Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity

Sakae Arimoto-Kobayashi,2, Yoshiko Ando, Yumi Horai, Keinosuke Okamoto, Hikoya Hayatsu, Jillian E. Lowe1 and Michael H.L. Green1

Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan and
1 School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb, Brighton BN2 4GJ, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite. NPRO has been reported to be nonmutagenic and noncarcinogenic. In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium. Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight. The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA. Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay. An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks. The formation of nitric oxide was observed in NPRO solution irradiated with UVA. We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm. Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm. The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation.

Abbreviations: carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylomodazoline-1-oxyl-3-oxide; EDTA, ethylenediamine-N,N,N',N'-tetraacetic acid; NMOR, N-nitrosomorpholine; NPRO, N-nitrosoproline; NPYR, N-nitrosopyrrolidine; 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine; RF, replicative form; Tris, Tris(hydroxymethyl)aminomethane; UV, ultraviolet radiation; UVA, ultraviolet-A radiation


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are many reports on the endogenous formation of N-nitroso compounds (1). Ohshima and Bartsch reported the formation of N-nitrosoproline (NPRO) in humans from sodium nitrite and proline, based on the finding of NPRO in the urine from male volunteers who had ingested proline and vegetable juice as a source of nitrite (2). NPRO was also reported to be present in cigarette tobacco at the level of 0.6–15 µg/cigarette (3). Greater amounts of NPRO are found in the smoker's urine at the level of 6 µg/24 h (4,5). Solar ultraviolet radiation (UV) is a causal factor for skin cancer in humans and ultraviolet-A radiation (UVA) (320–400 nm), although less DNA-damaging than UVB (280–320 nm), is the predominant source of radiant energy in the sunlight (6). UVA can reach to the subcutaneous area of the skin containing blood vessels. UVA is not absorbed very much by DNA, but tumor development was observed in the albino hairless mice exposed to UVA source (7).

Earlier studies of our laboratory have shown that N-nitrosodialkylamines can be converted into directly mutagenic compounds on UVA irradiation (8–13) and that oxidative species are produced during the photoreaction (10,11,13). N-Nitrosomorpholine (NMOR) was formed in vivo in mice that inhaled NO2 and were given morpholine in gavage, and the concentration of NMOR was highest in the skin (14). Although NPRO was reported to be noncarcinogenic and nonmutagenic in animal feeding experiments (15), perhaps because NPRO was not metabolized in vivo (16), we suspected that endogenous NPRO in skin might work as a photosensitizer of sunlight UVA and play a role in photocarcinogenicity.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
N-Nitrosoproline (NPRO) was a gift from Dr M.Mochizuki of Kyoritsu College of Pharmacy, who synthesized this compound as described by Lijinsky et al. (17) and checked the purity as >99% by high performance liquid chromatography. The NPRO was diluted with water and stored frozen at –20°C. Salmonella typhimurium TA1535 [hisG, rfa, {Delta}uvrB] was a gift from Dr B.N.Ames of the University of California, Berkeley, and phage M13mp2 was from Dr T.A.Kunkel (NIEHS, Research Triangle Park, NC). Other chemicals were commercial products of reagent grade.

Irradiation and the detection of the mutagenicity produced were performed as follows. The reaction mixtures (1 ml) of 40 mM NPRO in 20 mM sodium phosphate buffer (pH 7.4) were placed in an ice-cold tray (Nunc, Denmark) and exposed to light. UVC and the majority of UVB (<320 nm) in the sunlight were excluded by use of a glass cover. The intensity of light was measured by a black ray UV intensity meter (Ultraviolet Products, San Gabriel, CA) at 360 nm. Samples (300 µl each) were assayed for mutagenicity in S.typhimurium TA1535 without metabolic activation (18). The mutation assays were performed with four plates per datum point, and the numbers reported in this paper are the mean numbers of these plates.

The DNA single strand breaks were detected by irradiating phage M13mp2 replicative form DNA (RF I). Reactions were carried out using superhelical DNA of M13mp2 (3.3 µg/ml) in 2 mM phosphate (pH 7.0) with appropriate concentrations of NPRO with light for 20 min. The treated DNA sample was analyzed by agarose gel electrophoresis (0.7% gels in Tris–borate–EDTA buffer). Gels were run 1.5–2 h and stained with ethidium bromide. Single strand breaks give rise to the formation of the relaxed circular form (RF II), which migrates in the gel more slowly than RF I.

DNA damage was detected in human fibroblasts IBR3/9 treated with NPRO plus UVA (5.35 W/m2, 5 min). The slides mounted with cells were treated with NPRO for 60 min at 37°C and then irradiated with Atlas UVA 20 W tubes. The detection of comets was performed by a modified method of Singh (19,20). The results are shown in the averages of three independent experiments.

The measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA treated with NPRO (5 mM) plus simulated sunlight at 22.4 W/m2 for 20 min (Oriel solar simulator 81193, 320–400 nm) were carried out according to the previous work (13) in duplicate and the averages of data are shown.

Nitric oxide in the irradiated solution of NPRO was measured as NO2– (21). Briefly, NPRO was dissolved in 2 mM sodium phosphate buffer (pH 7.0) and irradiated by UVA lump as described above. The irradiated solutions (1 ml) were mixed with 0.1 ml of sulfanilamide (5 g/l), and the mixtures were allowed to stand for 3 min. N-(1-naphthyl)ethylenediamine dihydrochloride (1.2 g/l, 0.1 ml) was added and then samples were stood for 20 min. The absorption of the solutions was measured at 540 nm. The quantitative calibration was done with standard solutions of NaNO2. The experiments were done in triplicate.


    Results
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Figure 1aGo shows the direct-acting mutagenicity of sunlight-irradiated NPRO towards S.typhimurium TA1535. The mutagenesis was irradiation time-dependent. Figure 1bGo shows a photograph of an agarose gel of M13mp2 DNA, which was treated with NPRO (1–100 µM) plus sunlight. When superhelical DNA [replicative form I (RF I)] was irradiated with sunlight in the presence of NPRO, a new band for nicked circular DNA [replicative form II (RF II)] appeared (lanes 1, 3 and 5). Strand breaks occurred at an NPRO concentration as low as 10 µM. NPRO alone (lanes 2, 4, 6 and 8) or sunlight alone (lane 9) did not induce these single strand breaks. We examined whether NPRO (2–10 mM) can induce DNA strand breaks in human fibroblast cells on irradiation with UVA using a comet assay (Figure 1cGo). Positive comet tails were observed for the NPRO plus UVA group, but not for UVA alone or for the NPRO alone group. On UVA irradiation of calf thymus DNA in the presence of NPRO, the formation of 8-oxodGuo occurred (Figure 1dGo).



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Fig. 1. Genotoxic actions of NPRO plus light. The bar attached to each datum point indicates the standard deviation. *P < 0.05 and **P < 0.01; significantly different from the results in the dark–control (t-test). (a) Formation of direct-acting mutagenicity from sunlight-irradiated NPRO (40 mM). The intensity of the sunlight UVA was 3.7–4.7 W/m2 at 360 nm. The experiment was done from 12:35 to 13:35 h on 2nd March 2000. (b) Single strand breaks in M13mp2 DNA caused by NPRO plus sunlight. The intensity of UVA was 13.65–12.30 W/m2. The experiment was done from 12:30 to 12:50 h on 7th July 2000. The band of nicked circular DNA is indicated as RF II. (c) DNA damage detected by the Comet assay in human fibroblasts IBR3/9 treated with NPRO plus UVA (5.35 W/m2, 5 min). (d) Formation of 8-oxodG in calf thymus DNA treated with NPRO (5 mM) plus simulated sunlight.

 
As shown in Figure 2Go, the NPRO + UVA-mediated single strand breaks in M13mp2 DNA were decreased by adding either an OH-radical scavenger (mannitol), scavengers of singlet oxygen (histidine and NaN3) or an NO-radical scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylomodazoline-1-oxyl-3-oxide (carboxy-PTIO)] (22).



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Fig. 2. Effects of (a) mannitol, (b) histidine, (c) NaN3, and (d) carboxy-PTIO on the level of single strand breaks caused by NPRO (0.1 mM) plus UVA at 4.5 W/m2 for 30 min (National FLBL-B 15 W, 320–400 nm) in the replicative form of M13mp2 DNA.

 
Nitric oxide formation was observed in the NPRO solution irradiated with UVA and was dependent both on the NPRO concentration (Figure 3aGo) and on the irradiation time (Figure 3bGo). The yield of nitric oxide from NPRO was 2.4% based on the data shown in Figure 3aGo.



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Fig. 3. Formation of NO radical in the solution of NPRO irradiated with UVA. (a) Dependence on NPRO dose. UVA irradiation was for 20 min at 5.57 W/m2. (b) Dependence of irradiation time. The concentration of NPRO was 0.05 mM. The intensity of UVA was 5.75 W/m2.

 
To explore if NPRO acts as a sensitizer, the action spectrum of induced mutations and single strand breaks in M13mp2 DNA was determined (Figure 4Go). Both mutation frequencies and DNA breaks were highest at the absorption maximum of NPRO, 340 nm.



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Fig. 4. Action spectra. (a) Direct-acting mutagenicity of NPRO irradiated at different wavelengths of monochromatic light (•). Each value of [His+ revertants/10 kJ] was calculated from the dose–response curve of the mutagenicity versus radiation time. UV absorption spectrum of 2 mM NPRO in 20 mM NaPO4 at pH 7.4 is shown by the curve. (b) Single strand breaks of M13mp2 DNA treated with NPRO (0.1 mM) with monochromatic light at 300 nm, 320 nm, 340 nm, 360 nm, 380 nm and 400 nm (2.54 kJ/m2 each) (•) and UV absorption spectrum of NPRO (line).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A product of the UVA-exposed nitrosopyrrolidine (NPYR) in phosphate buffer had been identified as the phosphate ester of {alpha}-hydroxy-NPYR (12). The direct-acting mutagen formed with NPRO plus light may be a similar derivative of NPRO. We have also reported previously that the treatment with NPYR plus UVA generates oxidative damage in DNA, resulting in mutations (13). Nitric oxide was reported to have induced mutations in cultured human cells and in S.typhimurium (23). The observations shown in Figures 2 and 3GoGo suggest that reactive oxygen species and nitric oxide are also involved in the photo-process of NPRO. Therefore, it is likely that NPRO works as a photosensitizer in these reactions: a photon-excited NPRO molecule may be converted to a mutagenic compound, and the photo-energy may be transferred to other molecules to produce radicals.

An average value of 3.5–6.0 µg of NPRO was detected from urine samples varying in volume from 0.5–2.8 l per day, that is, the concentration of NPRO in the samples was at the average level of 0.01–0.1 µM (24). As tobacco smoke is a rich source of nitroso products (1), the concentration of NPRO in urine and in serum just after smoking might be higher than 0.1 µM. Formation of NO radical was observed in the 2.5 µM solution of NPRO irradiated with UVA (Figure 3aGo) and DNA strand breaks occurred with 10 µM NPRO plus sunlight UVA (Figure 1bGo). The intensity of UVA used in the UVA irradiation experiments is comparable with that in sunlight. UVA is the predominant source of radiant energy in sunlight (9) and NPRO may serve as endogenous photosensitizers for UVA. The results we have shown suggest an additional way in which UVA may be involved in the etiology of skin cancer. Carcinogenicity of NPRO in the presence of sunlight has not been examined. Our results imply the importance of such studies.


    Notes
 
2 To whom correspondence should be addressed Email: arimoto{at}cc.okayama-u.ac.jp Back


    Acknowledgments
 
This work was supported by the Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology (11672228), the NIBB Cooperative Research Program for the Okazaki Large Spectrograph (00–515 and 1–506). It was also supported by the UK Department of Health (121/6439) in part. The authors would like to thank Dr Y.Furusawa (National Institute of Radiation Science) for his help.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 4, 2002; revised May 20, 2002; accepted May 21, 2002.