ToxSci Advance Access originally published online on April 22, 2008
Toxicological Sciences 2008 104(2):274-282; doi:10.1093/toxsci/kfn081
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Combined Ascorbic Acid and Sodium Nitrite Treatment Induces Oxidative DNA Damage-Associated Mutagenicity In Vitro, but Lacks Initiation Activity in Rat Forestomach Epithelium
* Division of Pathology
Division of Genetics & Mutagenesis, National Institute of Health Sciences, Setagaya-Ku, Tokyo 158-8501, Japan
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183-8509, Japan
2 To whom correspondence should be addressed at Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-Ku, Tokyo 158-8501, Japan. Fax: +81-3-3700-1425. E-mail: nishikaw{at}nihs.go.jp
Received December 6, 2007; accepted April 11, 2008
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ABSTRACT |
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Combination treatment with sodium nitrite (NaNO2) and ascorbic acid (AsA) is well known to promote forestomach carcinogenesis in rats and weakly enhance esophageal carcinogenesis under acid reflux conditions. Nitric oxide generation and oxidative DNA damage are considered to be related to the enhancement of carcinogenesis. The purpose of the present study was to investigate whether oxidative DNA damage-associated genotoxicity and tumor initiating potential are involved in the carcinogenesis. In the bacterial reverse mutation assay using Escherichia coli deficient in the mutM gene encoding 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase, the combination with NaNO2 and AsA increased the mutation frequency dramatically, slight increase being evident in the parental strain. In vivo, a significant increase in 8-OHdG levels in the rat forestomach epithelium occurred at 24 h after combined treatment. Six-week-old F344 male rats were given drinking water containing 0.2% NaNO2 and a diet supplemented with 1% AsA in combination, or the chemicals individually or basal diet alone for 12 weeks. After an interval of 2 weeks, they received 1% butylated hydroxyanisole in the diet for promotion until the end of weeks 52 and 78. Although one squamous cell carcinoma was observed in the combined group, there was no significant variation in tumor development among the groups. The study indicated that the combination of NaNO2 with AsA induces genotoxicity due to oxidative DNA damage in vitro, and elevates 8-OHdG levels in the forestomach epithelium, but lacks initiating activity in the rat two-stage carcinogenesis model.
Key Words: ascorbic acid; nitrite; 8-hydroxydeoxyguanosine (8-OHdG); mutM-deficient E. coli strain; genotoxicity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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When antioxidants such as ascorbic acid (AsA) were simultaneously given with sodium nitrite (NaNO2) to rats, forestomach carcinogenesis was strongly enhanced after initiation with carcinogens, and forestomach papillomas were induced 51 weeks after the treatment without prior initiation, indicating carcinogenic potential for the combination treatment (Yoshida et al., 1994
; Okazaki et al., 2006). Interestingly, our recent study demonstrated that esophageal carcinogenesis was also enhanced by coadministration of AsA and NaNO2 after initiation of a carcinogen in a rat acid reflux esophagitis model (Kuroiwa et al., 2008). Thus, expansion of the target site to esophagus makes it imperative to elucidate the mode of action underlying the carcinogenesis in terms of the risk assessment for humans.
NaNO2 readily reacts with AsA to generate nitric oxide (NO) under acidic conditions (Iijima and Shimosegawa 2006; Samouilov et al., 1998) and we have shown generation of NO from NaNO2 with AsA using electron spin resonance (ESR) in vitro (Okazaki et al., 2006). It is generally accepted that excessive NO reacts with superoxide to form peroxynitrite, which has been shown to activate poly(ADP-ribose) polymerase and deplete cellular contents of NAD+ (nicotinamide adenine dinucleotide) and ATP, resulting in necrotic cell death (Pryor and Squadrito, 1995; Virag et al., 2003). In the rat forestomach, co-administrated AsA and NaNO2 induce ulcerative lesions in the mucosa, and regenerative hyperplasia in the neighboring epithelium (Okazaki et al., 2006). Therefore, it has been considered that cell proliferation following tissue injury causes tumor promotion in the forestomach and esophagus (Kuroiwa et al., 2007; Okazaki et al., 2006).
Peroxinitrite is also known to have a strong oxidative capacity and to induce oxidative DNA damage in cells, in addition to the action of nitration (Beckman and Koppenol, 1996; Ducrocq et al., 1999; Jay-Gerin and Ferradini, 2000; Radi et al., 1991), with production of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage (Szabó and Ohshima, 1997; Yermilov et al., 1996). In fact, increase in the level of nuclear 8-OHdG was observed in the rat forestomach epithelium after coadministration of NaNO2 and catechol/green tea catechin, and peroxinitrite was considered the cause of the 8-OHdG elevation in the catechol case (Ishii et al., 2006; Kuroiwa et al., 2007). Likewise, with NaNO2 and AsA in combination, a tendency for increase was also observed (Okazaki et al., 2006). 8-OHdG is one of the most stable products of oxidative damage to DNA, pairing with adenine during DNA replication, thereby causing G:C to T:A transversions (Cheng et al.,1992; Moriya, 1993; Shibutani et al., 1991; Wood et al., 1990). A variety of chemicals induce 8-OHdG in DNA due to generation of oxygen species (Kasai and Nishimura, 1991; Kasai et al., 1991) and it is postulated that this oxidized base is responsible for mutagenicity and carcinogenicity in some cases (Le Page et al., 1995; Nakae et al., 2002; Umemura et al., 2007). Thus, it is possible that the combination with antioxidant and nitrite has tumor initiation potential due to oxidative DNA damage.
The purpose of this study was to investigate whether tumor initiating potential through oxidative DNA damage participates in the forestomach carcinogenesis induced by the combination with NaNO2 and AsA. Firstly, in order to assess mutagenicity due to oxidative DNA damage caused by the combination of AsA and nitrite, we disrupted the mutM gene of Escherichia coli strain WP2 uvrA/pKM101 and compared the mutagenicity of the two chemicals between the mutM-deficient and -proficient strains. The mutM gene encodes 8-OHdG DNA glycosylase involved in repair of oxidized bases in DNA. Secondly, NO generation was determined by ESR analysis in the reaction of AsA with NaNO2 under the same pH condition as in the mutagenicity test. Thirdly, to confirm the sustained occurrence of DNA base oxidation, 8-OHdG levels were measured in the forestomach of rats treated with AsA and NaNO2 in combination for up to 2 weeks. Finally, to investigate in vivo tumor initiation activity of the combined treatment, we performed a two-stage rat forestomach carcinogenesis study using butylated hydroxyanisole (BHA) as a tumor promoter.
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MATERIALS AND METHODS |
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Construction of an E. coli strain deficient in the mutM gene.
The mutM gene of strain WP2 uvrA was disrupted by P1 transduction using strain E. coli BH20 carrying a mutM1::KmR allele as a donor (Miller, 1992
). Kanamycin resistant (KmR) colonies were selected on LB plates containing kanamycin and the resulting strain was named as YG5189. Plasmid pKM101, whose selection marker is ampicillin resistance (ApR), was introduced into strain YG5189 by conjugation from Salmonella typhimurium TA100. The resulting ApR and KmR conjugant was named YG5190, a mutM-deficient derivative of WP2 uvrA/pKM101.
Bacterial reverse mutation test (Ames test).
Mutagenicity of the combination with AsA and NaNO2 was assayed in a bacterial reverse mutation assay using E. coli WP2 uvrA/pKM101 and a mutM-deficient derivative, that is, YG5190. The test was conducted by the pre-incubation method in the presence or the absence of S9 mix with modification. Briefly, 0.05 ml of solution containing 100 µg of NaNO2, 0.5 ml of either 1/15M sodium potassium phosphate buffer (pH 7.4) or S9 mix and 0.1 ml of overnight culture of bacterial were mixed in a sterilized test tube. As a negative control, 0.05 ml of distilled water was added instead of NaNO2 solution. After incubation on ice for 20 min, 0.05 ml of solution containing various amounts of AsA was added and the mixture was incubated for 20 min at 37°C. The amounts of AsA added were 0, 2500, 5000, 7500, or 10,000 µg per tube (or plate). Top agar containing a limited amount of L-tryptophan was added to the tube and the whole mixture was spread on minimal agar plates. The plates were incubated for 2 days at 37°C for selection of Trp+ revertants. Experiments were repeated twice with two plates each, and the mean and the standard deviations (SD) were calculated. The pH of the bacterial culture media became 6.8 after addition of AsA (the final concentrations of AsA were approximately 20, 40, 60, and 80mM), and kept at 6.8 throughout the experimental period.
ESR analysis of NO generation by the reaction of NaNO2 with AsA.
Fe2+ complex with N-(dithiocarboxy)sarcosin(DTCS)-Na was used as an NO-specific trapping agent and NO formation was determined by detecting the characteristic triplet ESR signal (g = 2.04) resulting from the reaction with Fe(DTCS)2 complex and NO, as previously reported (Okazaki et al., 2006; Porasuphatana et al., 2001; Pou et al., 1999). ESR spectra were measured by a spectrometer JEOL JES-FA100 (JEOL, Tokyo, Japan) under following conditions: magnetic field, 329.7 ± 5 mT; power 9.0 mW; frequency, 9.4 GHz; modulation width, 0.06 mT; sweep time, 0.5 min; time constant, 0.03 s. The reaction procedure was as follows: one hundred micro liters of 1mM Fe(DTCS)2 complex were added to pH 6.8 sodium phosphate buffer, followed by addition of 100 µl of 100, 200, 300, or 400mM AsA, subsequently, 100 µl of 10mM NaNO2 was added. Each addition was performed at intervals of 15 s. This mixture was transferred to a quartz flat cell of 200 µl for ESR measurement after mixing for 10 min. The pH of the mixture was kept at 6.8 throughout the experimental procedure (data not shown).
Animals and chemicals for the in vivo studies.
Male 5-week-old F344 rats were purchased from SLC Japan (Shizuoka, Japan) and housed five animals per polycarbonate cage under specific pathogen-free standard laboratory conditions: room temperature, 23 ± 2°C; relative humidity, 60 ± 5%; with a 12:12-h light-dark cycle. After a 1-week acclimation period, the animals were used in the experiment. AsA, NaNO2, and BHA were purchased from Wako Pure Chemical Industries (Osaka, Japan), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). AsA and BHA were each mixed into Oriental CRF-1 powdered basal diet (Oriental Yeast, Tokyo, Japan) at 1 wt/wt % concentration and stored at 4°C in the dark before use. The diets were administered from a food containers replaced once a week. NaNO2 was dissolved in distilled water at 0.2 wt/vol% concentration immediately before use and the bottles were replaced twice a week. Food and water were available ad libitum. The doses of AsA and NaNO2 used in the present study were chosen based on previous studies (Okazaki et al., 2006; Yoshida et al., 1994).
Short-term study for measurement of forestomach 8-OHdG levels.
In the short-term study, a total of 76 rats at age 6 weeks were used. They were fasted for 16 h and then treated with basal diet alone, basal diet plus 0.2% NaNO2 in the drinking water, 1% AsA in the diet plus distilled water or 1% AsA in the diet plus 0.2% NaNO2 in the drinking water. Five animals each in the basal diet and NaNO2+AsA groups were sacrificed under ether anesthesia at 6 and 24 h, 3 days and 2 weeks of treatment. Rats in the single agent groups were also sacrificed at 24 h. In order to determine 8-OHdG levels, forestomach epithelium was collected by scraping with the back of a razor-edge from excised stomachs at stages of 6 and 24 h and 2 weeks, except at the 3-day stage when forestomach epithelium was expected to be lost by mucosal injury. Collected epithelium samples were frozen immediately in liquid nitrogen and stored at –80°C until measurement. To prevent 8-OHdG formation as a byproduct during DNA isolation (Kasai, 2002), nuclear DNA in forestomach epithelium was extracted by a slight modification of the method of Nakae et al. (1995). Briefly, nuclear DNA was extracted with a commercially available DNA Extractor WB Kit (Wako Pure Chemical Industries, Ltd.) containing an antioxidant NaI solution to dissolve cellular components. For further prevention of auto-oxidation in the cell lysis step, deferoxamine mesylate (Sigma Chemical Co.) was added to the lysis buffer (Helbock et al., 1998). As the amount of forestomach DNA was small, we used a precipitation carrier (GenTLE, Takara Bio, Inc., Shiga, Japan) at the ethanol precipitation step of DNA extraction. The DNA was digested to deoxynucleotides with nuclease P1 (Yamasa Co., Chiba, Japan) and alkaline phosphatase (Sigma Chemical Co.), and levels of 8-OHdG (8-OHdG/105 deoxyguanosine) were assessed by high-performance liquid chromatography with an electrochemical detection system (Coulochem II, ESA, Bedford, MA). To examine lesions contemporary with 8-OHdG measurement, stomachs of three animals each group were excised at 6, 24 h, 3 days and 2 weeks of treatment, and each stomach was inflated with formalin solution and later opened. Sections were cut from the forestomach, and stained routinely with Hematoxylin-Eosin (H-E) for histopathological assessment.
Two-stage carcinogenicity study.
In the two-stage carcinogenicity study, as shown in Figure 1, 120 rats at 6 weeks of age were divided into five groups each consisting of 25 animals and treated for 12 weeks as follows: group 1, basal diet for the negative control; group 2, 1% AsA; group 3, 0.2% NaNO2; group 4, 1% AsA plus 0.2% NaNO2; and group 5, single administration of MNNG (dimethylsulfoxide:water, 1:1) at a dose of 100 mg/kg body weight by gavage in the 1st week for a positive control. After an interval of two weeks, animals were given 1% BHA in diet for tumor promotion in the forestomach (Hirose et al., 1989). Totals of 10 animals in each group were necropsied under ether anesthesia just after the initiation treatment and at week 52 to confirm lesion development. The remaining fifteen animals in each group were sacrificed at week 78 of the experiment. The stomachs were excised and inflated with formalin solution and later opened. After fixation, the location and size of tumors were recorded and volumes of nodules were approximately calculated as spherical objects (formula: length x depth x height x 0.52). Sections were cut from the forestomach, and stained routinely with H-E for histopathological assessment. The grade of squamous hyperplasia observed in the forestomach was classified into mild, moderate and severe depending on the thickness of the mucosa (Hirose et al., 1989).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Bacterial Reverse Mutation Test
The results of the bacterial reverse mutation test are shown in Figure 2. In the WP2 uvrA/pKM101 strain (mutM+), the number of revertants slightly increased at 5000 µg of AsA per plate in the presence of 100 µg of NaNO2, but the increase over the control without AsA was not substantial (66 ± 25 revertants per plate versus 36 ± 5 revertants per plate). In the strain YG5190 (mutM-), the number of revertants was clearly increased at concentrations of 2500 and 5000 µg of AsA per plate in the presence of 100 µg of NaNO2. The number of revertants at a concentration of 5000 µg of AsA per plate was about threefold higher than that in the control without AsA (167 ± 61 revertants per plate vs. 61 ± 5 revertants per plate). The number of revertants decreased with 7500 and 10000 µg of AsA per plate, probably because of toxicity to the bacteria. The sharp increase and decrease in the number of revertants appeared to be caused by the combined effects of AsA plus NaNO2 because the number of revertants increased and decreased much more moderately in the absence of NaNO2. With S9 mix, the number of revertants of strain YG5190 (mutM-) increased in a dose-dependent manner until the highest concentration of AsA when NaNO2 was present. The number of revertants at a concentration of 10000 µg of AsA was about threefold higher than that in the control without AsA (188 ± 39 revertants per plate versus 69 ± 7 revertants per plate). The toxicity caused by the combination of AsA plus NaNO2 seemed to be compromised when S9 mix was present. When a similar assay was conducted at a twofold higher concentration of NaNO2 (200 µg per plate), the growth of bacteria was severely inhibited by the presence of NaNO2 itself and thus combined effects with AsA could not be examined (data not shown).
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ESR Analysis of NO Formation
Figure 3 illustrates ESR signals resulting from the reaction of Fe(DTCS)2 complex and NO following the mixture of AsA at several doses with NaNO2. Although ESR signals were not observed after treatment with 1.0mM NaNO2, or 40mM AsA alone, typical NO signals appeared after adding 10, 20, 30, and 40mM AsA to 1.0mM NaNO2 in a dose-dependent manner.
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Short-Term Study
Data for 8-OHdG levels of nuclear DNA in the forestomach epithelium of rats given combined treatment with 0.2% NaNO2 and 1% AsA at 6, 24 h and 2 weeks are summarized in Figure 4. Values for the combined treatment group were significantly elevated at 24 h as compared with the basal diet value, and the elevation remained at 2 weeks of treatment, albeit lacking statistically significance. In the single agent groups, slight elevation of 8-OHdG levels without statistically significance was observed in the NaNO2 group but there was no change in the AsA group. Table 1 summarizes data for histopathological lesions in the forestomachs of rats given 0.2% NaNO2 in the water and 1% AsA in the diet for up to 2 weeks. The forestomach of rats treated with the combination exhibited erosion of the squamous epithelium accompanied by strong inflammatory changes with edema in the submucosa. These changes gradually became more extensive from 24 h to 3 days of treatment (Fig. 5). However, regenerative hyperplasia eventually developed after week 2, and the above regenerative/necrotic lesions became obscure except for in areas of severe ulceration. In single agent groups, there were no apparent changes after 24 h of treatment.
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Two-Stage Carcinogenicity Study
Body weights at the end of the initiation phase, food and water consumption data in the initiation phase and AsA and NaNO2 intakes are shown in Table 2. The body weights were decreased in all the NaNO2 treated groups compared with the basal diet group. No obvious change was evident in food consumption among groups and average daily AsA intakes were 646 and 623 mg/kg/day. Intake of NaNO2 solution was lower than for distilled water and average daily NaNO2 intakes were 123 and 133 mg/kg/day. At week 46 of the experiment, one animal in the basal diet group was found dead, but the cause of death was unclear. Body weight at week 78, food and water consumption data in the promotion phase and BHA intakes are shown in Table 3. There were no differences in these data among groups.
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At the end of the initiation treatment, mild or moderate hyperplasias in the forestomach squamous epithelium were observed in all animals treated with AsA and NaNO2 in combination (Table 4). Ulceration in the glandular stomach was also found in one animal of the combination group (data not shown). No lesions were observed in the stomachs of basal diet, single agent or MNNG-treated groups.
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After BHA promotion, a few small tumors were grossly observed in the forestomachs of the MNNG-initiated positive control group at experimental weeks 52 and 78. The numbers and volumes of macroscopical tumors are given in Table 5. In the combination group, one large tumor was observed at week 78 but there were no tumors in the basal diet or single agent groups. Histopathologically, the lesions in the forestomach were classified into squamous hyperplasia, papilloma, and squamous cell carcinoma (SCC). As shown in Table 6, there were no differences in the incidence and degree of hyperplasia among all groups at both experimental time points. However, in the MNNG-treated group, incidences of SCC at 58 and 78 weeks, and papilloma at 78 weeks were clearly increased. In the combination group, one SCC, corresponding to the grossly observed tumor, was found at 78 weeks. There were no neoplastic lesions in the basal diet or single agent groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In E. coli, three genes, that is, mutM, mutY, and mutT, play important roles in synergistically preventing mutations induced by 8-OHdG in DNA and nucleotide pools (Michaels et al., 1991
, 1992; Maki and Sekiguchi, 1992). The product of mutM has DNA glycosylase activity and releases 8-OHdG from DNA (Tchou et al., 1991), deficiency in the mutM gene increasing G:C to T:A mutations induced by 8-OHdG (Cabrera et al., 1988). In fact, we have previously shown that S. typhimurium deficient in the analogous mutM gene was a maximum of 30 times more sensitive to the mutagenicity of several agents generating oxidative radicals such as photosensitizer and nitro compounds (Suzuki et al., 1997). Therefore, in the present study, we established strain YG5190, a mutM-deficient derivative of E. coli WP2 uvrA/pKM101, for examination of the combined mutagenicity of AsA plus NaNO2. We used E. coli WP2 uvrA/pKM101 as a background strain instead of S. typhimurium TA strains because E. coli WP2 uvrA/pKM101 is in general more tolerant to toxicity of chemicals compared with S. typhimurium TA strains. This may be related to the fact that S. typhimurium TA strains bear rfa mutations involved in lipopolysaccharide biosynthesis in the cell wall and therefore exhibit much higher permeability to chemical compounds as compared with E. coli strains. In this test system, despite NaNO2 or AsA alone being nonmutagenic, the combination of these agents clearly increased the mutation frequency (Fig. 2). In particular, the increment in the mutM-deficient strain was much higher than that in the parent strain.
It has been known that NaNO2 gave rise to NO and caused increases in the mutation frequency of E. coli WP2 uvrA/pKM101 under the acidic condition, but these findings were not observed at nearly neutral pH (Matsumoto et al., 2008; Samouilov et al., 1998). In fact, the present result revealing the absence of NO signal in NaNO2 alone at pH 6.8 was in line with these reports. Nevertheless, the addition of AsA to NaNO2 solution caused NO generation even at a concentration of 10mM of AsA, which was approximately half of the lowest dose examined in the Ames test. Thus, the ESR data allowed us to speculate the production of NO in the culture media following the mixture of AsA with NaNO2. Therefore, it is highly probable that 8-OHdG resulting from NO-mediated oxidative stress following chemical reaction of AsA with NaNO2 is responsible for the mutagenicity.
Although NaNO2 itself is a mutagenic compound in E. coli and S. typhimurium without S9 mix at high doses (Ishidate et al., 1984), it did not exhibit mutagenicity in E. coli WP2 uvrA/pKM101 (mutM+) and YG5190 (mutM–) strains under the conditions used here, i.e., at a dose of 100 µg per plate. Without S9 mixture, the mutation frequency in the mutM-deficient strain reached a peak with the middle dose of AsA, decreasing thereafter to the control level. This might have resulted from inhibition of bacterial growth at higher doses. In contrast, with S9 mixture, growth inhibition did not occur and maximum elevation of the mutation frequency appeared later as compared with the case without S9 mixture. These findings might be attributable to inhibition of NO/reactive oxygen species (ROS) generation by certain components in S9 mixture. In turn, the present data showing that the mutagenicity and the growth inhibition were more evident without metabolic activation might imply direct reaction of AsA and NaNO2.
In vivo, we previously showed non-significant elevation of 8-OHdG levels in the forestomach epithelium of rats treated with AsA and NaNO2 at 4 h after treatment (Okazaki et al., 2006). Therefore, along with the confirmation of 8-OHdG elevation, we performed measurement after a longer period of treatment to ascertain any sustained increase. As a result, 8-OHdG levels were significantly increased at 24 h. At 2 weeks after coadministration, the elevation of 8-OHdG levels was still observed, but without statistical significance. At this stage, the forestomach was covered with regenerative and hyperplastic squamous epithelium. Taking into consideration the fact that high expression levels of ROS-scavenging enzymes are detected in regenerating hyperproliferative epithelium during cutaneous wound repair (Steiling et al., 1999), the forestomach epithelium at 2 weeks might acquire resistance to oxidative stress.
In the present two-stage forestomach carcinogenicity study, tumor initiating potential of the combination with AsA and NaNO2 was not detected. In the combination group, one SCC was found, no such lesions being present in any other group except the MNNG-treatment group. However, there were no significant changes in incidences and multiplicities of neoplastic lesions among the compared groups, despite appreciable tumor induction observed in the MNNG-treated positive control group. It is well known that a certain period of time might be required for 8-OHdG to cause permanent mutations (Umemura et al., 2006). In the present short-term study, although in vitro bacterial mutation assay using E. coli strains was positive, elevation of 8-OHdG in vivo was moderate and significant accumulation of 8-OHdG did not found at week 2. Accordingly, transient and weak increment of 8-OHdG might be insufficient for inducing gene mutations derived from 8-OHdG formation. In contrast to clear evidence showing oxidative DNA damage and subsequent point mutations in genomic DNA, correlations between oxidative stress and tumor formation remain equivocal. Although both potassium bromate and ferric nitrilotriacetate cause oxidative DNA damage in kidneys (Kimoto et al., 2000; Umemura et al., 2004), resulting in increment of the mutation frequency in reporter gene assays (Jiang et al., 2006; Umemura et al., 2006), initiation activity was only found with the latter, but not the former (Toyokuni et al., 1998; Umemura et al., 2006). Further studies appear warranted to confirm whether oxidative DNA damage is directly contributed to tumor development.
Overall results from a series of studies suggest that the combination with AsA and NaNO2 generates NO and oxidative stress, and this is considered to promote carcinogenesis due to cytotoxicity and compensatory cell proliferation, but tumor initiating activity was not clear despite oxidative DNA damage. Humans consume large amounts of AsA in foods such as vegetables and fruits, and daily intake is estimated to be approximately 50–200 mg per person (Michels et al., 2001; Zhang et al., 1999). AsA and its derivatives have also found application as food additives, and higher doses of AsA have been used for medical and dietary supplements (Norman et al., 2003). Therefore, at least transiently, it is possible that the concentration of AsA in the stomach of humans reaches 7–14 mg/ml which is in the range of the concentration showing genotoxicity in the present study. On the other hand, the concentration of NaNO2 in the present mutagenicity study was approximately 140 µg/ml (2mM) and this is clearly higher than the level of estimated human exposure. Although nitrites are included in the human saliva due to reduction of entero-salivary recirculated dietary nitrate from meats, vegetables and tap water, the concentration in the stomach is reported to be 50µM or below (Tsuda and Kurashima, 1991). Therefore, the risk of genotoxicity by the combination with AsA and nitrite can be considered low in the stomachs of humans. However, it has been proposed that a high concentration of NO derived from salivary nitrite might be a cause of various diseases, including cancer at the human gastro-esophageal junction (Iijima and Shimosegawa, 2006; Iijima et al., 2003; McColl, 2005). Recently, we showed esophageal tumor promoting activity of the combination with AsA and NaNO2 in a surgically produced rat chronic acid reflux model (Kuroiwa et al., 2008), and it was speculated that NO derived from nitrite is responsible. Further studies are required to investigate the effects of NO derived from nitrite on the esophagus.
In conclusion, the present study demonstrated that the combination with NaNO2 and AsA exerts mutagenicity due to oxidative DNA damage in vitro, with elevation of 8-OHdG levels in the forestomach epithelium. However, our two-stage rat forestomach carcinogenicity study failed to demonstrate tumor initiating activity of the combination. Thus, oxidative stress-associated DNA damage dose not appear to be responsible, at least for initiation of forestomach carcinogenesis.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Health and Labour Sciences Research Grants (Research on Food Safety) from the Ministry of Health, Labour and Welfare, Japan (H16-Shokuhin-008).
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NOTES |
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1 Present address: Food Safety Commission, Cabinet Office Government of Japan, 6th Floor, Prudential Tower, 2-13-10 Nagata-cho, Chiyoda-ku, Tokyo 100-8989, Japan.
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ACKNOWLEDGMENTS |
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We thank Ms Machiko Maeda, Ayako Kaneko, and Fukiko Takagi for expert technical assistance in performing the animal experiments and processing histological materials.
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