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Carcinogenesis, Vol. 20, No. 8, 1485-1490, August 1999
© 1999 Oxford University Press

Molecular Epidemiology and Cancer Prevention

N-acetylcysteine, a cancer chemopreventive agent, causes oxidative damage to cellular and isolated DNA

Shinji Oikawa, Keitaro Yamada, Naruto Yamashita, Saeko Tada-Oikawa and Shosuke Kawanishi1

Department of Hygiene, Mie University School of Medicine, Mie 514, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although N-acetylcysteine is an antioxidant which has been expected to be a cancer chemopreventive agent, its safety and risk assessment have not been evaluated. N-acetylcysteine increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, whereas the amount of 8-oxodG in HP100, which is a hydrogen peroxide (H2O2)-resistant cell line derived from HL-60, was not increased. To clarify the mechanism of cellular DNA damage, we investigated DNA damage and its site specificity induced by N-acetylcysteine, using 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. N-acetylcysteine induced extensive DNA damage in the presence of Cu(II). The DNA cleavage was enhanced by piperidine treatment, suggesting that N-acetylcysteine plus Cu(II) caused not only deoxyribose phosphate backbone breakage but also base modification. N-acetylcysteine plus Cu(II) frequently modified thymine and guanine residues. Bathocuproine, a specific Cu(I) chelator, and catalase inhibited the DNA damage, indicating the participation of Cu(I) and H2O2 in the DNA damage. Typical hydroxyl radical scavengers did not inhibit N-acetylcysteine plus Cu(II)-induced DNA damage, whereas methional completely inhibited it. These results suggest that reactive species derived from the reaction of H2O2 with Cu(I) participates in N-acetylcysteine plus Cu(II)-induced DNA damage. The content of 8-oxodG in calf thymus DNA was increased by N-acetylcysteine in the presence of Cu(II). The present study has demonstrated that N-acetylcysteine could induce metal-dependent H2O2 generation and, subsequently, damage to cellular and isolated DNA. Therefore, it is reasonable to consider that N-acetylcysteine may have the dual function of carcinogenic and anti-carcinogenic potentials. This work requires further studies on safety and risk assessment of N-acetylcysteine.

Abbreviations: 8-oxodG, 8-oxo-7,8-dihydro-2'-deoxyguanosine (and also known as 8-hydroxy-2'-deoxyguanosine); DTPA, diethylenetriamine-N,N,N',N'',N''-pentaacetic acid; SOD, superoxide dismutase; H2O2, hydrogen peroxide; HPLC–ECD, high pressure liquid chromatography with electrochemical detection; OH·, hydroxyl free radical


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
N-acetylcysteine has been considered as one of the most promising cancer chemopreventive agents (1). Cancer prevention by N-acetylcysteine has been shown to be effective in several animal experiments (2–4). The oral administration of N-acetylcysteine completely prevented the induction of various DNA alterations in rat lung cells (5). In addition, N-acetylcysteine prevented the in vivo formation of carcinogen–DNA adducts, and suppressed the development of tumors in rodents (3). Thus, numerous studies indicate that N-acetylcysteine can prevent mutation and cancer through a variety of mechanisms (6–11).

Therefore, N-acetylcysteine is currently being clinically tested as a chemopreventive agent both in USA (National Cancer Institute) and in Europe (Project Euroscan). In an on-going study, Ponz de Leon and Roncucci (12) showed a 40% reduction of the reoccurrence of polyps (versus controls) in individuals given N-acetylcysteine. However, previous studies have reported that a number of antioxidants may have both anti-carcinogenic and carcinogenic effects (13,14). The unexpected results of increased lung cancer incidence in studies of cancer chemoprevention with ß-carotene and {alpha}-tocopherol suggest the importance of establishing the efficacy and safety of chemoprevention agents in carefully conducted clinical trials (15,16).

In the present study, to estimate possibility of carcinogenic effect of N-acetylcysteine, we investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in human leukemia cell line HL-60 and its hydrogen peroxide (H2O2)-resistant clone HP100 treated with N-acetylcysteine by using an electrochemical detector coupled to a high pressure liquid chromatograph (HPLC–ECD). A characteristic oxidative DNA lesion, 8-oxodG, has attracted much attention in relation to mutagenesis and carcinogenesis (17,18). HP100 cells were used to assess whether H2O2 participates in N-acetylcysteine-induced oxidative DNA lesion. Catalase activity of HP100 cells was 18 times higher than that of HL-60 cells (19). Furthermore, to investigate the mechanism of the cellular DNA damage, we examined the DNA damage and site specificity of DNA cleavage induced by N-acetylcysteine in the presence of Cu(II), using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. We also analyzed the 8-oxodG formation in calf thymus DNA by N-acetylcysteine in the presence of Cu(II) by using HPLC–ECD.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
Restriction enzymes (HindIII, AvaI, ApaI and PstI) and T4 polynucleotide kinase were purchased from New England Biolabs. Alkaline phosphatase from calf intestine was from Boehringer Mannheim GmbH. [{gamma}-32P]ATP (222 TBq/mmol) was from New England Nuclear. Calf thymus DNA, N-acetylcysteine, catalase (45 000 U/mg from bovine liver) and superoxide dismutase (SOD; 3000 U/mg from bovine erythrocytes) were from Sigma. Acrylamide, bisacrylamide and piperidine were from Wako (Osaka, Japan). Sodium formate, ethanol, D-mannitol and CuCl2 were from Nacalai Tesque (Kyoto, Japan). Diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were from Dojin (Kumamoto, Japan).

Measurement of 8-oxodG in cultured cells
HL-60 and HP100 cells were grown in RPMI 1640 supplemented with 6% FCS at 37°C under 5% CO2 in a humidified atmosphere. Cells (106 cells/ml) were incubated with N-acetylcysteine for 18 h at 37°C and immediately washed three times with PBS, and the DNA was extracted using a DNA Extractor WB Kit (Wako). The DNA was dissolved in water, and treated with 8 U nuclease P1 and then with 1.2 U bacterial alkaline phosphatase. The content of 8-oxodG was determined by the method described previously (20,21).

Preparation of 32P-5'-end-labeled DNA fragments
DNA fragments were obtained from the human p53 tumor suppressor gene (22) and the c-Ha-ras-1 protooncogene (23). The DNA fragment of the p53 tumor suppressor gene was prepared from pUC18 plasmid, ligated fragments containing exons of p53 gene amplified by the polymerase chain reaction (PCR) method. The singly 32P-5'-end-labeled 211 bp fragment (HindIII* 13972–ApaI 14182) was obtained according to a method described previously (24). The DNA fragment of the human c-Ha-ras-1 protooncogene was prepared from plasmid pbcNI, which carries a 6.6 kb BamHI chromosomal DNA segment containing the c-Ha-ras-1 gene. The singly labeled 337 bp fragment (PstI 2345–AvaI* 2681) was obtained according to a method described previously (25,26). Nucleotide numbering starts with the BamHI site (23). (The asterisk indicates the 32P label.)

Analysis of DNA damage by N-acetylcysteine in the presence of metal
The standard reaction mixture in a microtube (1.5 ml Eppendorf) contained N-acetylcysteine, 20 µM metal ion, 32P-5'-end-labeled DNA fragments and 10 µM per base of sonicated calf thymus DNA in 200 µl of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After incubation at 37°C for 1 h, the DNA fragments were precipitated with cold ethanol, followed by heating at 90°C for 20 min in 1 M piperidine. The DNA fragments recovered by the precipitation with cold ethanol were electrophoresed in 8% polyacrylamide–8 M urea gels. The autoradiograms were obtained by exposing X-ray film to the gels. The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam–Gilbert procedure (27) using a DNA sequencing system (LKB 2010 Macrophor). The relative amounts of oligonucleotides from the treated DNA fragments were measured with a laser densitometer (LKB 2222 UltroScan XL).

Analysis of 8-oxodG formation in calf thymus DNA by N-acetylcysteine plus Cu(II)
The amount of 8-oxodG was measured by a modified method of Kasai et al. (28). Calf thymus DNA fragments (50 µM/base) were incubated with N-acetylcysteine and 20 µM CuCl2 for 2 h at 37°C. After ethanol precipitation, DNA was digested to the nucleosides with nuclease P1 and calf intestine phosphatase and analyzed by HPLC–ECD (20).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Formation of 8-oxodG in human cultured cells by N-acetylcysteine
To investigate cellular induction of oxidative DNA damage, we measured the content of 8-oxodG, a relevant indicator of oxidative base damage, in HL-60 cells treated with N-acetylcysteine. Production of 8-oxodG in DNA extracted from the treated HL-60 cells was increased in a dose-dependent manner (Figure 1Go). The content of 8-oxodG of DNA in HL-60 cells treated with 1 mM N-acetylcysteine was significantly increased in comparison with no treated cells. However, N-acetylcysteine did not increase the amount of 8-oxodG in HP100 cells (Figure 1Go). It was reported that HP100 cells were ~340-fold more resistant to H2O2 than the parent cell-line, HL-60 (19). These results suggest that N-acetylcysteine can cause oxidative DNA damage in human cultured cells and that generation of H2O2 plays a critical role in N-acetylcysteine-mediated DNA damage.



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  		Fig. 1. Contents of 8-oxodG in DNA of HL-60 cells treated with N-acetylcysteine. The HL-60 (•) and HP100 ({circ}) cells (1.0x106 cells/ml) were incubated with various concentrations of N-acetylcysteine for 18 h and the treated DNA was extracted immediately. The extracted DNA was subjected to enzyme digestion and analyzed by HPLC–ECD as described in the Materials and methods. Results are expressed as means ± SE of values obtained from six independent experiments. The asterisk indicates significant differences compared with control by t-test (P < 0.01).

 
Damage of 32P-labeled DNA fragments by N-acetylcysteine in the presence of Cu(II)
Figure 2Go shows an autoradiogram of DNA fragment treated with N-acetylcysteine plus Cu(II). Oligonucleotides were detected on the autoradiogram as a result of DNA damage. In the presence of Cu(II), N-acetylcysteine induced a significant increase of DNA damage with a concentration as low as 50 µM. At a higher concentration of N-acetylcysteine (100 µM), the DNA damage was decreased. Even without piperidine treatment (Figure 2AGo), oligonucleotides were formed by N-acetylcysteine in the presence of Cu(II), indicating breakage of the deoxyribose phosphate backbone. The amount of oligonucleotides was increased by piperidine treatment (Figure 2BGo). Since the altered base is readily removed from its sugar by the piperidine treatment, it is considered that the base modification was induced by N-acetylcysteine in the presence of Cu(II). N-acetylcysteine did not cause DNA damage in the presence of other metal ions [Fe(III), Co(II), Ni(II), Mn(II) or Mg(II); data not shown].



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  		Fig. 2. Autoradiogram of 32P-labeled DNA fragments treated with N-acetylcysteine in the presence of Cu(II). The reaction mixture contained the 32P-5'-end-labeled 211 bp fragment, 10 µM per base of sonicated calf thymus DNA, the indicated concentrations of N-acetylcysteine, 20 µM CuCl2 and 5 µM DTPA in 200 µl of 10 mM phosphate buffer at pH 7.8. After the incubation at 37°C for 60 min, followed by piperidine treatment (B) or without piperidine treatment (A), the treated DNA fragments were electrophoresed on an 8% polyacrylamide–8 M urea gel (12x16 cm), and the autoradiogram was obtained by exposing X-ray film to the gel.

 
Effects of hydroxyl free radical (OH·) scavengers, catalase and bathocuproine on DNA damage induced by N-acetylcysteine in the presence of Cu(II)
Figure 3Go shows the effects of OH· scavengers, catalase and bathocuproine on DNA damage induced by N-acetylcysteine in the presence of Cu(II). Typical OH· scavengers, ethanol (lane 3), mannitol (lane 4) and sodium formate (lane 5), showed little or no inhibitory effect on the DNA damage. Methional (lane 6) inhibited the DNA damage. Inhibition of the DNA damage by catalase (lane 7) and bathocuproine (lane 8), a Cu(I)-specific chelator, suggests the involvement of H2O2 and Cu(I). SOD did not inhibit DNA damage by N-acetylcysteine plus Cu(II) (lane 9).



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  		Fig. 3. Effects of OH· scavengers, SOD, catalase and bathocuproine on DNA damage induced by N-acetylcysteine in the presence of Cu(II). The reaction mixture contained the 32P-5'-end-labeled 211 bp fragment, 10 µM per base of sonicated calf thymus DNA, 50 µM N-acetylcysteine, 20 µM CuCl2, and scavenger in 200 µl of 10 mM sodium phosphate buffer at pH 7.8 containing 5 µM DTPA. Scavenger was added where indicated. After the incubation at 37°C for 60 min, followed by the piperidine treatment, the DNA fragments were analyzed by the method described in Figure 2Go. Lane 1, control; lane 2, no scavenger; lane 3, 0.8 M ethanol; lane 4, 0.1 M mannitol; lane 5, 0.1 M sodium formate; lane 6, 1.0 M methional; lane 7, 30 U catalase; lane 8, 50 µM bathocuproine ; lane 9, 30 U SOD.

 
Site specificity of DNA cleavage by N-acetylcysteine in the presence of Cu(II)
The patterns of DNA cleavage induced by N-acetylcysteine in the presence of Cu(II) were determined with DNA sequences by the Maxam–Gilbert procedure (27). The relative intensity of DNA cleavage obtained by scanning the autoradiogram with a laser densitometer is shown in Figure 4Go. N-acetylcysteine induced piperidine-labile sites frequently at thymine residues especially located 5' and/or 3' to guanine in DNA fragments obtained from the human p53 tumor suppressor gene (Figure 4AGo) and the c-Ha-ras-1 protooncogene (data not shown). Furthermore, when 50 µM Cu(II) in carbonate buffer was used, piperidine-labile sites occurred preferentially at the 5' site of GG sequence in addition to thymine residues (Figure 4BGo). Similar buffer effects were observed with DNA damage by endogenous reductants in the presence of Cu(II) (29).



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  		Fig. 4. Site specificity of DNA cleavage induced by N-acetylcysteine in the presence of Cu(II). (A) The 32P-5'-end-labeled 211 bp fragment obtained from the human p53 in 200 µl of 10 mM sodium phosphate buffer at pH 7.8 containing 5 µM DTPA and 10 µM per base of sonicated calf thymus DNA was incubated with 50 µM N-acetylcysteine plus 20 µM CuCl2 at 37°C for 60 min. (B) The 32P-5'-end-labeled 226 bp fragment obtained from the c-Ha-ras-1 protooncogene in 200 µl of 10 mM sodium carbonate buffer at pH 7 containing 5 µM DTPA and 10 µM per base of sonicated calf thymus DNA was incubated with 100 µM N-acetylcysteine plus 50 µM CuCl2 at 37°C for 60 min. After the piperidine treatment, the DNA fragment was electrophoresed on an 8% polyacrylamide–8 M urea gel and the autoradiogram was obtained by exposing film to the gel.

 
Formation of 8-oxodG in calf thymus DNA by N-acetylcysteine in the presence of Cu(II)
8-oxodG is considered to be one of the DNA products generated by the reaction with reactive oxygen species. In the presence of Cu(II), the amount of 8-oxodG increased with N-acetylcysteine concentration as low as 50 µM (Figure 5Go). The amount of 8-oxodG decreased at 100 µM N-acetylcysteine. The formation of 8-oxodG was increased ~3.7-fold with DNA denaturation (Figure 5Go).



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  		Fig. 5. Formation of 8-oxodG induced by N-acetylcysteine in the presence of Cu(II). Calf thymus DNA (50 µM per base) was incubated with N-acetylcysteine and 20 µM CuCl2 in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA at 37°C for 2 h. For the experiment with denatured DNA, calf thymus DNA was treated at 90°C for 10 min and quickly chilled before the addition of N-acetylcysteine and Cu(II). After ethanol precipitation, the DNA was subjected to enzyme digestion and analyzed by HPLC–ECD as described in Materials and methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we have demonstrated that the content of 8-oxodG in HL-60 cells was increased by the N-acetylcysteine treatment. On the other hand, the content of 8-oxodG in HP100 cells was not increased by the N-acetylcysteine treatment. The catalase activity of HP100 cells was higher than that of HL-60 cells (19). Therefore, it is considered that generation of H2O2 plays an important role in N-acetylcysteine-induced 8-oxodG formation. Numerous studies have indicated that the formation of 8-oxodG causes misreplication of DNA that may lead to mutation or cancer (17,18). The formation of 8-oxodG in cellular DNA induced by N-acetylcysteine is noteworthy in relation to the report that 8-oxodG results in GT transversions which are frequently found in tumor relevant genes (30).

To clarify the mechanism of cellular DNA damage, we investigated DNA damage induced by N-acetylcysteine using 32P-labeled DNA fragments and calf thymus DNA. DNA damage including base modification such as 8-oxodG was efficiently induced by N-acetylcysteine in the presence of Cu(II). N-acetylcysteine plus Cu(II) induced piperidine-labile sites frequently at thymine and guanine residues, although the site specificity of DNA damage depended on reaction conditions. The site specificity cannot be explained by OH·. It is generally considered that OH· causes DNA cleavage at every nucleotide with no marked site specificity (29,31,32). Catalase and bathocuproine completely inhibited DNA damage induced by N-acetylcysteine plus Cu(II), indicating the participation of H2O2 and Cu(I) in the DNA damage. Typical OH· scavengers showed little or no inhibitory effect on the DNA damage, suggesting that OH· might not play an important role. Methional, however, inhibited the DNA damage, because it can scavenge not only OH· but also other radicals such as metal–oxygen complexes (33). On the basis of these results, a possible mechanism of DNA damage by N-acetylcysteine in the presence of Cu(II) has been proposed in Figure 6Go. It is reasonable to speculate that N-acetylcysteine undergoes Cu(II)-mediated autoxidation to generate Cu(I) and the thiyl radical of N-acetylcysteine. Cu(I) reacts with O2 to generate O2– and subsequently H2O2. Generated Cu(I) binding to DNA interacts with H2O2, resulting in the formation of a reactive complex, such as DNA–Cu(I)OOH. The complex DNA–Cu(I)OOH may be considered to be a bound hydroxyl radical, which can release OH· causing DNA damage. The OH· released from a bound hydroxyl radical immediately attacks an adjacent constituent of DNA before it can be scavenged by OH· scavengers (34). Therefore, it is considered that H2O2 is activated by endogenous metal ions such as Cu(I) to cause damage to cellular DNA.



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  		Fig. 6. A possible mechanism of oxidative DNA damage induced by N-acetylcysteine in the presence of Cu(II).

 
The binding of copper to DNA and/or protein in chromatin is proposed to serve physiological functions (35), whereas copper bound to DNA and/or protein may provide an adventitious site for deleterious redox reactions (36). Recently, Chiu et al. (37) have reported that copper ion binds to non-histone proteins, leading to ascorbate-mediated DNA damage, which is much stronger than that in the case of iron. It is reported that Cu(II) induced endogenous reductant-dependent DNA damage more efficiently than Fe(III) (29,34,38). The present work suggests that copper is an important factor to oxidative DNA damage induced by N-acetylcysteine.

A recent epidemiological investigation in USA and Finland failed to confirm effective chemoprevention with antioxidants (15,16). The incidence of lung cancer in male smokers was unaffected by {alpha}-tocopherol supplementation and was unexpectedly increased by ß-carotene supplementation. In addition, several studies employing laboratory animals have also demonstrated that vitamin A can induce or promote tumor formation (39,40). Several reports have indicated that {alpha}-tocopherol can act as a carcinogen, at both the initiation and promotion stages (41–44), and can induce metal-mediated DNA damage (45).

After failure in cancer chemoprevention by ß-carotene and {alpha}-tocopherol, N-acetylcysteine is considered to be the most efficacious cancer chemopreventive agent and it is currently undergoing clinical trial for the prevention of cancer (1,11,46). Izzotti et al. (47) reported that N-acetylcysteine was quite efficient in inhibiting oxidative DNA modifications. However, numerous studies have suggested that many antioxidants can exhibit prooxidant behavior under certain conditions (13,45,48). Furthermore, a number of antioxidants may have both anti-carcinogenic and carcinogenic effects (13–15,45). In the present study, we have found that N-acetylcysteine induces oxidative DNA damage not only in isolated DNA but also in cellular DNA. Relevantly, Sprong et al. (49) have reported that low-dose N-acetylcysteine protects against endotoxin-mediated oxidative stress by scavenging H2O2, while higher doses may have the opposite effect. N-acetylcysteine concentrations tested in this study can be considered high relative to concentrations likely to result from the use of the compound in chemoprevention trials to humans (600 mg/daily). However, the concentration of N-acetylcysteine (1 mM) causing oxidative DNA lesion is lower than the concentration (10 mM) which is reported to protect against oxidative stress in the cells (8) and may be considered roughly comparable with the concentrations showing the chemopreventive effect of N-acetylcysteine in animals (1–2 g/kg body weight) (5,6,8). There is growing evidence that compounds that are antioxidants at some concentrations become prooxidants at other concentrations. Similarly, it is reasonably considered that N-acetylcysteine may have the dual function of carcinogenic and anti-carcinogenic potentials. This work requires further studies on safety and risk assessment of N-acetylcysteine.


   Acknowledgments
 
This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and a Grant-in-Aid from the Mie Medical Research Foundation.


   Notes
 
1 To whom correspondence should be addressed E-mail: kawanisi{at}doc.medic.mie-u.ac.jp Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 22, 1999; revised April 26, 1999; accepted April 27, 1999.


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