Near ultraviolet radiation-mediated reaction between N-nitrosoproline and DNA: Isolation and identification of two new adducts, (R)- and (S)-8-(2-pyrrolidyl)-2′-deoxyguanosine

Article abstract of DOI:10.1016/j.jphotochem.2013.11.009

To explore possible genotoxicity of N-nitrosoproline (NPRO), we investigated near-ultraviolet radiation (UVA)-mediated chemical reaction of NPRO with 2′-deoxyguanosine (dG). An acidic solution containing NPRO and dG was irradiated with UVA and products we

Full text of DOI:10.1016/j.jphotochem.2013.11.009

Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Near ultraviolet radiation-mediated reaction between
N-nitrosoproline and DNA: Isolation and identification of two new
adducts, (R)- and (S)-8-(2-pyrrolidyl)-2ꢀ-deoxyguanosine
Shuhei Aoyamaa, Kayoko Sanoa, Chiharu Asahia, Masaki Machidaa, Keinosuke Okamotoa,
Tomoe Negishia, Sachiko Kimurab, Toshinori Suzukic, Tsutomu Hatanoa,
Sakae Arimoto-Kobayashia,∗
a Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan
b School of Human Science and Environment, University of Hyogo, Shinzaike-hon-machi, Himeji, Hyogo 670-0097, Japan
c School of Pharmaceutical Sciences, Shujitsu University, Nishigawara, Naka-ku, Okayama 703-8516, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To explore possible genotoxicity of N-nitrosoproline (NPRO), we investigated near-ultraviolet radiation
(UVA)-mediated chemical reaction of NPRO with 2ꢀ-deoxyguanosine (dG). An acidic solution containing
NPRO and dG was irradiated with UVA and products were analyzed by HPLC. In addition to the three
known modified guanine nucleosides, i.e., 2ꢀ-deoxyxanthosine, 8-oxo-7,8-dihydro-2ꢀ-deoxyguanosine
and 2ꢀ-deoxyoxanosine, we found two products. These new products were isolated and identified as (R)-
and (S)-8-(2-pyrrolidyl)-2ꢀ-deoxyguanosine (G1 and G2, respectively). Experiments using monochro-
matic UVA in the range 300–400 nm were performed to determine the relation between products-yield
and wavelength, and the highest yields of both G1 and G2 were found to occur at 340 nm. This wave-
length coincided with the absorption maximum of NPRO. We propose that the NO radical produced from
NPRO by photolysis triggers the formation of these products. Further investigation using calf thymus
DNA showed that G1 and/or G2, probably both, could be produced by UVA irradiation of DNA + NPRO as
well.
Received 3 September 2013
Received in revised form 26 October 2013
Accepted 20 November 2013
Available online 1 December 2013
Keywords:
Photoreaction
N-nitrosoproline
DNA adducts
8-Oxo-deoxyguanosine
NO radical
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
However, we have detected the direct mutagenicity of NPRO
plus natural sunlight toward Salmonella typhimurium [11]. Our pre-
Recently, the International Agency for Research on Cancer
(IARC) announced that it has classified outdoor air pollution as
carcinogenic to humans (Group 1) [1]. Occurrence of carcino-
genic N-nitroso compounds in the human environment including
air pollution and tobacco smoke has been extensively studied
[2–4]. Secondary amines and amino acids can react with nitrite
to form N-nitroso derivatives [4,5]. Endogenous formation of N-
nitrosoproline (NPRO) from l-proline by nitrite in humans has been
reported by Ohshima and Bartsch [6] and its excretion has been
used as an index for endogenous nitrosation [7,8]. NPRO was not
carcinogenic in a long-term study in rats [9]. In addition, NPRO was
non-mutagenic in the Ames test [10]. Thus, NPRO is regarded as
non-mutagenic and non-carcinogenic for humans [5–8].
vious studies have also shown that DNA exposed to NPRO plus
UVA results in strand breaks [11,12]. We observed that UVA irradi-
ation of NPRO in aqueous solution generates OH radical, singlet
oxygen and nitric oxide (NO). By using inhibitors against these
active components, we showed that DNA strand breaks were
caused by these three species [11]. We also found that 8-oxo-
7,8-dihydro-2ꢀ-deoxyguanosine (8-oxodG), a mutagenic adduct,
was formed in DNA on treatment with NPRO plus simulated
sunlight [11]. In addition, Suzuki et al. have shown that treat-
ment of dG with nitric oxide in acetate buffer (3.0 M, pH 3.7)
produces 2ꢀ-deoxyoxanosine (dO), 2ꢀ-deoxyxanthosine (dX) and
2-nitro-deoxyinosine in addition to 8-oxodG [13]. Stefan and
Bolton [14] reported that the reaction rate of the photolysis of N-
nitrosodimethylamine (NDMA) at pH 3 is greater than at pH 7. We
have suspected that other modified nucleosides may also be pro-
duced during the photoreaction of NPRO with dG and DNA. We
therefore investigated the photoproducts from an UVA-irradiated
mixture of NPRO and dG under acidic conditions. Here, the isola-
tion and identification of two new photoproducts generated from
NPRO and dG are described. Formation of these new adducts in
Abbreviations: NPRO, N-nitrosoproline; UVA, near-ultraviolet light; dG, 2ꢀ-
deoxyguanosine; NO, nitric oxide; 8-oxodG, 8-oxo-7,8-dihydro-2ꢀ-deoxyguanosine;
dO, 2ꢀ-deoxyoxanosine; dX, 2ꢀ-deoxyxanthosine.
∗
Corresponding author. Tel.: +81 86 251 7947; fax: +81 86 251 7947.
E-mail address: arimoto@cc.okayama-u.ac.jp (S. Arimoto-Kobayashi).
1010-6030/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2013.11.009

2
S. Aoyama et al. / Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
calf thymus DNA on exposure to UVA + NPRO at pH 7 is also
reported.
with authentic specimen was performed with 10 ␮L of irradiated
sample mixed with 1 nmol of dX, 1 nmol of dO or 2 nmol of 8-oxodG.
1H NMR spectra of samples dissolved in dimethylsulfoxide-d6 were
recorded on a Unity INOVA AS600 spectrometer (Agilent Technolo-
gies, Tokyo) at 600 MHz.
2. Materials and methods
2.1. Materials
2.3. Relation between photoproducts-yield and UVA wavelength
N-Nitrosoproline (NPRO) was a gift from Dr. M. Mochizuki of
Tokyo University of Science, who synthesized this compound as
described by Lijinsky et al. [15]. 2ꢀ-Deoxyguanosine (dG) and 8-oxo-
7,8-dihydro-2ꢀ-deoxyguanosine (8-oxodG) were purchased from
Wako Pure Chemicals (Osaka, Japan). 2ꢀ-Deoxyxanthosine (dX) and
2ꢀ-deoxyoxanosine (dO) were synthesized as described [13] both
with purities >99%. All other materials used were of reagent grade
obtained from commercial sources.
Monochromatic irradiation was performed at the Spectrogra-
phy and Bioimaging Facility, National Institute of Basic Biology
(Okazaki, Aichi Prefecture) using the Large Spectrograph. The inten-
sity of radiation at 300, 320, 340, 360, 380 and 400 nm employed
was 5.5, 13.5, 19.4, 24.0, 22.5 and 21.3 W/m2, respectively. By
adjusting the time of irradiation, the UV-dose at each wavelength
was made equal. Mixtures containing 10 mM NPRO and 10 mM
dG in sodium acetate buffer (3 M, pH 3.7) were exposed to these
monochromatic radiations at 25 ◦C. Immediately after irradiation,
samples were taken and stored at −80 ◦C. The amount of each
photoproduct formed was determined by LC–MSMS analysis. The
experiments were performed in triplicate.
2.2. Characterization of photoproducts formed in the irradiation
of dG with NPRO
dG (10 mM) was irradiated in sodium acetate buffer (3 M, pH
3.7) in the presence of NPRO (100 mM) in a chamber kept at 4 ◦C.
Continuous mixing was done during the reaction. UVA irradia-
tion was performed for 12 h with 20-W black-light bulbs emitting
radiation in the range 300–400 nm (Panasonic, Japan). Use of a 4-
mm thick glass plate between the light source and the reaction
solution excluded radiation of wavelength <320 nm. The UVA dose
rate at 360 nm on the surface of the solution, measured using a
black-ray UV intensity meter (Ultraviolet Products, San Gabriel,
CA), was 0.98 0.060 mW/cm2. Irradiated samples were fraction-
ated using HPLC (TSK-GEL ODS-80Ts, 21.5 mm × 300 mm) equipped
with a photodiode array detector SPP-M 10Avp (Shimadzu, Kyoto).
These fractions were analyzed by LC–MSMS (API3000 and API4000,
AB SCIEX, MA, USA) with positive mode ESI. The column used
was Inertsil ODS-3 (1.0 mm × 150 mm), and the eluent was 5 mM
ammonium acetate with 0–40% methanol. Co-chromatography
Fig. 2. Co-chromatographic profiles. NPRO (100 mM) and dG (10 mM) were used.
(A) Chromatographic profile of the sample of NPRO and dG after UVA irradiation for
12 h as shown in Fig. 1A. (B) Co-chromatographic profile of irradiated sample (10 ␮L)
with dX (1 nmol). The arrow indicates the retention time of authentic dX. (C) Co-
chromatographic profile of irradiated sample (10 ␮L) with 8-oxodG (2 nmol). The
arrow indicates the retention time of authentic 8-oxodG. (D) Co-chromatographic
profile of irradiated sample (10 ␮L) with dO (1 nmol). The arrow indicates the reten-
tion time of authentic dO.
Fig. 1. HPLC profiles of UVA-dG-NPRO reaction. (A) A solution containing 10 mM dG
and 100 mM NPRO at pH 3.7 was incubated at 4 ◦C for 12 h under UVA (320-400 nm)
irradiation. (B) 10 mM dG-100 mM NPRO, pH 3.7, incubated at 4 ◦C for 12 h without
UVA. (C) 100 mM NPRO, pH 3.7, incubated at 4 ◦C for 12 h under UVA without dG.
Peaks 1–5 indicate products to be identified.

S. Aoyama et al. / Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
3
2.4. Analysis of products formed in the UVA irradiation of DNA
plus NPRO
used was Inertsil ODS-3 (4.6 mm × 250 mm). Eluent from 19 to
22 min, which corresponded to that of G1 and G2, was collected
and evaporated to dryness. The residue was dissolved in water and
analyzed by positive mode LC–MSMS with a constant neutral scan,
monitoring the precursor/product ion transitions m/z 337/221.
A mixture of calf thymus DNA (0.5 mg/mL) and NPRO (50 mM)
in 15 mM sodium chloride–1.5 mM sodium citrate buffer (pH 7.0)
was irradiated with UVA for 4 h at 4 ◦C. UVA irradiation was per-
formed with 20-W black-light bulbs, with an inserted glass plate
(see above). Irradiated DNA was dialyzed against 1 mM Tris–HCl –
0.1 mM EDTA buffer (pH 8.0). The DNA (0.1 mg) was then digested
with nucleaseP1 (Sigma, St. Louis, MO) followed by alkaline phos-
phatase (Sigma) into nucleosides. The mixture was centrifuged
with a filter (Amicon Ultra free-MC, Milipore, Billerica, MA) and
the filtrate was subjected to HPLC, with 20 mM triethylammonium
acetate (pH 7.0) and 2–30% methanol as elution media. The column
3. Results
3.1. UVA-mediated reaction of 2ꢀ-deoxyguanosine (dG) and
N-nitrosoproline (NPRO)
Fig. 1A shows HPLC fractionation pattern of the UVA-dG-NPRO
reaction, Fig. 1B that of dG-NPRO incubation without UVA, and
Fig. 1C that of UVA-NPRO incubation without dG. As can be seen,
Fig. 3. Identification of peak 4 as (R)-8-(2-pyrroldyl)dG (G1). UV and NMR spectra, and the chemical structure.

4
S. Aoyama et al. / Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
five peaks numbered 1–5 (Fig. 1A) were the products to be iden-
tified. Without dG, recovery of NPRO after 12 h irradiation was
12% (Fig. 1C). Without NPRO, the recovery of dG after 12 h irradia-
tion was 99% and no new peaks were observed (data not shown).
The retention times (Fig. 1A) and UV-absorption profiles (data not
shown) of the product in peaks 1–3 (hereafter referred to as P1, P2
and P3, respectively) were identical with those of authentic dX, 8-
oxodG and dO, respectively. The yields of dX, 8-oxodG and dO from
dG (1 mM) with NPRO (10 mM) irradiated in the acidic solution
were 1.3–2.0%, 2.0–5.0% and 0.5–2.5% of original dG, respectively,
Fig. 4. Identification of peak 5 as (S)-8-(2-pyrrolidyl)dG (G2). UV and NMR spectra, and the chemical structure.

S. Aoyama et al. / Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
5
with 12 h UVA irradiation (triplicate experiments). Co-injection of
P1 with dX, P2 with 8-oxodG, and P3 with dO gave respectively a
single peak having areas coincident to the sum of the peak areas
of P1 and dX, P2 and 8-oxodG, and P3 and dO, respectively (Fig. 2).
The ESI-MS spectra of P1 and P3 showed protonated molecular ion
([MH]+) m/z 269, and their major MS/MS product ion was m/z 153
that could be formed by a loss of deoxyribose (116 amu) from dX
and dO (mw = 268). The ESI-MS spectra of P2 showed [MH]+ m/z
284, and the major product ion was m/z 168 that could be formed
by a loss of deoxyribose from 8-oxodG (mw = 283). 1H NMR analysis
of P1, P2, P3, and authentic specimens of 8-oxodG and dO dissolved
in dimethylsulfoxide-d6 were performed (summarized in Suppor-
ting information), and the 1H NMR profiles of P1, P2 and P3 were
identical with those of authentic specimens of dX, 8-oxodG and
dO, respectively [13,16]. Thus, P1, P2 and P3 were identified as dX,
8-oxodG and dO, respectively.
0.5
0.4
0.3
0.2
0.1
0
2
1.5
1
0.5
0
300 320 340 360 380 400
Wavelength (nm)
Fig. 5. UV absorption spectrum of 5 mM NPRO at pH 3.7 (line) and amounts of
products (G1 by circles and G2 by triangles) formed on irradiation at individual
wavelength. Peak area values are those of the integrator reading.
We attempted to determine the structures of compounds in
peaks 4 and 5 (hereafter referred to as G1 and G2, respectively). UV
spectra of G1 and G2 seemed highest at about 260 nm (Figs. 2 and 3),
making us to suspect that G1 and G2 might be guanine derivatives.
With positive mode LC–MS/MS analysis under a constant neutral
scan that should release a fragment of 116 amu, we observed an
ion (m/z 337) from both G1 and G2. With the product ion scan from
the precursor ion (m/z 337) of G1 and G2, we found two peaks; one
major MS/MS product ion (m/z 221) that could be formed by loss of
deoxyribose (116 amu) from the protonated molecule ion ([MH]+)
(m/z 337), and one minor product ion (m/z 152) that seems to rep-
resent the protonated guanine ion formed by a loss of the pyrrolidyl
moiety (69 amu) from the product ion (m/z 221).
MRM mode by monitoring the precursor/product ion transitions
m/z 337/221 ([MH]+/[MH−116]+, the loss of deoxyribose) a signal
for detecting G1 and G2. Indeed a peak was observed at reten-
tion time 25.7 min, which corresponded to the authentic G1 and
G2 (Fig. 6B). We concluded that G1 and/or G2, probably both, were
formed in DNA by the action of UVA-NPRO.
4. Discussion
Although N-nitroso compounds are thermally stable in aqueous
solutions, Chow et al. described that nitroso-␣-amino acid includ-
ing NPRO are photolyzed to give CO2 and piperidonoxime with
UVC (Scheme, Reaction 1) [17]. In 1967, Chow observed that the
formation of piperidinium radical from an acidic aqueous solution
(pH 2) of N-nitrosopiperidine on photolysis, and proposed that a
radical disproportion occurs to form HNO and tetrahydropyridine
(Scheme, Reaction 2) [18].
The present findings show that new modified nucleosides, (R)-
and (S)-8-(2-pyrrolidyl)-2ꢀ-deoxyguanosine can be formed by UVA-
irradiation of NPRO-dG and of NPRO-DNA in aqueous solutions. Our
finding that the plots of G1 and G2 formation match the absorption
curve of NPRO, both being highest at 340 nm (Fig. 5), suggests that
the rate limiting step of G1 and G2 formation is photo-cleavage
of the N N bond in NPRO. Thus, a possible mechanism of the G1
and G2 formation may involve release of NO from NPRO result-
ing in the formation of an aminium radical, and the latter reacting
with NO radical to create an aminium cation. Subsequent nucleo-
philic attack of the C8 moiety of dG by the cation would give CO2
The 1H NMR spectrum of G1 showed signals at ı 1.68–2.10 (2H,
multiple, H␤a,b; 2H, m, H␥a,b), 2.01 (1H, m, H2ꢀa), 2.79 (2H, m,
H␣ and H-␦a), 2.87 (1H, ddd, H2ꢀb), 2.95 (1H, ddd, H-␦b), 3.50
(5H, m, H5ꢀa,b), 3.78 (1H, dd, H4ꢀ), 4.34 (1H, br, H3ꢀ), 5.45 (1H, s,
3ꢀ-OH), 6.29 (1H, dd, H1ꢀ) and 6.76 (2H, s, NH2) (Fig. 3). The cou-
plings were confirmed by COSY measurements (Fig. 3). The 1H NMR
spectrum of G2 showed signals at ı 1.57–1.79 (2H, m, H␥a,b), 1.96
(1H, m, H␤a), 2.06 (1H, m, H2ꢀ), 2.17 (1H, m, H␤b), 2.81 (1H, m,
H-␦a), 2.90 (2H, m, H2ꢀ and H-␦b), 3.26 (1H, dd, H5ꢀa), 3.34 (1H,
dd, H5ꢀb), 3.78 (1H, dd, H4ꢀ), 4.31 (1H, t, H␣), 4.37 (1H, br, H3ꢀ),
5.43 (1H, s, 3ꢀ-OH), 6.31 (1H, t, H1ꢀ) and 6.76 (2H, s, NH2) (Fig. 4).
The couplings were confirmed by COSY measurements (Fig. 4). The
proton signal of the H8 position in the dG residue at 7.90 ppm was
absent in either profiles of G1 or G2, indicating that substitution
had occurred at the C8 position of dG. The 1H NMR signal of the H␣
proton of G1 at 2.79 ppm was upfield to that of G2 at 4.31 ppm. In
the R-conformation of 8-(2-pyrrolidyl)dG, the H␣ proton must have
become close to protons of the deoxyribose moiety, thereby being
shielded by steric compression; hence the signal of the H␣ pro-
ton in the R-configuration would be shifted upfield. We concluded
that G1 must be (R)-8-(2-pyrrolidyl)-2ꢀ-deoxyguanosine, and G2
(S)-8-(2-pyrrolidyl)-2ꢀ-deoxyguanosine.
Experiments using monochromatic UVA in the range
300–400 nm were performed to determine the relation between
products-yield and wavelength. As Fig. 5 shows, the highest yields
of both G1 and G2 were found to occur at 340 nm. This wavelength
coincided with the absorption maximum of NPRO, suggesting that
sensitization of NPRO by UVA triggers the formation of both G1
and G2.
3.2. Formation of G1 and G2 in DNA-NPRO-UVA
DNA was treated with NPRO plus UVA under neutral conditions.
The DNA was then digested into nucleosides and the mixture frac-
tionated by HPLC. As the retention times of authentic G1 and G2
were 19.5 and 21.5 min, the eluent from 19 min to 22 min was col-
lected (Fig. 6A). This fraction was analyzed with LC-MSMS in the
Fig. 6. Identification of G1 and G2 in DNA treated with NPRO plus UVA. (A) HPLC
profile of nucleosides produced by enzymatic digestion of DNA that had been treated
with NPRO and UVA. (B) LC–MSMS profile of the fraction pooled from 19 to 22 min
in (A).

6
S. Aoyama et al. / Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
Reaction 1
h
+
CO2
N
OH
N
H
COOH
N
N
O
Reaction 2
nucleophilic attack
O+
H
h
+
+
HNO
NH
NO
NH
N
N
Reaction 3
O
N
COOH
H+
COOH
NH
COOH
H
N
COOH
CO2
N
N
UVA
O
NH
NH2
HNO
NO
H+
N
N
N
NH
HO
O
N
O
NPRO
N
N
NH
NH2
HO
NO
Type I or Type II
photoreaction
dR
dG
dG
O
H
N
O
NH
O
O
N
N
N
N
NH2
O
N
N
dR
NH
O
dR
N
NH2
N
H
dX
dR
8-oxodG
dO
Scheme 1. Mechanisms of the reactions.
and 8-(2-pyrrolidyl)-dG adducts (Scheme, Reaction 3). Whether the
sequence of intermediates, i.e., the decarboxylation, is as shown or
at an earlier step is not clear. Chow et al. [17] demonstrated that a
carboxylic acid located at an ␣-position of nitrosamines could act
as a proton donor causing the nitrosamino group to become pho-
tosensitive and decompose in neutral or acidic solutions without
much difference in quantum efficiency.
UVA-sensitized furocoumarins with a mechanism involving singlet
oxygen is known [23].
We propose that endogenous NPRO might have a role in the
photogenotoxicity of sunlight. Further studies are required to gain
a deeper understanding of the phototoxicity of NPRO.
In our earlier studies, we have shown that irradiation of NPRO
with UVA or sunlight can produce mutagenic activity and that
the activity of irradiated NPRO with DNA involves alkylation and
oxidation of DNA [11]. The present results show that photoreac-
tion of NPRO with dG produces 8-oxodG as well as dX and dO.
We also observed nitric oxide formation in the solution of UVA-
irradiated NPRO [12]. dX and dO were isolated from a reaction of
dG with nitric oxide [13]. Therefore, photoproduction of dX and
dO from dG-NPRO could occur by the attack of the released NO.
Our present work shows that NPRO can facilitate UVA-induced NO
release to create an aminium ion that can give 8-(2-pyrrolidyl)-dG
adducts.
The role of UVA in the phototoxicity and photogenotoxicity of
carcinogens and chemicals has been investigated by many research
groups. For example, UVA-induced reaction of benzo(a)pyrene with
DNA in vitro results in single-strand breaks and benzo(a)pyrene-
nucleoside adducts in vitro [19]. Reactive oxygen species in CHO
cells formed by UVA radiation of benzo(a)pyrene causes increased
level of micronuclei and 8-oxodG legions [20]. UVA-sensitized
riboflavin degrades purine and pyrimidine bases in DNA [21]. Wier-
ckx et al. reported that UVA-irradiated 2-nitrofluorene becomes
covalently bound to RNA to form a guanine C8-adduct [22].
Photooxidation of guanine base in DNA and RNA treated with
Conflicts of interest
The authors declare that no financial or personal conflicts of
interest exist.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scien-
tific Research (C) from the Ministry of Education, Culture, Sports,
Science and Technology (20510064 and 23234567) and the NIBB
Cooperative Research Program for the Okazaki Large Spectrograph
(11-509, 12-511 and 13-510). The authors wish to thank Dr. M.
Mochizuki (Tokyo University of Science) for the gift of NPRO. The
authors also would like to thank Dr. H. Hayatsu (Okayama Univer-
sity) for invaluable advice.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
jphotochem.2013.11.009.

S. Aoyama et al. / Journal of Photochemistry and Photobiology A: Chemistry 276 (2013) 1–7
7
References
by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogeni-
city, Carcinogenesis 23 (2002) 1537–1540.
[12] S. Arimoto-Kobayashi, K. Sano, M. Machida, K. Kaji, K. Yakushi, UVA activation
of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well
as oxidative damages in DNA, and inducing mutations in the Ames test, Mutat.
Res. 691 (2010) 47–54.
[13] T. Suzuki, R. Yamaoka, M. Nishi, H. Ide, M. Makino, Isolation and charac-
terization of a novel product, 2ꢀ-deoxyoxanosine, from 2ꢀ-deoxyguanosine,
oligodeoxynucleotide, and calf thymus DNA treated by nitrous acid and nitric
oxide, J. Am. Chem. Soc. 118 (1996) 2515–2516.
[1] IARC, IARC Scientific Publication No. 161, Air Pollution and Cancer, IARC, Lyon,
2013.
[2] IARC, A Review of Human Carcinogens. E. Personal Habits and Indoor Combus-
tions, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans,
vol. 100 (E), IARC, Lyon, 2012.
[3] IARC, Smokeless Tobacco and some Tobacco-Specific N-Nitrosamines, IARC
Monographs on the Evaluation of Carcinogenic Risks to Human, vol. 89, IARC,
Lyon, 2007.
[4] S.R. Tannenbaum, Endogenous formation of N-nitroso compounds: a current
perspective, in: H. Bartsch, I.K. O’Neill, R. Schulte-Hermann (Eds.), IARC Sci-
entific Publications No. 84, The Relevance of N-Nitroso Compounds to Human
Cancer: Exposures and Mechanisms, IARC, Lyon, 1987, pp. 292–296.
[5] K.D. Brunnemann, J.C. Scott, N.J. Haley, D. Hoffmann, Endogenous formation
of N-nitrosoproline upon cigarette smoke inhalation, in: I.K. O’Neill, R.C. von
Borstel, C.T. Miller, J. Long, H. Bartsch (Eds.), IARC Scientific Publications No. 57,
N-Nitroso Compounds, IARC, Lyon, 1984, pp. 819–828.
[6] H. Ohshima, H. Bartsch, Quantitative estimation of endogenous nitrosation in
humans by monitoring N-nitrosoproline excreted in the urine, Cancer Res. 41
(1981) 3658–3662.
[7] K. Cope, H. Seifried, R. Seifried, J. Milner, P. Kris-Etherton, E.H. Harrison, A
gas chromatography–mass spectrometry method for the quantitation of N-
nitrosoproline and N-acetyl-S-allylcysteine in human urine: application to a
study of the effects of garlic consumption on nitrosation, Anal. Biochem. 394
(2009) 243–248.
[14] M.I. Stefan, J.R. Bolton, UV direct photolysis of N-nitrosodimethylamine
(NDMA): kinetic and product study, Helv. Chim. Acta 85 (2002)
1416–1426.
[15] W. Lijinsky, L. Keefer, J. Loo, The preparation and properties of some
nitrosamino acids, Tetrahedron 26 (1970) 5137–5153.
[16] R.C. Moschel, L.K. Keefer, Preparation of 2ꢀ-deoxyxanthosine by nitrosative
deamination of 2ꢀ-deoxyguanosine under alkaline aqueous conditions, Tetra-
hedron Lett. 30 (1989) 1467–1468.
[17] Y.L. Chow, D.P. Horning, J. Polo, Photochemistry of N-nitroso-N-alkyl amino
acids: a facile oxidative decarboxylation, Can. J. Chem. 58 (1980) 2477–2483.
[18] Y.L. Chow, Photochemistry of nitroso compounds in solution. V. Photolysis of
N-nitrosodialkylamines, Can. J. Chem. 45 (1967) 53–62.
[19] G.F. Stmiste, E. Martinez, A.M. Martinez, R.J. Brake, Photo-induced reactions of
benzo(a)pyrene with DNA in vitro, Cancer Res. 40 (1980) 245–252.
[20] C. Botta, C. Di Giorgio, A.-S. Sabatier, M. De Meo, Effects of UVA and visible light
on the photogenotoxicity of benzo[a]pyrene and pyrene, Environ. Toxicol. 24
(2009) 492–505.
[8] K. Lin, W. Shen, Z. Shen, Y. Wu, S. Lu, Dietary exposure and urinary excretion
of total N-nitroso compounds, nitrosamino acids and volatile nitrosamine in
inhabitants of high- and low-risk areas for esophageal cancer in southern China,
Int. J. Cancer 102 (2002) 207–211.
[21] P.C. Joshi, T.C. Keane, Investigation of riboflavin sensitized degradation of
purine and pyrimidine, derivatives of DNA and RNA under UVA and UVB,
Biochem. Biophys. Res. Commun. 400 (2010) 729–733.
[22] F.C.J. Wierckx, G.M.J. Beijiersbergen van Henegouwen, L.T. van den Broek, H. de
Vries, J.H.N. Meerman, G.J. Mulder, Photoactivation of 2-nitrofluorene in vitro
and in the rat in vivo. UVA-induced formation of reactive intermediates that
bind covalently to RNA and protein, Carcinogenesis 13 (1992) 1759–1762.
[23] W.G. Wamer, W.C. Timmer, R.R. Wei, S.A. Miller, A. Kornhauser, Furocoumarin-
photosensitized hydroxylation of guanosine in RNA and DNA, Photochem.
Photobiol. 61 (1995) 336–340.
[9] S.S. Mirvish, O. Bulay, R.G. Runge, K. Patil, Study of the carcinogenicity of large
doses of dimethylnitramine, N-nitroso-l-proline, and sodium nitrite adminis-
tered in drinking water to rats, J. Natl. Cancer Inst. 64 (1980) 1435–1442.
[10] D.R. Stoltz, N.P. Sen, Mutagenicity of five cyclic N-nitrosamines: assay with
Salmonella typhimurium, J. Natl. Cancer Inst. 58 (1977) 393–394.
[11] S. Arimoto-Kobayashi, Y. Ando, Y. Horai, K. Okamoto, H. Hayatsu, J.E. Lowe,
M.H.L. Green, Mutation, DNA strand cleavage and nitric oxide formation caused