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Acrylonitrile (ACN) is widely used as a monomer in the polymer industry. Studies on carcinogenicity in rats exposed to ACN showed increased incidences...
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Chem. Res. Toxicol. 2001, 14, 1421-1427

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Acrylonitrile Enhances H2O2-Mediated DNA Damage via Nitrogen-Centered Radical Formation Mariko Murata, Shiho Ohnishi, and Shosuke Kawanishi* Department of Hygiene, Mie University School of Medicine, Tsu, Mie 514-8507, Japan Received April 19, 2001

Acrylonitrile (ACN) is widely used as a monomer in the polymer industry. Studies on carcinogenicity in rats exposed to ACN showed increased incidences of tumors including glial cell tumors of central nervous system and increased production of 8-oxo-7,8-dihydro-2′deoxyguanosine (8-oxo-dG) in glial cells. Using a high performance liquid chromatograph equipped with an electrochemical detector, we revealed that ACN enhanced the formation of 8-oxo-dG induced by H2O2 and Cu(II) whereas ACN itself did not cause DNA damage. The enhancing effect of ACN was much more efficient in the double-stranded DNA than that in the single-stranded DNA. Experiments with 32P-labeled DNA revealed that addition of ACN enhanced the site-specific DNA damage at guanines, particularly at 5′-site of the GG and GGG sequences while H2O2/Cu(II) induced piperidine-labile sites at thymine, cytosine, and guanine residues. An electron spin resonance spectroscopy using R-(4-pyridyl-1-oxide)-N-tert-butylnitrone showed that a nitrogen-centered radical was generated from ACN in the presence of H2O2 and Cu(II). It is considered that ACN enhances H2O2-mediated DNA damage via nitrogen-centered radical formation. We will discuss the mechanism of the enhancing effect on oxidative DNA damage in relation to expression of ACN carcinogenicity.

Introduction Acrylonitrile (ACN)1 is a monomer used in the synthesis of acrylic fibers, resins, rubbers, and plastics, and it is also present in cigarette smoke and has been detected at low levels in ambient air and water (1). Concerning carcinogenicity of ACN to humans, previous cohort studies have provided the controversial results (1-9). Occupational exposure to ACN in viscose rayon plant induced considerable genotoxic consequences (10). Studies on carcinogenicity in rats exposed to ACN showed increased incidences of glial cell tumors of the central nervous system, tumors of the gastrointestinal tract, malignant mammary tumors and Zymbal gland carcinomas (1). ACN is mutagenic, especially after bioactivation by a microsomal system (1, 2). ACN is metabolized to the reactive cyanoethylene oxide by P450 2E1 and other forms of human P450 (11, 12). Since formation of DNA adducts with ACN in vitro is strongly increased by formation of its epoxide, it is likely that the genotoxicity of ACN is mediated by the epoxide. Cyanoethylene oxide possesses strong electrophilic properties and is reactive toward nucleophilic sites of cellular macromolecules such as proteins, DNA, and RNA (13). However, DNA alkylation in vivo was not found or present only at very low levels in liver of rats treated with ACN (14). Therefore, clear evidence for the DNA adduct formation has not been provided, and it is speculated that alkylation by cyanoethylene oxide does not occur readily in vivo (15). On the * To whom correspondence should be addressed. Phone and Fax: +81 (59) 231-5011. E-mail: [email protected]. 1 Abbreviations: ACN, acrylonitrile; MeCN, acetonitrile; HPLC, high-performance liquid chromatography; HPLC-ECD, HPLC equipped with an electrochemical detector; 8-oxo-dG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; Fpg, formamidopyrimidine-DNA glycosylase; ESR, electron spin resonance; DTPA, diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid; POBN, R-(4-pyridyl-1-oxide)-N-tert-butylnitrone.

other hand, many studies revealed the induction of oxidative stress by ACN, as shown by increased production of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in rat brain and in glial cells (16-19). These studies suggest the possibility that ACN-induced tumors may arise from a mode of action involving 8-oxo-dG. However, the mechanisms of inducing 8-oxo-dG formation remain to be clarified. As one of the methods to approach clarifying the mechanisms of oxidative DNA damage by ACN, we investigated formation of 8-oxo-dG in calf thymus DNA using a high-performance liquid chromatograph equipped with an electrochemical detector (HPLC-ECD). Formation of 8-oxo-dG is a relevant indicator of oxidative base damage and causes DNA misreplication that might lead to mutation or cancer (20). We also investigated sequencespecificity of DNA damage by ACN in the presence of H2O2 and Cu(II) using 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. The p53 gene is known to be the targets for chemical carcinogens (21). Furthermore, to clarify the mechanism of DNA damage, we examined the generation of radicals from ACN in comparison with MeCN by an electron spin resonance (ESR) spectroscopy.

Materials and Methods Materials. Restriction enzymes (HindIII, AvaI, and PstI) and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). Restriction enzymes (SmaI, EcoRI, ApaI, and StyI) and calf intestine phosphatase were from Roche Molecular Biochemicals (Mannheim, Germany). [γ-32P]ATP (222 TBq/mmol) was from New England Nuclear (Boston, MA). Diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were from Dojin Chemicals Co. (Kumamoto, Japan). MeCN, copper(II) chloride dihydrate, ethanol, dCMP, and dTMP were from Nacalai Tesque, Inc. (Kyoto,

10.1021/tx010081s CCC: $20.00 © 2001 American Chemical Society Published on Web 09/27/2001

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Japan). Calf thymus DNA, dAMP, and dGMP were from Sigma Chemical Co. (St Louis, MO). Nuclease P1 was from Yamasa Shoyu Co. (Chiba, Japan). ACN and R-(4-pyridyl-1-oxide)-N-tertbutylnitrone (POBN) were from Tokyo Kasei Co. (Tokyo, Japan). Acrylamide, bisacrylamide, piperidine, and H2O2 (30%) were from Wako Chemicals Co. (Osaka, Japan). Formamidopyrimidine-DNA glycosylase (Fpg, 20 000 units/mg from Escherichia coli) was from Trevigen Inc. (Gaithersburg, MD). Analysis of 8-oxo-dG Formation in Calf Thymus DNA. Calf thymus DNA (100 µM/base) was incubated with indicated concentrations of H2O2 and ACN in the presence of CuCl2 in 4 mM sodium phosphate buffer (pH 7.8) at 37 °C for the indicated duration. After ethanol precipitation, the DNA was digested to the nucleosides by incubation with nuclease P1 and alkaline phosphatase and analyzed with an HPLC-ECD as described previously (22). Preparation of 32P-5′-End-Labeled DNA Fragments. DNA fragments were obtained from the human p53 tumor suppressor gene (23) and the c-Ha-ras-1 protooncogene (24). The DNA fragment of the p53 tumor suppressor gene was prepared from pUC18 plasmid. A singly 32P-5′-end-labeled 343-bp fragment (StyI 13160-EcoRI* 13507) was obtained according to the method described previously (25). DNA fragments were also prepared from plasmid pbcNI, which carries a 6.6-kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene. A singly labeled 337-bp fragment (PstI 2345AvaI* 2681) was obtained according to the method described previously (26). The asterisk indicates 32P-labeling. Detection of Damage to 32P-5′-End-Labeled DNA. The reaction mixture in a 1.5-mL microtube contained indicated concentrations of ACN, H2O2, CuCl2, and 32P-5′-end-labeled DNA fragment in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After incubation at 37 °C for the indicated duration, the DNA was treated with 6 units of Fpg protein in 10 µL of the reaction buffer [10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA, and 0.1 mg/mL BSA] at 37 °C for 2 h. Fpg protein catalyzes the excision of 8-oxo-7,8dihydro-2′-deoxyguanine (27). In certain experiments, the DNA fragments were heated at 90 °C in 1 M piperidine for 20 min, instead of Fpg treatment. After ethanol precipitation, the DNA fragments were electrophoresed and the autoradiogram was obtained by exposing X-ray film to the gel as described previously (26). Determination of Site Specificity of Damage to 32P-5′End-Labeled DNA. 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 (28) using a DNA-sequencing system (LKB 2010 Macrophor). Oligonucleotides cleaved at thymine and cytosine residues (T + C) and those at guanine and adenine residues (G + A) were produced by the chemical reactions of DNA fragments with hydrazine and formate, respectively. The (T + C) and (G + A) as standard markers and ACN-treated DNA samples were simultaneously electrophoresed using a DNAsequencing system, and the autoradiogram was obtained. A laser densitometer (LKB 2222 UltroScan XL) was used for the measurement of the relative amounts of oligonucleotides from treated DNA fragments. The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides and standard markers. ESR Spectra Measurements. ESR spectra were measured at room temperature (25 °C) by using a JES-TE-100 (JEOL, Tokyo, Japan) spectrometer with 100-kHz field. Spectra were recorded with microwave power of 4 mW, modulation amplitude of 0.02 mT, receiver gain of 32, time constant 0.3 s and sweep time 8 min. The magnetic fields were calculated by the splitting of Mn(II) in MgO (∆H3-4 ) 8.69 mT). POBN was used as a radical trapping reagent. In certain experiment, mononucleotides were added to the reaction mixtures.

Figure 1. Formation of 8-oxo-dG by H2O2 and Cu(II) in the presence and absence of ACN. The reaction mixture contained calf thymus DNA (100 µM/base), 20 µM CuCl2 and indicated concentrations of H2O2 and ACN in 400 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 2 µM DTPA. After incubation at 37 °C for 30 min, DNA fragment was enzymatically digested into nucleosides, and 8-oxo-dG formation was measured with an HPLC-ECD as described in the Materials and Methods.

Results Formation of 8-oxo-dG by ACN in the Presence of H2O2 and Cu(II). Using HPLC-ECD, we measured 8-oxo-dG content in calf thymus DNA treated with various concentrations of H2O2 plus Cu(II) in the presence and absence of ACN (Figure 1). The amount of Cu(II)mediated 8-oxo-dG formation increased with increasing concentration of H2O2. The addition of ACN enhanced the formation of 8-oxo-dG by H2O2 plus Cu(II) in a dosedependent manner. Comparison of Enhancing Effects of ACN and MeCN on 8-oxo-dG Formation in Double- and SingleStranded DNA. To examine the relation of DNA conformation to the enhancing effect of 8-oxo-dG formation by ACN, both double- and single-stranded DNA were used (Figure 2). In single-stranded DNA, H2O2/Cu(II) induced 8-oxo-dG formation more efficiently than that in native double-stranded DNA when ACN was absent. However, the enhancing effect of ACN was much more efficient in the double-stranded DNA, and the content of 8-oxo-dG was higher than that in the single-stranded DNA by addition of 0.5 and 1.0% ACN. When MeCN (CH3-CtN) was used instead of ACN (CH2dCH-CtN), the enhancing effect on 8-oxo-dG formation was also observed, especially in double-stranded DNA, but MeCN was less effective than that of ACN. Damage to 32P-Labeled DNA Fragments Induced by ACN in the Presence of H2O2 and Cu(II). The extent of damage to isolated DNA induced by ACN followed by Fpg treatment, was estimated by gel electrophoretic analysis (Figure 3). Oligonucleotides were detected on the autoradiogram as a result of DNA cleavage. ACN alone did not caused DNA damage even in the presence of Cu(II) (data not shown). H2O2 slightly caused Cu(II)-mediated DNA damage under the condition used. However, addition of ACN markedly enhanced DNA damage by H2O2 and Cu(II). The DNA damage increased with increasing concentration of ACN. MeCN also enhanced it but a little. In the cases of hot piperidine treatment, enhancing effects of ACN on H2O2/Cu(II)induced DNA damage were also observed (data not shown).

Acrylonitrile Enhances H2O2-Mediated DNA Damage

Figure 2. Comparison of effects between ACN and MeCN on the formation of 8-oxo-dG by H2O2 and Cu(II). The reaction mixture contained calf thymus DNA (100 µM/base), 20 µM H2O2, 20 µM CuCl2 and indicated concentrations of ACN (squares) and MeCN (circles) in 400 µL of 4 mM phosphate buffer (pH 7.8) containing 2 µM DTPA. Native double-stranded DNA (open) and denatured single-stranded DNA (crossed) were used to examine an effect of DNA conformation. For DNA denaturation, DNA was treated at 90 °C for 5 min and quickly chilled on ice before incubation. After incubation at 37 °C for 30 min, DNA fragment was enzymatically digested into nucleosides, and 8-oxo-dG formation was measured with an HPLC-ECD as described in the Materials and Methods.

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ments treated with H2O2, CuCl2, and ACN, followed by piperidine or Fpg treatments were electrophoresed, and an autoradiogram was obtained. Scanning the autoradiogram with a laser densitometer gave the results in Figures 4 and 5. Followed by piperidine treatment, H2O2 and Cu(II) induced weakly DNA cleavage at thymine, cytosine, and guanine residues under the condition used (Figure 4A). Addition of ACN strongly enhanced piperidine-labile sites at guanine residues, especially at the 5′ site of GG, GGG, and at the second guanine of GGGG sequences (Figure 4B). When ACN was added into the mixture of H2O2 and CuCl2, Fpg-catalyzed DNA cleavage was observed at the guanines particularly at the 5′-site of the GG and GGG sequences in double-stranded DNA (Figure 5A). The DNA damage decreased in singlestranded DNA (Figure 5B). Formation of the Radicals from ACN and MeCN in the Presence of H2O2 and Cu(II). An ESR spectrum of spin adduct was observed when POBN was added to a mixture solution of H2O2, Cu(II), and ACN (Figure 6C). It is estimated by reference to the reported constants (29) that this spectrum is a nitrogen-centered adduct of POBN with splitting constants aN ) 1.53 mT, aH(β) ) 0.22 mT, and aN(β) ) 0.22 mT. When one of three was omitted, the signal was not observed (Figure 6, panels A and B). To examine the reactivity of the radicals to DNA, the effects of mononucleotides on the radical formation were observed. Addition of dGMP induced a decrease in the signal (Figure 6D), whereas dAMP, dTMP, and dCMP had no significant effects on the intensity of the signals (data not shown). By the addition of MeCN, a similar but small signal was observed (Figure 6E). On the basis of this result and effects of ACN and MeCN on 8-oxo-dG formation, it may be concluded that the radicals observed here are responsible for oxidative DNA damage.

Discussion

Figure 3. Autoradiogram of 32P-DNA fragments incubated with ACN in the presence of H2O2 and Cu(II). The reaction mixture contained 32P-5′-end-labeled 343-bp DNA fragments, indicated concentrations of ACN or MeCN, 20 µM CuCl2 and 20 µM H2O2 in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA was incubated at 37 °C for 10 min. After ethanol precipitation, DNA was treated with 6 units of Fpg protein at 37 °C for 2 h as described in the Materials and Methods. Then, the DNA fragments were electrophoresed and the autoradiogram was obtained.

Effect of ACN on Site Specificity of H2O2 Plus Cu(II)-Induced DNA Damage. The DNA 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 (28), as shown in Figures 4 and 5. The 32P-labeled DNA frag-

This study revealed that ACN enhanced oxidative DNA damage induced by H2O2 and Cu(II), especially in doublestranded DNA. ESR showed that nitrogen-centered radicals were generated from ACN in the presence of H2O2 and Cu(II). The mechanism for the formation of the radicals can be envisioned as follows. Since Cu(I)OOH, which is formed from H2O2 and Cu(II), has a property like ‚OH, Cu(I)OOH may react with ACN (CH2dCHCtN) to form CH2dCH-C(OH)dN‚. Like ACN, when MeCN (CH3-CtN) reacted with Cu(I)OOH, CH3-C(OH)d N‚ could be formed, but only in small amounts. This difference may be explained by assuming that the vinyl group can stabilize the nitrogen-centered radicals more efficiently than the methyl group. The extent of 8-oxodG formation was increased by ACN added to the reaction system of H2O2/Cu(II) more efficiently than that by MeCN. Therefore, it is speculated that the nitrogencentered radicals participate in the increase of 8-oxo-dG formation. Furthermore, experiments with 32P-labeled DNA revealed that addition of ACN enhanced the DNA damage at guanines, particularly at the 5′-site of GG and GGG sequences while H2O2/Cu(II) induced piperidinelabile sites at thymine, cytosine, and guanine residues. Because of piperidine-resistant property of 8-oxo-dG, it is speculated that the damaged guanine residues are piperidine-labile lesions such as oxazolone, another oxidative product of guanine (30). Recently, it is demonstrated that Fpg proteins can recognize and excise

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Figure 4. Effects of ACN on site specificity of DNA cleavage induced by H2O2/Cu(II). The reaction mixture containing the 32P-5′end-labeled 337-bp DNA fragment, calf thymus DNA (10 µM/base), 100 µM H2O2, and 20 µM CuCl2 (A) or 100 µM H2O2, 20 µM CuCl2, and 0.5% ACN (B) in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA was incubated at 37 °C for 1 h. The DNA fragments were treated with 1 M piperidine at 90 °C for 20 min and then DNA fragments were electrophoresed with the (T + C) and (G + A) standard markers, which were produced by the chemical reactions of the Maxam-Gilbert procedure and the autoradiograms were scanned with a laser densitometer. The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides. The horizontal axis shows the nucleotide number of the human c-Ha-ras-1 protooncogene.

oxazolone in addition to 8-oxo-7,8-dihydro-2′-deoxyguanine (31). The experiment treated with Fpg protein revealed that the radicals from ACN participated in the oxidative guanine lesions including 8-oxo-7,8-dihydro-2′deoxyguanine and oxazolone, predominantly at the 5′site of GG and GGG sequences in double-stranded DNA. When denatured single-stranded DNA was used, the intensity of DNA damage decreased. Recently, molecular orbital calculations have revealed that a large part of highest occupied molecular orbital (HOMO) is located on the 5′-G of GG and GGG sequences in double-stranded DNA (32, 33), and therefore, this guanine is more likely to be oxidized than single guanine residue in double-

stranded DNA and any guanine residue in singlestranded DNA. HOMO distribution could explain the polyguanine-specific DNA damage induced by less reactive species such as nitrogen-centered radicals. Organic radicals are less reactive than ‚OH and cause DNA damage in site-specific manner, as we previously reported (34-36). ‚OH can react with all biomolecules, and the lifetime of ‚OH is very short (37), whereas the nitrogencentered radicals may have longer lifetime than ‚OH. This may be supported by the report that the radicals formed from amines have long lifetimes such as 5-50 µs (38). The enhancing effect of ACN on DNA damage by H2O2 and Cu(II) can be explained by assuming that the

Acrylonitrile Enhances H2O2-Mediated DNA Damage

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Figure 5. Site specificity of DNA cleavage induced by H2O2, Cu(II), and ACN, followed by Fpg treatment. The reaction mixture containing the 32P-5′-end-labeled 343-bp DNA fragment, 20 µM CuCl2, 1% ACN, and 20 µM H2O2 in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA was incubated at 37 °C for 10 min, and treated with Fpg protein by the method described in the legend to Figure 3. Native double-stranded DNA (A) and denatured single-stranded DNA (B) were used to examine an effect of DNA conformation. The preferred cleavage sites were determined by the method described in the legend to Figure 4. The horizontal axis shows the nucleotide number of the human p53 tumor suppressor gene.

nitrogen-centered radicals have more opportunities for damaging DNA than ‚OH. There are many mechanistic ideas proposed for ACNcarcinogenesis in rats. Concerning DNA adduct formation, ACN and its metabolite cyanoethylene oxide interacted with DNA resulting in DNA alkylation (14). With respect to the oxidative DNA modification by ACN, the formation of reactive oxygen species and subsequent

oxidative damage may occur mainly through three pathways. One is that cyanide produced from ACN interfered with electron flow through respiratory chain in mitochondria, leading to production of reactive oxygen species and lipid peroxidation (18, 39). The second is the idea concerning inhibitory effects of ACN on antioxidant enzymes such as catalase, SOD (17, 18). The third is that antioxidant macromolecules such as GSH, cysteine, and

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Figure 6. ESR spectra of radicals derived from ACN and MeCN in the presence of H2O2 and Cu(II). The reaction mixture contained 100 mM POBN, 5 mM H2O2, 20 µM CuCl2, 10% ACN, or MeCN in 100 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA, and the spectra were recorded by ESR spectroscopy, immediately after mixture. Mononucleotides (500 µM) were added to the reaction mixture, where indicated. (A) H2O2 + ACN, (B) Cu(II) + ACN, (C) H2O2 + Cu(II) + ACN, (D) dGMP + H2O2 + Cu(II) + ACN, (E) H2O2 + Cu(II) + MeCN.

vitamin E, were consumed by ACN and/or its metabolites for their detoxication (16-18, 40). All of these toxicities of ACN result in failure of protection against noxious effects of reactive oxygen species generated in vivo. We showed here that ACN enhanced H2O2-mediated DNA damage via nitrogen-centered radical formation in vitro. The concentrations of ACN, Cu(II) and H2O2 used in this study were high when extrapolated to in vivo. However, this study may serve another novelty explanation for the mechanism of 8-oxo-dG formation. Reactive oxygen species are ubiquitous and occur naturally in all aerobic species, coming from both endogenous and exogenous sources (41-43). Especially, mitochondrial oxidative phosphorylation is the major source of the reactive oxygen species (44). Therefore, ACN may enhance endogeous oxidative stress in addition to other cytotoxicity, though ACN itself dose not have ability of inducing oxidative DNA damage. Enhancing effects of ACN on oxidative DNA damage via nitrogen-centered radical formation is noteworthy in relation to its carcinogenic effects to rats.

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