution was introduced immediately afterwards.
Transformation of Escherichia coli strain MBM7070 with
the treated plasmids allowed analysis of mutation frequencies and the types of mutations induced in the target supF
gene. The mutation frequency resulting from NO2 exposure
was not different from that of the control. However, N2O3
produced a substantial number of mutations. The mutation
frequency and the types of mutations were found to depend
on the length of time that the gases were allowed to incubate
in the syringe prior to introduction into the 1 l flasks
(mutation frequency was maximal at ~2 min). The most
prevalent mutations were AT→GC transitions (68%), followed by GC→AT transitions (30%), similar to previous
findings when pure NO was bubbled through pSP189
solutions. These results suggest that it is N2O3, rather than
NO2, that is the most likely source of mutagenic potential
from gaseous nitrogen oxides.
a different set of mutations in DNA than does NO released in
aerobic solution (9,10). Aerobic gaseous NO was shown to
generate primarily AT→GC transitions, whereas aqueous NO,
derived from NO donor compounds, generated largely GC→AT
transitions.
The identity of the species generating the DNA damage
from aerobic gas phase exposures remained unclear. Aerobic
NO gas undergoes two reactions to generate species that we
postulated might react with DNA. The initial reaction of NO
with oxygen in the gas phase results in the production of
nitrogen dioxide (NO2) (equation 1). NO2 has been shown to
generate single-strand breaks in DNA (11,12), but we have
found no data on its ability to alter the DNA bases. Equation
1 is essentially an irreversible reaction and, with sufficient
oxygen, all NO will be converted to NO2. NO2 can also
dimerize (equation 2). Thus, air/NO mixtures with air in
substantial excess should primarily contain an equilibrium
mixture of NO2 and N2O4. In the case where oxygen is limited,
NO can react with NO2 to form dinitrogen trioxide (N2O3)
(equation 3) and such a preparation would contain, therefore,
NO, NO2, N2O3 and N2O4.
2NO 1 O2 → 2NO2
(1)
(2)
2NO2 ≥ N2O4
NO 1 NO2 ≥ N2O3
(3)
Given that aerobic gaseous NO generates mutations in DNA,
it is likely that either NO2/N2O4 or N2O3 is responsible for
the mutations. Several authors have proposed that the mutations
generated by aerobic NO might be attributable to N2O3 (13,14).
By exposing DNA to the two gas preparations (i.e. to NO2/
N2O4 or to N2O3), as well as to air alone, we have obtained
results that are consistent with the proposed mutagenicity of
N2O3 and indicate that N2O3 is responsible for the AT→GC
mutations observed earlier by Routledge et al. (9) when this
plasmid was exposed in aqueous buffer to bubbles of NO gas.
Materials and methods
Introduction
Nitric oxide (NO) has received much attention in the last
several years (for reviews see 1–3). The majority of this
research has been centered upon the endogenous generation
of NO and its functions and effects within the body. NO has
been shown to mediate such diverse functions as cardiovascular
relaxation, platelet aggregation, immune response and neurotransmission. However, man is also exposed to NO from
exogenous sources, such as cigarette smoke (4,5) and diesel
exhaust (6). The chemistry of NO in the gas phase is different
from that in solution (7,8) and NO in the gas phase may lead
to biological effects that differ from those of endogenous NO.
Previous work has shown that aerobic gaseous NO generates
*Abbreviations: X-GAL, 5-bromo-4-chloro-3-indolyl β-D-galactoside; IPTG,
isopropyl β-D-thiogalactoside; TE, Tris–EDTA buffer; LB, Luria–Bertani
medium; PAH, polycyclic aromatic hydrocarbons.
© Oxford University Press
5-Bromo-4-chloro-3-indolyl β-D-galactoside (X-GAL*) and isopropyl β-Dthiogalactoside (IPTG) were obtained from Amersham (Arlington Heights,
IL). Nitric oxide (99.9%) was obtained from Potomac Air Gas (Potomac,
MD). To remove NO2, N2O4 and N2O3, the NO gas was bubbled through
2 M KOH before use. The plasmid pSP189 and Escherichia coli strain
MBM7070 were gifts from Michael M.Seidman (Oncor Pharmaceuticals,
Gaithersburg, MD).
Gas treatments
In all experiments, a gas-tight syringe was used to introduce the gas into a 1
l flask sealed with a rubber septum. The septum was wrapped in wire to
prevent gas leakage. Plasmid DNA (10 µg in 1 ml 1 M HEPES), adjusted to
pH 7.4 with NaOH, was then introduced into the flasks by syringe and the
site of introduction was sealed with silicone grease immediately afterwards.
After 10 min the flasks were evacuated to halt the reaction and were filled
with argon before opening the flask to the atmosphere.
The air control and NO2 flasks were filled with ambient air before
introduction of any gas. However, the N2O3 flasks, where air was to be
limited, were alternately evacuated and filled with argon (six times) in order
to remove as much oxygen as possible. The NO2 gas was formed by drawing
4 ml NO into a gas-tight syringe, then pulling 20 ml air into the syringe. The
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D.J.Kelman et al.
gases were allowed to react for 0.1–10 min before being introduced into the
flask, giving a final NO2 concentration of 4000 p.p.m. The plasmid solution
was then introduced by syringe. N2O3 was formed in a similar fashion, by
drawing 10 ml NO into a gas-tight syringe and then pulling 10 ml air into
the syringe. The approximate maximal concentration of N2O3 upon introduction
into the flask was 500 p.p.m.
Treated plasmid solution was pipetted from the flask. The buffer was
removed by spinning on Ultrafree MC centrifugal filters (30000 NMWL;
Millipore Corporation, Bedford, MA) and was stored at –40°C prior to analysis
for nitrate and nitrite. The plasmid was then washed twice on the filter with
Tris–EDTA (TE) buffer, pH 8.0, and stored in 50 ml of the same TE buffer
at 4°C.
Nitrate and nitrite were analyzed by capillary zone electrophoresis, performed on a Beckman P/ACE System 5510 equipped with a diode array
detector and System Gold data station. Detection was at 214 nm using a
polyacrylamide-coated column (column dimensions: total length 57 cm;
detection length 50 cm; internal diameter 100 mm) and 10 µM phosphate
buffer, pH 3.2, containing 0.1% Brij-30 (Calbiochem, La Jolla, CA).
Mutation assay
Escherichia coli strain MBM7070 was transformed with the treated plasmids
by electroporation and plated on Luria–Bertani (LB) plates containing 50 µg/
l ampicillin, 80 µg/l X-GAL and 200 µg/l IPTG. White and pale blue colonies
were picked off the plates and streaked onto fresh plates. White and pale blue
colonies were picked from those plates and grown overnight in LB medium
with 50 µg/l ampicillin. The plasmids were isolated and purified by means of
a Wizard Miniprep kit (Promega, Madison, WI). The purified plasmids were
sequenced by means of a dye terminator DNA sequencing kit and an automated
sequencer (Perkin Elmer, Foster City, CA).
Table I. Mutation frequencies from four trials of treatments with air, NO2
and N2O3a
Mutation frequency (3104)
Treatment
Run 1
Run 2
Run 3
Run 4
Average
Air (control)
NO2
N2O3
0.1
0.2
1.9
,0.1
,0.1
0.1
,0.1
0.2
0.8
0.1
1.3
5.8
0.1
0.4
2.1
aDetermined
by dividing the number of proven mutants by the number of
colonies obtained. A minimum of 200 000 colonies were examined in each
case.
Table II. Types of mutations observed as a result of treatment with N2O3
Transitions
AT→GC
GC→AT
Transversions
GC→TA
Number
Percent
99
43
68
30
3
2
Results
In concert with previous work (15), no increase in mutation
frequency was seen when plasmid pSP189 was exposed to
anaerobic NO gas (data not shown). In the present study,
plasmid samples in buffer were exposed to either air or the
NO2 or N2O3 preparations for 10 min. Following the treatment,
the buffer was filtered off and analyzed for nitrate/nitrite
concentration by means of capillary zone electrophoresis.
Analysis of the buffers showed that the NO2 and N2O3
treatments generated different amounts of nitrate and nitrite.
NO2, in the form of N2O4, reacts with water to form equimolar
quantities of nitrate and nitrite (equation 4). N2O3 should
generate solely nitrite (equation 5) (7). As expected, NO2 gave
approximately equal quantities of nitrate and nitrite (33 and
41 mM respectively). Though N2O3 alone should have led to
the formation of only nitrite, there was one third as much
nitrate as nitrite present in the buffer (19 and 62 mM respectively). Because N2O3 is in equilibrium with NO2 and NO in
the gas phase (equation 2), NO2 is present in the N2O3 system.
Dimerization of this NO2 presumably gave rise to the nitrate
observed in the N2O3 system.
N2O4 1 H2O → NO2– 1 NO3– 1 2H1
(4)
N2O3 1 H2O → 2NO2– 1 2H1
(5)
The mutation frequencies generated from four exposures
each of NO2, N2O3 and air controls are shown in Table I. NO2
did not consistently increase the mutation frequency over that
of the control. N2O3, on the other hand, induced a higher
mutation frequency than the air control, with a mean mutation
frequency of 2.1310–4. However, there was substantial variability in the mutation frequency of DNA treated with N2O3 and
this resulted in only a borderline level of significance for the
comparison of the aggregate N2O3 data with those of air
(Table I).
In order to determine the source of the variability of mutation
frequency from treatment with N2O3, NO and air were mixed
and allowed to incubate in a gas-tight syringe for either 0.1,
1, 2, 3, 5 or 10 min. The gas was then introduced into the
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Fig. 1. Time dependence of N2O3 mutation frequency. The time that NO
and air were allowed to incubate before treating the plasmid DNA was
varied to determine the effect on mutation frequency.
reaction vessel and the plasmid solution was added immediately
afterwards. As seen in Figure 1, mutation frequency increased,
reaching a maximum around the 2–3 min time points, then
declined. This indicates that a mutagenic species in the gas
requires time to form, then decays within a few minutes.
The types of mutations generated by the N2O3 preparation
are shown in Table II. The predominant mutations were
AT→GC transitions, with GC→AT transitions present to a
much lower extent. However, the types of mutations that form
do appear to be time dependent. Figure 1 shows the percentage
of the mutations that were AT→GC transitions at each time
point. These mutations only occurred at the 2 and 3 min time
points, the times when the mutation frequency was highest.
At all other time points, the only point mutations observed
were GC→AT transitions. This indicates that the mutations
generated by the N2O3 preparation were AT→GC transitions
and that the mutagen was a transient species in this system;
its concentration reached a maximum around 2–3 min, then
decreased.
Mutagenicities of gaseous nitrogen oxides
Fig. 2. Mutation spectrum resulting from N2O3 treatment of the supF gene. * Locations of hotspots for mutation derived in a previous study (9).
The mutation spectrum for the N2O3 treatment is shown in
Figure 2. Several sites were mutated frequently, three or more
times, and are considered to be hotspots for mutation (sites at
which five or more times the expected number of mutations
were observed). Hotspots in the present work were at positions
112, 121, 130, 145 and 151; two of these (112, 121) were also
hotspots in the previous gaseous NO study (9).
Discussion
Routledge et al. (9) demonstrated that DNA that had been
treated with aerobic NO gas developed mutations. The dominant type of mutation was the AT→GC transition, observed in
75% of the mutagenic events. The GC→AT transition was the
second most prevalent mutation observed, comprising ~23%
of the mutations. The mutations obtained using the N2O3
preparation in this experiment mirror the previous results.
Indeed, both of the hotspots for mutations reported in the
previous study were also hotspots in this study. Therefore, it
seems likely that the mutagen generated by Routledge et al.
was also present in our N2O3 preparations.
Since neither air nor NO alone nor the NO2 preparation
were clearly mutagenic, it seems likely that none of the species
present in these three preparations are by themselves mutagenic.
The most obvious component present in the N2O3 preparation
that was not present in the other systems is N2O3 itself and,
therefore, this is a good candidate for the unknown mutagen.
Other possible candidates, such as the ONOOd radical, would
be expected to be formed in both the NO2 and N2O3 preparations. An important property of the mutagen was its transient
existence (Figure 1). Although scenarios in which N2O3 would
exhibit a similar transient existence could be envisaged, we
cannot unambiguously identify N2O3 as this mutagen. However,
N2O3 is clearly a prime candidate and the findings above
indicate that NO2, N2O4 and other species present in the
inactive as well as active preparations could not be responsible
for the mutagenicity.
Christen et al. (16) exposed a series of Salmonella strains
to NO 1 O2 gas mixtures, in order to determine the mutagenicity of N2O3, and found that the GC→AT transition was
the most pronounced mutation observed in their system,
followed by the AT→TA transversion and the CG→GC transversion. However, their gases were dynamically mixed and
flowed through a chamber containing the bacterial plates at a
rate of gas flow of 3 l/min. Given the volume of the chamber
in which the plates were exposed (2.5 l), it is likely that the
bacteria were exposed to NO and O2 that had been mixed for
,1 min. This, and our results for the time dependence of the
types of mutations observed, provide one possible explanation
for the lack of AT→GC transitions in their study. Indeed, at
the 1 min time point in our study, the predominant mutation
in the system was also the GC→AT transition.
Interestingly, there was a uniformity of mutation at the
hotspots. Every base change at a given site was always the
same. This is in contrast to the mutation spectra observed for
polycyclic aromatic hydrocarbons (PAHs) in this mutation
system. These compounds generated a number of different
mutations at the same location in the gene (17). One possible
explanation for this phenomenon is that the bulky PAH
adducts are interpreted as a non-informational base by DNA
polymerase, so that there is no clear instruction as to what
bases should be added opposite the adduct. However, the bases
damaged by the N2O3 preparation may form an informational
base that leads to the addition of a specific base across
from it, in a manner similar to O6-methylguanine mispairing
consistently with T (18).
There have been a number of reports that NO2 is mutagenic
(11,12,19), but that was not the case in the pSP189 system.
However, all the systems reporting mutagenic activity of NO2
have used live cell culture exposures or there have been other
compounds added to the DNA that have been essential for
DNA damage to occur. The present data show that NO2 does
not react directly with DNA to an extent that would result in
a mutation under the conditions employed here.
Analysis of mutations in the p53 gene of human tumors
showed that 11% of all human tumors had mutant p53
containing AT→GC mutations (20). However, certain types of
tumors were more likely to possess this mutation than others.
Most notably, 17 and 16% of tumors of the pharynx/larynx
and of the oral cavity respectively had AT→GC mutations in
the p53 gene. While 11% of the lung tumors of smokers had
AT→GC mutations in the p53 gene, there were none reported
from lung tumors of non-smokers. These are sites in the body
that would be expected to be exposed to gaseous NO as the
result of cigarette smoking. Although NO levels in vivo may
differ substantially from these used here, these data suggest
that NO may play a role in the formation of tumors in
these tissues.
Acknowledgements
We thank Ms Marilyn Powers for her technical support with the automated
sequencing. This research was supported in part by the National Cancer
Institute, DHHS, on a contract with ABL.
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Received on November 26, 1996; revised on January 22, 1997; accepted on
January 28, 1997
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