Potassium Bromate Enhances N-Ethyl-N-Hydroxyethylnitrosamine–Induced Kidney Carcinogenesis Only at High Doses in Wistar Rats: Indication of the Existence of an Enhancement Threshold

Abstract

As susceptibility to carcinogens varies considerably among different strains of experimental animals, evaluation of dose-response relationships for genotoxic carcinogen in different strains is indispensable for risk assessment. Potassium bromate (KBrO₃) is a genotoxic carcinogen inducing kidney cancers at high doses in male F344 rats, but little is known about its carcinogenic effects in other strains of rats. The purpose of the present study was to determine dose-response relationships for carcinogenic effects of KBrO₃ on N-ethyl-N-hydroxyethylnitrosamine (EHEN)–induced kidney carcinogenesis in male Wistar rats. We found that KBrO₃ showed significant enhancement effects on EHEN-induced kidney carcinogenesis at above 250 ppm but not at doses of 125 ppm and below when evaluated in terms of induction of either preneoplastic lesions or tumors in male Wistar rats. Furthermore, KBrO₃ significantly increased the formation of oxidative DNA damage at doses of 125 and above but not at doses of 30 ppm and below in kidneys. These results demonstrated that low doses of KBrO₃ exert no effects on development of EHEN-initiated kidney lesions and induction of oxidative DNA damage. Taking account of previous similar findings in male F344 rats, it is strongly suggested that a threshold dose exists for enhancement effects of KBrO₃ on kidney carcinogenesis in rats.

Introduction

Exposure to environmental carcinogens is a very significant cause of human cancer. Dose-response studies to define the relationship between exposure to a carcinogen and the probability of exerting a carcinogenic effect is therefore of paramount importance. Estimates of risk associated with exposure to low levels of genotoxic carcinogens are generally obtained by linear extrapolation from experimental animal bioassays, although the dose levels used in such studies are generally orders of magnitude higher than actual human exposure levels. At present, the dose-response relationship for genotoxic carcinogens is generally assumed to be linear without a threshold dose below which carcinogenic effects are absent, and so genotoxic carcinogens are generally considered to pose some risk at any level of exposure (linear nonthreshold theory).

However, there is increasing evidence that genotoxic carcinogens do not show carcinogenic effects at low doses. For example, N-nitrosodiethylamine (NDEA), a well-known geno-toxic carcinogen, has been shown to have no effects on liver (Fukushima et al. 2002) at low doses in rats. 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx), a potent genotoxic carcinogen detected in cooked meat and fish, has also undergone a series of dose-response studies using different liver carcinogenesis models and rat strains (Fukushima et al. 2003; Hoshi et al. 2004; Kang et al. 2006; Kushida et al. 2005; Wanibuchi et al. 2006; Wei et al. 2006). One recent in vivo mutagenicity study revealed that low doses of MeIQx did not induce mutations in rat liver (Hoshi et al. 2004). With respect to a genotoxic colon carcinogen, it was also reported that low doses of 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine (PhIP) lack both of initiation and promotion activities in the rat (Doi et al. 2005; Fukushima et al. 2004). These findings suggest the existence of no-effect levels for genotoxic carcinogens. To establish a balance-of-evidence for analysis of carcinogenic effects of low-dose genotoxic carcinogens, data on examples targeting other organs are clearly a high priority.

Potassium bromate (KBrO₃) is an oxidizing agent that was once widely used as a flour improver. Although no data are available as to the potential carcinogenicity of KBrO₃ to humans, it is classified as a category 2B carcinogen (possibly carcinogenic to humans) based on sufficient evidence of kidney carcinogenicity in rats by the International Agency for Research on Cancer (IARC). Furthermore, KBrO₃ has been classified as a genotoxic carcinogen based on positive results in the Ames (Ishidate et al. 1984), chromosome aberration (Ishidate and Yoshikawa 1980), and micronucleus tests (Hayashi et al. 1988). KBrO₃ has now been banned from use in food products in most countries. It is, however, allowed to be applied in manufacture of bread in Japan under the condition that no detectable residue is present in the final baked products, and it is allowed in the manufacture of bread and malting of barley in the United States under prescribed conditions. More importantly, it is also known that bromate is generated as a by-product during ozone disinfection of drinking water (Cavanagh et al. 1992). Although the concentrations of bromate in drinking water are generally extremely low, they still constitute a potential hazard, and there have been concerns regarding low-dose carcinogenic effects.

So far, several dose-response analyses of KBrO₃ have been conducted in F344 rats. In a 2-year carcinogenicity study using doses from 15 to 500 ppm, increased incidences and number of renal cell tumors (RCT) were observed at doses of 125 ppm and higher but not at 60 ppm and below (Kurokawa et al. 1986). In a two-stage kidney carcinogenesis study using the same doses as the above study, KBrO₃ increased N-ethyl-N-hydroxyethylnitrosamine (EHEN)–induced preneoplastic lesion development at doses of 30 ppm and higher but not at 15 ppm, and an increase in renal tumors was only observed at 500 ppm in male F344 rats, possibly due to the low incidences without any enhancement (Kurokawa et al. 1985). These results suggested that no-effect levels of KBrO₃ for carcinogenicity lie between 15 and 30 ppm for kidneys of male F344 rats (Umemura and Kurokawa 2006). Since susceptibility to carcinogens varies considerably among different strains of rat, dose-response curves may differ with the strain. The Wistar rat is known to be more susceptible to EHEN-induced renal carcinogenesis than F344 rats (Konishi et al. 1994). However, renal carcinogenicity of KBrO₃ in Wistar rats has hitherto not been investigated.

The present investigation was conducted to determine the dose-response relationship of enhancement effects of KBrO₃ on EHEN-induced kidney carcinogenicity in Wistar rats, particularly at low doses. To cast light on mechanisms underlying the enhancement effects of KBrO₃, we also examined formation of oxidative DNA damage and expression of DNA repair and oxidative responsive genes in the kidneys of Wistar rats given EHEN followed by various doses of KBrO₃.

Material and Methods

Chemicals

KBrO₃ was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) with a purity of greater than 99.5%. EHEN was purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan).

Animals and Experimental Design

The animal experiment protocols were approved by the Institutional Animal Care and Use Committee of Osaka City University Medical School.

Male Crj:Wistar rats, 5 weeks old, were purchased from Charles River Japan, Inc. (Hino, Shiga, Japan) and housed in an animal room with a targeted temperature of 22 ± 3 °C, relative humidity of 55 ± 5%, and a 12-hr light/dark cycle. They were acclimated for 7 days before the start of treatment. Basal diet (CE2 pellet, Oriental Yeast Co., Tokyo, Japan) and drinking water (with or without KBrO₃) were available ad libitum during the study. As the concentration of BrO₃ ⁻ in the tap water in Osaka City is about 0.002 ppm (according to Osaka City Waterworks Bureau Database) and no differences were observed in mutation frequency, oxidative DNA damage, and cell proliferation activity in kidneys between rats given distilled water and tap in a previous in vitro mutagenicity study (Yamaguchi et al. 2008), KBrO₃ was dissolved in the tap water in the present study. Body weights, food intake, and water consumption were measured weekly.

In experiment 1, since no data are available on carcinogenicity of KBrO₃ in Wistar rats or on the higher susceptibility of this strain of rats to EHEN than Fisher rats that might mask the effects of low doses of KBrO₃ in a longer-term study, we used the same 26-week, two-stage treatment protocol as was used in an earlier F344 rat study (Kurokawa et al. 1985). As the concentration of BrO₃ ⁻ in the tap water in Osaka City is about 0.002 ppm, 0.02 ppm was selected as the lowest dose in terms of actual human exposure levels. Doses of KBrO₃ used were from 0.02 to 500 ppm as in the previous in vitro mutagenicity study using Big Blue rats mentioned above. A total of 240 male Wistar rats at 6 weeks of age were divided into 8 groups of 30 rats each. All were administered 500 ppm EHEN in the drinking water for the first 2 weeks, and then groups 1 through 8 were given KBrO₃ at the concentrations of 0 (control group), 0.02, 0.2, 2, 8, 30, 125, 500 ppm, respectively, in the drinking water for 24 weeks. As 11 of 30 rats died by week 12 in the 500 KBrO₃ ppm group due to toxicity of KBrO₃, the dose was reduced to 250 ppm from week 12. At the end of experimental week 26, all surviving rats were killed under ether anesthesia, and then kidneys were excised and weighed. After recording all suspected neoplastic lesions for their number, size, and location, each kidney was cut transversely into 6 to 8 semiserial slices and fixed in 10%buffered formalin. After 3 days’ fixation, kidney tissues were processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin for histological examination. Areas of kidney sections examined were measured using an Image Processor for Analytical Pathology (IPAP; Sumica Technos, Osaka, Japan).

In experiment 2, a total of 48 male Wistar rats at 6 weeks of age were divided into 8 groups of 6 rats each. Because of increased mortality in the 500 ppm KBrO₃ group in experiment 1, the highest dose of KBrO₃ was reduced to 250 ppm. All rats were treated with 500 ppm ENEN in the drinking water for the first 2 weeks, followed by treatment with KBrO₃ at concentrations of 0 (control group), 0.02, 0.2, 2, 8, 30, 125, and 250 ppm, respectively, in the drinking water for 2 weeks. At the end of experimental week 4, all rats were killed under ether anesthesia. At necropsy, kidneys were excised, half of each being snap-frozen in liquid nitrogen and stored at −80°C for examination of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and gene expression. The remaining kidney tissues were fixed in 10%%buffered formalin and processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin for histological examination.

Detection of 8-OHdG Formation in Kidney DNA

The levels of 8-OHdG formation in rat kidneys in experiment 2 were measured by the method described previously (Wei et al. 2002). Briefly, DNA was extracted from frozen kidney tissues from 6 rats of each group using a DNA Extractor WB Kit (Wako Pure Chemical Industries, Kyoto, Japan), and then digested into deoxynucleosides by combined treatment with nuclease P1 (Tamasa Shoyu, Chiba, Japan) and alkaline phosphatase (Sigma, St Louis, MO, USA). After filtration through an Ultrafree-MC filter unit 100,000 (Millipore Co., Bedford, MA, USA), the levels of 8-OHdG in each sample were determined by high-performance liquid chromatography (HPLC) with electrochemical detection.

TaqMan Real-Time Quantitative PCR

The mRNA expression levels of genes that might be involved in KBrO₃-induced kidney carcinogenesis were evaluated in kidneys of experiment 2 by TaqMan real-time quantitative PCR. These genes included 6 examples that have been reported to be up-regulated and 2 examples that have been reported to be down-regulated in kidneys of F344 rats treated with 400 ppm KBrO3 for 52 weeks (Delker et al. 2006; Geter et al. 2006). The 6 up-regulated genes mentioned above are 8-oxoguanine DNA glycosylase (Ogg1), glutathione S-transferase M1 (GSTM1), glutathione S-transferase P1 (GSTP1), glutathione peroxidase 2 (GPX2), glutamate cysteine ligase (GCL), and cyclin G1. The 2 down-regulated genes mentioned above are solute carrier family 12 member 3 (Slc12a3) and solute carrier family 26 member 4 (Slc26a4). We also examined expression levels of the following genes: cell cycle regulator cyclin D1; DNA damage repair genes including MYH, MTH1, growth arrest and DNA damage-inducible protein 45 (GADD45); oxidative response genes, including heme oxygenase 1 (HOX1), HSP90, HSP70; and genes involved in xenometabolism, including CYP1A1, CYP1B1, CYP2B1, CYP2E1. Sequence-specific primers and probes (Taqman Gene Expression Assay) were purchased from Applied Biosystems, Inc Foster City, CA, USA. ß-actin was employed as an internal control. Briefly, total RNAs of kidney were isolated using an Isogen RNA Isolation Kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s instructions. cDNA synthesis was performed with 600 ng of RNA using an Advantage RT-for-PCR kit (Takara Bio, Inc., Japan), and then cDNA solutions were diluted to a final volume of 100 μ1 by adding 80 μ1 DEPC-treated H₂O. PCR reactions were performed in a 25 μ1 reaction mixture containing 5 μ1 cDNA, 1 μ1 of Taqman Gene Expression Assay Mix, and 12.5 μ1 TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Inc., Foster City, CA, USA) under the following conditions: 95°C for 20 s, then 40 cycles at 95°C for 3 s and 60°C for 30 s using a 7500 Fast Real-Time PCR System (Applied Biosystems, Inc., Japan). Serial dilution standard cDNAs were included in each Taqman PCR reaction to create standard curves. The amounts of gene products in the test samples were estimated relative to the respective standard curves. Values for target genes were normalized to those for ß-actin.

Statistical Analysis

All mean values are reported as means ± SDs. Statistical analyses were performed using the Statlight program (Yukms Co., Ltd., Tokyo). Homogeneity of variance was tested by the Bartlett test for body and tissue weights, numbers of kidney lesions, 8-OHdG formation, and mRNA expression among the groups. Differences in mean values between the control and KBrO₃-treated groups were evaluated by 2-tailed Dunnett test when variance was homogeneous and the 2-tailed Steel test when variance was heterogeneous. Differences in incidences of tumor and eosinophilic body were analyzed by the Fisher’s exact test. p-values less than .05 were considered significant.

Results

General Observation (Experiment 1)

Final numbers, final body weights, kidney weights, and data for total intake of KBrO₃ by EHEN-initiated rats are summarized in Table 1. Mortality before the end of study (week 26) in the groups administered from 0 (control group) to 125 ppm KBrO₃ was only observed during weeks 2 to 3 except for 1 rat in the control group at week 11 and 1 rat in the 0.2 ppm group at week 18. Since no rats died after week 18, the effective numbers of animals in the present study were calculated as the numbers of rats alive at the end of the study (week 26). No macroscopic tumors were observed in any of the rats dying during the experiment.

Final body weights were significantly decreased in the group-administered 500→250 ppm KBrO₃ after EHEN initiation compared with the EHEN-initiation-alone group. The absolute weight of the kidneys was decreased in the 500→250 KBrO₃ ppm group, but this was not statistically significant. The weights of kidneys relative to the body weights were significantly increased in the 500→250 KBrO₃ ppm group, and covariant analysis indicate this change was due to decreased body weight.

Water and food consumption was significantly decreased in rats administered 500→250 ppm compared with the EHEN-initiation-alone group from week 4 (data not shown), and this meant that intake of KBrO₃ in this group was lower than anticipated. Nevertheless, the average total intake of KBrO₃ was increased roughly in a dose-dependent manner (Table 1).

Development of Kidney Lesions (Experiment 1)

Kidney lesions were classified into atypical tubular hyper-plasia (ATH) (Figure 1A) and RCT (Figure 1B–C) and evaluated in terms of incidence and number per cm² area examined (Kurokawa et al. 1985, 1990). Both adenomas and adenocarcinomas were classified as RCT, as in previous studies (Kurokawa et al. 1985), since there has been controversy as to the definition of adenocarcinoma in rat kidneys due to lack of any characteristic cellular atypia, growth pattern, or local invasion. Atypical tubular hyperplasia was defined as tubules with stratified layers of atypical tubular epithelial cells characterized by various degrees of cellular and nuclear pleomorphism. They may show a solid structure or a multilayered structure in dilated tubules. RCT is differentiated from ATH by the characters of larger size, compression of the adjacent parenchyma, and signs of neovascularization.

Incidences and numbers of ATH and RCT in EHEN-initiated rats at the end of week 26 are shown in Table 2. Incidences of spontaneous RCT in control male Wistar rats in 2-year studies were reported to be 0%to 2.5%(Poteracki and Walsh 1998). Therefore, the kidney tumors observed in the present studies can be attributed to the carcinogenic effects of EHEN and KBrO₃, although there was not a nontreatment group in the present study. There were no significant differences in incidences of ATH and RCT between KBrO₃-treated groups and the control group, which may in part be due to the high incidences of both lesions in the control group. Significant increases in the numbers of both ATH and RCT were observed in the 500→250 ppm KBrO₃ group compared to the control group. However, no significant differences in either number of ATH or RCT were observed in groups administered 125 ppm and below to the control group.

General Condition (Experiment 2)

All the rats survived in good condition until the scheduled sacrifice. Final body weight, kidney weights, and total intake of KBrO₃ in EHEN-initiated rats are summarized in Table 3. There were no significant differences in body and kidney weights among the groups (Table 3). Food consumption was similar in all postinitiation KBrO₃ treatment groups as well as the EHEN-initiation-alone group (data not shown). Water consumption was slightly decreased in rats administered 250 ppm KBrO₃ compared with the EHEN-initiation-alone group during the 2-week KBrO₃ treatment period (data not shown) so that intake of KBrO₃ was lower than expected. Nevertheless, the average total intake of KBrO₃ was increased in a dose-dependent manner (Table 3).

Histopathological Examination (Experiment 2)

Increased formation of eosinophilic bodies characterized by large rounded droplets stained strongly with eosin in the cytoplasm of proximal tubular epithelial cells is a well-known KBrO₃ treatment-related change in rat kidneys (Kurokawa et al. 1990). It is also known to be associated with accumulation of alpha-2u globulin and related to KBrO₃-induced cell proliferation and toxicities in rat kidneys (Umemura and Kurokawa 2006). In experiment 2, eosinophilic bodies were observed in 3 of 6 (50%) and 5 of 6 rats (83%, p < .05 vs. EHEN-alone group) in groups given postinitiation KBrO₃ at doses of 125 and 250 ppm, respectively, but not at 30 ppm and below. No necrotic or regenerative tubules, atypical tubules, or fibrosis were observed in any of the groups.

8-OHdG Formation in Kidney DNA (Experiment 2)

8-OHdG formation levels in kidney DNA are shown in Figure 2. Significant increase was noted in the 125 and 250 ppm KBrO₃ postinitiation treatment groups but not in the 30 ppm and lower doses groups compared to the EHEN-initiation-alone group.

Gene Expression Analysis (Experiment 2)

mRNA expression level of oxidative DNA damage repair genes including Ogg1 (Figure 3A), MTH1, MYH and GADD45, cell cycle regulators including cyclin D1 (Figure 3B) and cyclin G1, oxidative response genes including HSP70 (Figure 3C), HSP90, GPX2, GSTM1, GSTP1, GCL and HOX1, ion transport genes including SLC12a3 (Figure 3D) and SLC26a4, xenometabolism genes including CYP1A1, CYP1B1, CYP2B1, and CYP2E1 were examined in kidneys of rats. However, none of them were significantly changed in postinitiation KBrO3 treatment groups compared to the EHEN-initiation-alone group (data not shown except Ogg1, cyclin D1, HSP70, and SLC12a3).

Discussion

KBrO₃ has been classified as a genotoxic carcinogen which is positive in the Ames (Ishidate et al. 1984), chromosome aberration (Ishidate and Yoshikawa 1980), and micronucleus tests (Hayashi et al. 1988). Although dose-response relationships for genotoxic carcinogens are generally assumed to be linear without a threshold dose below which carcinogenic effects are absent, the results of the present studies demon strated that KBrO₃ at levels of 125 ppm and below had no significant enhancement effects on EHEN-induced kidney carcinogenesis when evaluated in terms of induction of either preneoplastic lesions or tumors in Wistar rats. In F344 rats, it was reported previously that 15 ppm of KBrO₃ had no influence on development of EHEN-induced preneoplastic lesions in the kidney (Kurokawa et al. 1985). As summarized in Table 4, evidence from different studies using different strains of rat clearly indicates that KBrO₃ exerts enhancement effects only at high doses, with no observed effect levels of enhancement effects of KBrO₃ on kidney carcinogenicity in rats. However, it should be noted that 125 ppm KBrO₃ demonstrated enhanced ATH and RCT multiplicities when administered to Wistar rats for a prolonged promotion period, based on the finding that 125 ppm KBrO₃ significantly increased 8-OHdG formation in rat kidneys in experiment 2.

Understanding the mode of action of carcinogens will facilitate not only clarification of dose-response relationships below the experimental observable range of neoplastic lesions but also how carcinogenic effects may relate to the human situation. Although the precise mechanisms underlying KBrO₃ carcinogenesis remain to be elucidated, available data suggest formation of oxidative DNA damage and consequent gene mutation and chromosomal anomalies are most likely involved in rats (Moore and Chen 2006; Umemura and Kurokawa 2006). Formation of 8-OHdG, a well-established marker for oxidative DNA damage, has been investigated in kidney DNAs of different kinds of KBrO₃-treated rats. It was reported that KBrO₃ significantly increased 8-OHdG in kidney DNA at 250 ppm and above in both F344 rats (Umemura et al. 2004) and gpt delta transgenic rats that have an SD genetic background and harbor the gpt gene of E. coli and the red/gam genes of lambda phage (Umemura et al. 2006) and at 500 ppm in Big Blue transgenic rats that have a F344 genetic background and harbor the lacI gene of E. coli (Yamaguchi et al. 2008) but had no effects at 125 ppm and below in all three kinds of rats. Both gpt delta and Big Blue transgenic rats have been used to evaluate the in vivo mutagenicities using respective incorporated marker genes and carcinogenicities of chemicals. In line with the earlier findings, in the present Wistar rat study, no-effect levels of KBrO₃ on 8-OHdG formation were observed at 30 ppm and below. Furthermore, the no-effect level of KBrO₃ for induction of oxidative DNA damage is lower than that for carcinogenicity, as 125 ppm significantly increased 8-OHdG formation but not the development of ATH or RCT in the kidney. With respect to induction of gene mutations, recent in vivo mutagenic studies revealed that KBrO₃ significantly increased mutations only at 500 ppm in the rat kidneys of both Big Blue (Yamaguchi et al. 2008) and gpt delta rats (Umemura et al. 2006), with no observed effect levels of 125 and 250 ppm, respectively. These above findings for different-strain rats (Table 4) indicate that no-effect levels of KBrO₃ exist for induction of oxidative DNA damage and mutation, as well as enhancement effects on kidney carcinogenesis in rats.

We also examined effects of 2-week KBrO₃ treatment on mRNA expression levels of 18 genes implicated in DNA damage repair, oxidative response, or cell cycle in the kidneys of EHEN-initiated Wistar rats, including 7 genes (Ogg1, GSM1, GSTP1, GPX2, GCL, HOX1, cyclin G1) that are reported to be overexpressed in kidneys of F344 rats treated with 400 ppm KBrO₃ in the drinking water for 52 weeks (Delker et al. 2006). However, none of them were significantly altered. The Ogg1 gene encodes a DNA repair enzyme (DNA glycosylase) that excises 8-OHdG, and its mRNA expression level and activity are considered to be correlated (Kim et al. 2001). The lack of any effect of KBrO₃ on its mRNA expression in the present study might be due to the short treatment duration. However, similar results were reported in a recent gpt delta rat study in which 13 weeks’ treatment with 500 ppm KBrO₃ significantly increased 8-OHdG formation in the kidney but had no effect on the mRNA expression level of Ogg1 (Umemura et al. 2006). Lack of significant changes in other genes investigated can also be explained at least in part by the short treatment duration. Another possibility is that effects of KBrO₃ on expression of the above genes were masked or modified by the effects of initiation treatment of EHEN. Nevertheless, these results do suggest that alterations of the genes in question are not early events even if important in the present mode. To further understand the mode of action of KBrO₃, further studies to identify KBrO₃-responsive genes in contexts of with or without prior initiation treatment are necessary.

In summary, the present study demonstrated that KBrO₃ shows enhancement effects and induces oxidative DNA damage only at high dose but not at low dose. Furthermore, the no-effect level of KBrO₃ for induction of oxidative DNA damage is lower than that for enhancement effects. Combining available data on carcinogenicity and mutagenicity of KBrO₃ from studies using different strain rats (Kurokawa et al. 1990; Umemura and Kurokawa 2006; Yamaguchi et al. 2008), it is reasonable to suggest that a threshold dose for carcinogenicity of KBrO₃ exists in the rat kidney. There is increasing evidence suggesting the existence of no-effect levels for genotoxic carcinogens such as NDEA (Fukushima et al. 2002), MeIQx (Fukushima et al. 2003; Hoshi et al. 2004; Kang et al. 2006; Kushida et al. 2005; Wanibuchi et al. 2006; Wei et al. 2006), and PhIP (Doi et al. 2005; Fukushima et al. 2004); and several threshold mechanisms have been suggested, including induction of detoxification processes, cell cycle delay, DNA repair, and apoptosis and the suppression of neoplastically transformed cells by the immune system (De Flora 1984; Kirsch-Volders et al. 2003; Lutz and Kopp-Schneider 1999; Sofuni et al. 2000). Dose-response relationships for low-dose genotoxic carcinogens, however, are still controversial within the field of carcinogen risk assessment at present. In view of the weight of evidence, further data accumulation should be promoted to facilitate not only understanding low-dose carcinogenic effects of genotoxic carcinogens but also establishment of accurate means of risk assessment.