Carcinogenesis vol.18 no.12 pp.2429–2433, 1997
Mutation and formation of methyl- and hydroxylguanine adducts
in DNA caused by N-nitrosodimethylamine and
N-nitrosodiethylamine with UVA irradiation
Sakae Arimoto-Kobayashi2, Keiko Kaji,
Gavain M.A.Sweetman1 and Hikoya Hayatsu
Faculty of Pharmaceutical Sciences, Okayama University, Tsushima,
Okayama 700, Japan and 1MRC Toxicology Unit, Hodgkin Building,
Leicester University, Lancaster Road, Leicester LE1 9HN, UK
2To
whom correspondence should be addressed
Previously we reported that when Escherichia coli was
treated with N-nitrosodialkylamine under irradiation with
near UV light, mutagenesis of the bacteria took place:
there was no requirement for metabolic activation. We
have now studied the spectra of mutations caused by
N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) with UVA (320–400 nm) irradiation, using
standard tester strains for identifying types of mutations.
Induced mutations by NDMA F UVA were the transition
GC→AT and transversions GC→CG, GC→TA and
AT→TA. NDEA F UVA induced mainly the GC→CG
transversion. In both cases no frameshift mutations were
observed. When O6-alkylguanine-DNA alkyltransferasedeficient strains of E.coli and Salmonella typhimurium were
used, the mutation levels with both NDMA F UVA and
NDEA F UVA became remarkably higher than those
observed with the proficient strains. We measured the O6methylguanine (O6-meG) level in calf thymus DNA treated
with NDMA F UVA. The O6-meG level was increased as
a function of NDMA concentration and irradiation time.
We also detected N7-methylguanine in DNA treated with
NDMA F UVA. In our previous work we found formation
of 8-oxodeoxyguanosine (8-oxodG) in DNA treated with
N-nitrosomorpholine F UVA. The 8-oxodG/dG ratio in
DNA treated with NDMA F UVA increased up to 42-fold
over that of the untreated control and that in DNA treated
with NDEA F UVA increased up to 67-fold. 8-OxodG
formation was not affected by replacing H2O in the reaction
mixture with D2O, suggesting that singlet oxygen is not the
rate limiting factor in this photoactivation. We conclude
that both alkylation and oxidation are involved in mutations
induced by NDMA F UVA and NDEA F UVA.
Introduction
N-Nitrosodialkylamines have been shown to cause a wide
range of tumors in all animal species so far tested (1) and are
considered to be a potential health hazard to humans (2).
These compounds are present in the workplace, processed
meats and cigarette smoke. UVA has been reported to be
genotoxic and carcinogenic (3,4). Early studies showed that
metabolic activation was required to convert N-nitrosodialkylamines into alkylating agents (5). We have found that
*Abbreviations: NDMA, N-nitrosodimethylamine; NDEA, N-nitrosodiethylamine; 8-oxodG, 8-oxodeoxyguanosine; O6-meG, O6-methylguanine; N7meG, N7-methylguanine; dG, deoxyguanosine; LCMS, liquid chromatography–
mass spectrometry.
© Oxford University Press
N-nitrosodialkylamines can be activated by irradiation with
near-UV light (UVA, 320–400 nm). Thus, mutagenesis in
Escherichia coli took place when a mixture of N-nitrosodimethylamine (NDMA*) and the bacteria in saline was
irradiated with UVA, without any metabolic activation (6).
N-Nitrosodiethylamine (NDEA) 1 UVA also induced
mutations in E.coli. NDMA and NDEA can become directly
clastogenic towards cultured Chinese hamster lung fibroblasts
on UVA irradiation (7).
Alkylating agents are generated from N-nitrosodialkylamines
with metabolic activation and they can modify almost any of
the nucleophilic oxygens and nitrogens of nucleic acid bases
(8). For NDMA 1 UVA we have shown that when an aromatic
amine is simultaneously present in the reaction mixture, the
amine is oxidized into its nitro derivative (9). Furthermore,
treatment of DNA with N-nitrosomorpholine 1 UVA results
in formation of 7,8-dihydro-8-oxo-29-deoxyguanosine
(8-oxodG) in the DNA (10). These observations suggest that
UVA irradiation of N-nitrosodialkylamines generates oxidative
species. However, it is also possible that alkylating species
are simultaneously generated in this process.
In the present study we treated Salmonella typhimurium and
E.coli with NDMA/NDEA 1 UVA and examined the induced
mutagenesis and its spectrum. Effects of alkyltransferase (ada
and ogt) deficiencies on the mutation frequency were also
investigated. Furthermore, we analyzed the formation of O6methylguanine (O6-meG), N7-methylguanine (N7-meG) and
8-oxodG in calf thymus DNA treated with NDMA/NDEA 1
UVA.
Materials and methods
Reagents and strains
NDMA (CAS. 62-75-9) and NDEA (CAS. 55-18-5) were purchased from
Kanto Chemicals (Tokyo). The N-nitrosamines were diluted with water and
the solutions were stored frozen at –20°C. O6-meG (CAS. 20535-83-5) and
calf thymus DNA were purchased from Sigma (St Louis, MO). N7-meG
(CAS. 578-76-7) was obtained from Dr P.B.Farmer (MRC Toxicology Unit,
Leicester, UK). 7,8-Dihydro-8-oxo-29-deoxyguanosine (CAS. 88847-89-6)
was a gift from Dr H.Kasai (University of Occupational and Environmental
Health, Kitakyushu, Japan). Other reagents were commercial products of
reagent grade.
Salmonella typhimurium TA1535 (hisG rfa ∆uvrB) was a gift of Dr
B.N.Ames (University of California, Berkeley) (11). Salmonella typhimurium
YG7100 (as TA1535 but ∆adast::Kmr), YG7104 (as TA1535 but ∆ogtst::Cmr)
and YG7108 (as TA1535 but ∆adast::Kmr, ∆ogtst::Cmr) were given by
Dr M.Yamada (National Institute of Hygienic Sciences, Tokyo) (12,13).
Escherichia coli CC101–CC111, which are derivatives of strain P90C [ara,
∆(lac proB)XIII] carrying an F9 (lacI–, Z–, proB1) episome (14), and E.coli
CC101–CC111, containing plasmid pGW1700 carrying mucAB, were gifts of
Dr M.Watanabe (Institute of Environmental Toxicology, Tokyo) (15). Each
strain carries a different lacZ– allele and strains CC101–CC106 are designed
to differentiate six types of base substitutions: CC101 for the AT→CG
transversion, CC102 for the GC→AT transition, CC103 for the GC→CG
transversion, CC104 for the GC→TA transversion, CC105 for the AT→TA
transversion and CC106 for the AT→GC transition. The five strains CC107–
CC111 can detect reversions by frameshift. Escherichia coli CC102 (ada–,
ogt–) and CC106 (ada–, ogt–) were gifts from Dr K.Ihara (University of
Kyushu, Japan). Escherichia coli YG5113 [ara–, ∆(gpt–lac)5, mutM::cat/
F9(lacI378, lacZ461, proAB)] was a gift from Dr T.Nohmi (National Institute
2429
S.Arimoto-Kobayashi et al.
of Hygienic Sciences, Tokyo), which is a derivative of E.coli CC104 carrying
the 8-oxoG DNA glycosylase deficiency (10).
UVA irradiation
Two 20 W black light bulbs (National, Japan), set in parallel at a distance of
18 cm above the mutagenesis solution, were used as a source of near-UV
irradiation, emitting light with a wavelength range of 300–400 nm. Reaction
mixtures were placed in a sterile microtiter tray (Nunc, Denmark). The tray
was covered with a 4 mm thick glass plate, which excluded light of wavelength
,320 nm. The intensity of light was 4.5 W/m2, as measured by a black ray
UV intensity meter (Ultraviolet Products, San Gabriel, CA) (16). This intensity
may be compared with that of sunlight on a sunny day at noon (October 22,
1993) in the grounds of Okayama University, which was 24 W/m2.
Mutagenesis experiments using E.coli and S.typhimurium
An overnight culture of E.coli was centrifuged at 3500 r.p.m. for 10 min at
4°C. The pellet was resuspended in cold 0.9% NaCl and the absorbance at
600 nm of the suspension was adjusted to 1.5 for E.coli CC101–CC106 and
to 0.8 for E.coli CC107–CC111. The suspensions were stored on ice until
use. A bacterial suspension (0.3 ml) and a solution of N-nitrosodialkylamine
(0.3 ml) were mixed and the mixture was irradiated in a chamber maintained
at 37°C, with continuous mixing. An aliquot of 0.15 ml of the irradiated
sample was poured onto a lactose plate. After incubation for 48 h at 37°C
lac1 revertants formed were scored. Another portion of the sample was diluted
to 10–6 with phosphate-buffered saline and poured onto LB plates to determine
survival (15). After 48 h incubation the resulting colonies were counted.
The procedure for S.typhimurium was similar to that for E.coli, except that
a minimal glucose plate was used for mutation detection (11) and an NB
plate, consisting of 8 mg/ml Nutrient Broth (Difco), 5 mg/ml NaCl and
15 mg/ml agar, was used for survival assay. As a control experiment nonirradiated samples were prepared by wrapping the plates in aluminum foil
and placing them in the chamber for a desired period of time. Duplicate
experiments were performed for each assay.
Analysis of O6-meG in DNA with fluorescence detection
Calf thymus DNA (0.05 mg) and NDMA were dissolved in 16 mM sodium
phosphate, pH 7, in a total volume of 1.2 ml and the solution was irradiated
in a chamber maintained at 37°C with continuous mixing. The DNA was then
dialyzed against water and precipitated by ethanol. The precipitate was freed
of ethanol under reduced pressure and then dissolved in 0.1 ml 0.1 N HCl.
The mixture was heated at 70°C for 30 min, cooled on ice and 2 vol. ethanol
were added to it. Precipitates were removed from the mixture by centrifugation
and the supernatant obtained was concentrated to dryness under reduced
pressure. The residue was dissolved in 0.1 ml water and analyzed using HPLC
on a Waters system coupled to a fluorescence detector. HPLC was performed
with a column of ODS-80Ts (4.63250 mm; Tosoh, Japan): column temperature
40°C, eluent 0.1 M NH4OAc, pH 5.0, containing 15% methanol, flow rate
0.8 ml/min. O6-meG was detected by fluorescence (287 nm excitation and
362 nm emission) and deoxyguanosine (dG) by 247 nm absorbance.
Analysis of N7-meG and O6-meG in DNA by liquid chromatography–mass
spectrometry (LCMS)
LCMS was performed on a Quattro BQ tandem quadrupole mass spectrometer,
fitted with an electrospray interface (Micromass, Manchester, UK). This was
coupled to an HPLC system consisting of a Varian 9012 solvent delivery
system (Varian, Walton-on-Thames, UK), a Rheodyne 7125 injector (Cotati,
CA) fitted with a 20 µl loop and a Phase Sep S5 ODS2 (2.03250 mm)
column (Phase Separations Ltd, Deeside, UK). A 10 min isocratic separation
was achieved using 10 mM ammonium acetate, pH 5.4, and 25% methanol
at a flow rate of 200 µl/min. The column eluent was split roughly 2:1 with
~65 µl/min passing to the mass spectrometer.
To achieve the best sensitivity, single ion recording was used to detect
guanine (m/z 252.1) and the two methylguanines (m/z 166.1). Calibration lines
were constructed using peak areas of authentic standards of guanine, N7-meG
and O6-meG and quantification of the samples was based on these calibration
lines. All samples and calibration points were analyzed in duplicate. The
limits of detection for this system were better than 1 pmol/ml for O6-meG
and 15 pmol/ml for N7-meG (this is equivalent to 20 and 300 amol loaded
on column).
Analysis of 8-oxodG in DNA
The irradiated mixture of calf thymus DNA (0.05 mg) and NDMA in 16 mM
sodium phosphate, pH 7.0, was dialyzed against water. The treated DNA was
isolated by ethanol precipitation. DNA, dissolved in 10 mM Tris–HCl, pH 8.0,
was digested with DNase I (0.06 mg/ml) at 37°C for 2 h and then with a
mixture of 0.015 mg/ml alkaline phosphatase and 0.06 mg/ml snake venom
phosphodiesterase in 20 mM Tris–HCl, pH 8–9, at 37°C for 2 h. After
incubation 3 vol. ethanol were added and the mixture was allowed to stand
at –80°C for 30 min and then centrifuged at 4°C for 10 min at 15 000
2430
Fig. 1. Mutagenesis of S.typhimurium TA1535 (s), YG7100 (j), YG7104
(u) and YG7108 (d) induced by N-nitrosodialkylamine 1 UVA.
(a) Irradiation time 30 min; (b) irradiation time 10 min.
r.p.m. The supernatant was taken and analyzed using HPLC coupled to an
electrochemical detector (EICOM, Japan). The HPLC was performed with a
column of Waters Nova-Pak C18 (3.93300 mm): column temperature 40°C,
eluent 10 mM NaH2PO4 containing 8% methanol, flow rate 0.7 ml/min.
8-OxodG was detected at 550 mV versus Ag/AgCl by electrochemical
amperometry and dG was detected by 260 nm absorbance. The molar ratio
of 8-oxodG to dG in each DNA sample was determined based on the peak
height, in comparison with those of known amounts of authentic 8-oxodG
and dG (17).
Results
Mutagenesis and mutational spectra
Mutation induction for S.typhimurium was determined. The
results given in Figure 1a show that the mutagenicity of
S.typhimurium TA1535 increased in a dose-dependent manner
for NDMA. A dose dependence was also observed with respect
to UVA. At longer irradiation times (.60 min for NDMA and
.30 min for NDEA) a decline in survival was observed (data
not shown). With O6-alkylguanine alkyltransferase-deficient
strain YG7104 (an ogt– strain) the NDMA 1 UVA treatment
gave ~2 times greater mutation frequencies compared with
those found with the parent strain, TA1535. Further enhancement of mutation frequency was found for the double deficiency
mutant YG7108 (an ada–, ogt– strain). In the case of NDEA 1
UVA a 6 times higher mutation frequency was obtained with
the ogt– strain compared with the parental strain at a dose of
40 mM NDEA (Figure 1b). The double deficient strain
(ada–, ogt–) gave a 4 times higher mutation frequency than
that observed with the wild-type strain.
Figure 2a shows the mutational spectra induced by NDMA 1
UVA. The base substitutions GC→AT (CC102), GC→CG
(CC103), GC→TA (CC104) and AT→TA (CC105) were
observed, but no frameshifts were detected. Mutation by
NDEA 1 UVA was observed only for the GC→CG substitution
(Figure 2b). It is well established that O6-meG mispairs
with thymine and O4-methylthymine with guanine and these
methylations cause GC→AT and AT→GC transitions (18,19).
When an alkyltransferase (ada and ogt)-deficient derivative of
CC102 was used the induced mutation GC→AT was dramatically increased both for the NDMA 1 UVA and NDEA 1
UVA treatments (Figures 3 and 4). The transition AT→GC in
CC106 was also elevated in the ada, ogt double deficient
strain. The mutation frequencies in CC102 (ada–, ogt–) were
significantly greater compared with those in CC106 (ada–,
ogt–) both for the NDMA 1 UVA and NDEA 1 UVA
N-nitrosodimethylamine, N-nitrosodiethylamine and UVA
Fig. 2. Mutation spectra induced by N-nitrosodialkylamine 1 UVA in E.coli
containing plasmid pGW1700. Dependence on NDMA (a) and NDEA (b)
concentrations. The Lac1 revertant numbers/plate caused by UVA only were
14 for E.coli CC101, 20 for CC102, 1 for CC103, 9 for CC104, 7 for
CC105 and 1 for CC106. The irradiation time was 4 h.
Fig. 5. Formation of O6-meG and 8-oxodG in DNA. Dependence on
irradiation time. Calf thymus DNA was treated with NDMA 1 UVA (d,
j) or UVA without NDMA (s, u). The concentration of NDMA was
32 mM.
Fig. 6. Formation of O6-meG and 8-oxodG in DNA. Dependence on the
NDMA dose. Calf thymus DNA was treated with NDMA 1 UVA (d, j)
or with NDMA in the dark (s, u). The irradiation time was 2 h.
Fig. 3. Effect of alkylguanine alkyltransferase deficiency (ada–, ogt–) on
mutation frequency of E.coli induced by NDMA 1 UVA (4 h): u, E.coli
CC102; j, CC102(ada–, ogt–); s, CC106; d, CC106(ada–, ogt–).
Fig. 4. Effects of ada–, ogt– on the mutation frequency induced by
NDEA 1 UVA (4 h): u, E.coli CC102; j, CC102(ada–, ogt–); s, CC106;
d, CC106(ada–, ogt–).
treatments. When an 8-oxoG DNA glycosylase (mutM)-deficient derivative of CC104 was used, in comparison with its
proficient parent CC104 no significant increase in induced
mutation was observed for the NDMA 1 UVA treatment. The
mutation frequencies were (310–7): in YG5113 (mutM) 5.4
(UVA only), 2.5 (5 mM NDMA 1 UVA), 4.4 (50 mM
NDMA 1 UVA) and 8.3 (250 mM NDMA 1 UVA); in CC104
Table I. Formation of O6-meG and N7-meG in DNA treated with NDMA 1
UVA: quantitation with LCMS
NDMA
(nM)
UVA
(h)
O6-meG/105 dG
N7-meG/105 dG
32
64
64
2
2
0
0.251
0.954
0a
17.4
39.0
0a
aThe detection limit
N7-meG/105 dG.
was ,0.0263 for O6-meG/105 dG and ,3.95 for
0.99 (UVA only), 0.95 (5 mM NDMA 1 UVA), 1.7 (50 mM
NDMA 1 UVA) and 1.1 (250 mM NDMA 1 UVA).
Formation of O6-meG and 8-oxodG in DNA
We explored the formation of O6-meG in calf thymus DNA
treated with NDMA 1 UVA. The analysis was first performed
by HPLC coupled with fluorescence detection. The treatment
yielded high amounts of O6-meG, formation being dependent
both on irradiation time (Figure 5a) and NDMA concentration
(Figure 6a). At higher amounts of NDMA (.32 mM) O6meG production reached a plateau. At these concentrations of
NDMA the reaction limiting factor may not be the amount of
NDMA but the dose of UVA. We also analyzed formation of
N7-meG and O6-meG by LCMS (Table I). The authentic
specimen of guanine (m/z 152.1) was eluted at a retention time
of 3.5 min, that of N7-meG (m/z 166.1) at 4.3 min and that of
O6-meG (m/z 166.1) at 7.1 min. In the DNA sample obtained
2431
S.Arimoto-Kobayashi et al.
Fig. 7. 8-OxodG formation in DNA. (a) Dose dependence for NDEA 1
UVA (m) or for NDEA in the dark (n). The irradiation time was 2 h.
(b) Dependence on irradiation time with NDEA (m) or without NDEA (n).
The concentration of NDEA was 32 mM.
Table II. Effect of D2O on formation of 8-oxodG in DNA treated with
NDMA/NDEA 1 UVA
8-oxodG/105 dG
Untreated
UVA (2 h)
NDMA (2 mM)
NDMA (2 mM) 1 UVA (2 h)
NDEA (8 mM)
NDEA (8 mM) 1 UVA (2 h)
In H2O
In D2O
1.5
2.6
1.4
62.6
1.4
99.8
1.8
3.0
4.6
77.2
1.0
95.3
on treatment with NDMA 1 UVA we detected peaks at 3.4
min (m/z 152.1), 4.3 min (m/z 166.1) and 6.9 min (m/z 166.1).
8-OxodG formation in DNA was also measured (Figures
5b, 6b and 7). After 1 h treatment with 32 mM NDMA 1
UVA an 8-oxodG content as high as 1 residue per 2200 dG
residues in DNA was obtained (Figure 5b). With a longer
period of irradiation and with higher concentrations of NDMA
the amount of 8-oxodG decreased (Figures 5b and 6b). In the
NDEA 1 UVA treatment 8-oxodG was produced in DNA in
a similar manner.
If 8-oxodG formation by treatment with NDMA/NDEA 1
UVA is mediated by singlet oxygen, the amount of 8-oxodG
produced may be expected to increase with the use of D2O
instead of H2O as the medium of the reaction solution (20).
However, as the results in Table II show, this was not the case.
Discussion
The 30 min irradiation with UVA of S.typhimurium in the
presence of NDMA caused significant mutation induction
(Figure 1a). In the case of NDEA only a 10 min irradiation
was sufficient to induce mutation (Figure 1b). In view of the
fact that the intensity of UVA used in this study was only
~25% of that of sunlight, these highly sensitive mutagenic
responses suggest that this phenomenon could have biological
relevance.
It was reported that the mutation induced by metabolically
activated NDMA and NDEA is predominantly the base pair
transition GC→AT (21,22). In mutagenesis by NDMA/
NDEA 1 UVA various base substitutions appear to occur:
GC→AT, GC→CG, GC→TA and AT→TA (Figure 3a). The
GC→AT transition may be caused by the formation of O6meG (21) and the GC→TA transversion by 8-oxoG (23). The
2432
E.coli strains used have a Tcr pBR322-derived plasmid carrying
the mucAB operon that can enhance error-prone DNA repair.
Therefore, the presence of unrepaired alkylated bases in the
genome may signal induction of the SOS response and thus
SOS mutagenesis may account for the elevated GC→CG and
AT→TA transversions (24). It was also reported that E.coli
deficient in the ada, ogt DNA methyltransferases display
spontaneously occurring base pair changes of the types
GC→AT, AT→GC, GC→CG, AT→GC and AT→TA (24).
Photoactivated mutation of E.coli with NDMA needs a longer
irradiation time (1–4 h) compared with that of S.typhimurium
(10–30 min). This could be due to increased permeability of
S.typhimurium, which carries an rfa phenotype, as compared
with that of the E.coli strains used.
A deficiency in the repair function gene ogt elevated
mutation of S.typhimurium induced by NDMA 1 UVA. In
contrast, a deficiency in another repair function, ada, did not
affect mutagenesis (Figure 1a). However, the double deficiency
ada, ogt resulted in a remarkably elevated level of NDMA 1
UVA induced mutations, i.e. a level significantly greater than
that observed for the ogt-deficient strain. Therefore, we deduce
that DNA adducts are formed upon NDMA 1 UVA treatment,
the repair of which may require ada. With NDEA 1 UVA
treatment increased mutability of an ogt-deficient strain was
also observed (Figure 1b). However, the double deficiency
(ada, ogt) gave no further enhancement of mutation frequency.
This apparent lack of effect of ada suggests that repair of
ethylated guanine in DNA is dependent mostly on the activity
of ogt in this strain of Salmonella. Abril et al. (25) reported that
the contribution of ogt-encoded alkyltransferase to resistance to
chloroethylnitrosourea in E.coli is greater than that of the adaencoded enzyme and that the mechanism of protection is likely
to be removal of the chloroethyl group from the O6 position
of guanine. The ogt-encoded enzyme may play a greater role
in repair of DNA damage derived from treatment with NDEA 1
UVA than the ada enzyme.
Oxidative metabolism of nitrosamines is mediated by
cytochrome P450 enzymes and is presumed to involve the
formation of α-hydroxyalkylnitrosamines (26). We have
isolated
a
direct-acting
mutagen
formed
from
N-nitrosopiperidine 1 UVA in sodium phosphate buffer and
have identified it as a phosphate ester of α-hydroxy-N-nitrosopiperidine (27). This compound can be decomposed by an
esterase. A number of α-acetoxy-N-alkylnitrosamines have
been synthesized which will give rise to α-hydroxy-N-alkylnitrosamines on treatment with esterases, and the resulting
α-hydroxy-N-alkylnitrosamines decompose spontaneously
(28). Therefore, it is probable that an unstable phosphate ester
of α-hydroxy-N-dimethylamine is formed in the reaction of
N-nitrosodimethylamine with UVA, which then decomposes
to give an alkydiazonium ion via α-hydroxy-N-nitrosodimethylamine. We have detected and quantified the alkyl adducts
in DNA treated with NDMA 1 UVA. The study reported here
has established that alkylation of DNA occurs with UVAactivated NDMA. The level of O6-meG found by LCMS was
similar but somewhat smaller than that detected by HPLC
fluorescence. It may be partly due to some fluorescent contaminants co-eluted with O6-meG in the HPLC (work in
progress). The amount of N7-meG formed in DNA was 40–
70 times higher than that of O6-meG. These results are
consistent with reports showing that the N7-alkyl adduct of
guanine is the most abundant among alkylated base products
(29).
N-nitrosodimethylamine, N-nitrosodiethylamine and UVA
We also detected 8-oxodG in DNA treated with NDMA/
NDEA 1 UVA. The amounts quantified were similar to those
found previously in the DNA of phage M13mp2 treated with
N-nitrosomorpholine 1 UVA (10). At high concentrations of
N-nitrosodialkylamine and with longer irradiation periods the
amounts of 8-oxodG decreased. Buchko et al. (30) reported that
8-oxodG can be oxidized further by photodynamic reactions.
8-OxodG produced by UVA activation of NDMA/NDEA is
likely to undergo further oxidative degradation during the
reaction. As formation of 8-oxodG was not enhanced in D2O,
singlet oxygen will not be involved in this DNA damage. Kasai
et al. (31) showed that in riboflavin-mediated photooxidation of
guanine electron transfer from the guanine moiety to the
triplet-excited riboflavin is likely to lead to formation of a
guanine radical cation. The radical cation can then react with
molecular oxygen to form 8-oxodG. Since in our present
reaction singlet oxygen does not appear to be involved, it is
possible that here again oxidation of guanine to form 8-oxodG
occurs via a guanine radical cation. In ‘N-nitrosomorpholine 1
UVA’ mutagenesis we observed a higher level of mutation of
phage M13mp2 in a mutM-deficient host (10). However, in
the present experiments no stimulation by mutM deficiency
was observed in the NDMA 1 UVA-induced mutation in
E.coli. As the spontaneous mutation frequency in mutMdeficient strain YG5113 was 5 times higher than that of the
proficient parent strain CC104, additional formation of 8-oxoG
by NDMA 1 UVA could have made only an insignificant
contribution to mutagenesis.
It is almost an established fact that sunlight causes human
skin cancer (3). Possible co-mutagenic and co-toxic actions of
NDMA/NDEA 1 UVA forming alkyl and oxidative adducts
in DNA are of considerable interest in relation to health hazards.
Acknowledgement
The authors wish to thank Dr P.B.Farmer for invaluable advice and support.
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Received on March 28, 1997; revised on July 8, 1997; accepted on July 31, 1997
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