Existence of a Threshold for the Genotoxic Carcinogens: Evidence ...

Genes and Environment, Vol. 31, No. 2 pp. 33–36 (2009)

Review

Existence of a Threshold for the Genotoxic Carcinogens: Evidence from Mechanism-based Carcinogenicity Studies1 Shoji Fukushima2,4, Anna Kakehashi3, Min Wei3 and Hideki Wanibuchi3 2Japan

Bioassay Research Center, Japan Industrial Safety and Health Association, Kanagawa, Japan of Pathology, Osaka City University Medical School, Osaka, Japan

3Department

(Received November 8, 2008; Revised January 14, 2009; Accepted January 15, 2009)

food were identiˆed. In general, environmental carcinogens are detected in experiments at high doses, including the maximum tolerated dose. In the risk assessment, theoretically, the carcinogenic dose response curve for non-genotoxic carcinogens has been thought to have no-response level at low dose, indicating the carcinogenic threshold. However, for genotoxic carcinogens it has always been thought to reach zero. This statement forms the basis of ``non-threshold concept'' in the ˆeld of risk assessment which means the absence of threshold in exerting carcinogenic potential. The ``non-threshold concept'' is a putative theory, since it appeared to be very di‹cult to prove experimentally that genotoxic carcinogens are able to exert carcinogenicity at low doses. In case of the risk assessment of carcinogen exposure to humans, whether it is appropriate or not to extrapolate the eŠects of exposure to high doses to low doses is still a major question of interest. The ``non-threshold concept'' for carcinogenicity means that even very low doses of carcinogens could have an in‰uence on humans. Therefore, in cancer risk assessment it is very important to verify scientiˆcally whether this concept is true. When studying the mechanisms of chemical carcinogenesis, indirect carcinogens which are genotoxic, particularly mutagenic, were found to be metabolized to ultimate carcinogens and to bind to DNA in target organ cells forming DNA adducts, leading to mutations, and thus exerting carcinogenicity. The carcinogen-induced DNA damage is e‹ciently repaired, however, some of adducts give rise to miss repair, resulting in ˆxation of mutations and appearance of mutated cells (1). Furthermore, apoptosis as well as the DNA repair system helps maintain the normal condition of cells. This sequence of events is thought to occur during the initiation stage in

The hepatocarcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx), a carcinogen contained in fried meat and ˆsh, was examined in the mediumterm rat liver carcinogenicity bioassay. Induction of DNAMeIQx adducts occurred with low doses of the chemical, followed by increasing doses by elevation of 8-hydroxy-2'deoxyguanosine (8-OHdG) formation in DNA and lacI gene mutations, which might have been related to the initiation of carcinogenesis by MeIQx. Further elevation of the MeIQx dose was shown to cause the formation of glutathione S -transferase placental form (GST-P) positive foci in the liver, a well-known preneoplastic marker in rat hepatocarcinogenesis. In studies with N -nitrosocompounds such as N -nitrosodiethylamine and N -nitrosodimethylamine, no induction of GST-P positive foci was observed after their administration at low doses. When the carcinogenicity of a well-known colon carcinogen 2-amino-1-methyl-6phenylimidazo[4,5-b ]pyridine (PhIP) was examined, PhIPDNA adduct formation was observed after treatment with low doses, while only high doses of the chemical were found to induce aberrant crypt foci (ACF), which are a surrogate marker of preneoplastic lesions in the colon. In experiments with potassium bromate, carcinogenicity, mutagenicity, and 8-OHdG formation in the rat kidney were observed only after administration at high dose. From these results, the genotoxic carcinogens were concluded to have a threshold, at least practical, with respect to their carcinogenicity. Key words: genotoxic carcinogens, carcinogenic threshold, heterocyclic amines, N -nitrosocompounds, potassium bromate

Introduction Until today, chemical carcinogens have been basically identiˆed using the epidemiological methods. For instance, in factories cancer induction was observed due to exposure to high concentrations of chemicals. On the other hand, it was found that in a 2-year carcinogenicity tests performed in rats and mice, carcinogens dosedependently induced cancer. Using these experimental methods, all environmental carcinogens contained in  The Japanese Environmental Mutagen Society

1Presented

at the International Symposium on Genotoxic and Carcinogenic Thresholds, Tokyo, July 22/23, 2008 4Corresponding to: Shoji Fukushima, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Hirasawa 2445, Hadano, Kanagawa 257-0015, Japan, Tel: ＋81-463-82-3911, Fax: ＋ 81-463-82-3860, E-mail: s-fukushima＠jisha.or.jp 33

Shoji Fukushima et al.

higher than 1 ppm (unpublished data). Next, we examined the mutagenicity of MeIQx in terms of the mutation level of the lacI gene in the liver of Big Blue rats of F344 background (4). Signiˆcant elevation of lacI gene mutation level was detected after 16 weeks of treatment with MeIQx at a dose of 10 ppm, while a marked increase was found at 100 ppm. On the other hand, in the MeIQx – treated rats, the formation of GST-P positive foci was signiˆcantly induced at a dose of 100 ppm. From these results, the existence of a no-eŠect level for the mutagenicity of MeIQx has been demonstrated and it was suggested that this level (the highest dose of MeIQx at which there is no eŠect) is lower than that observed for the MeIQx carcinogenicity in terms of induction of rat liver preneoplastic lesions. When initiation activity of MeIQx was examined in a 2-stage carcinogenesis model using 850, 21-day-old male F344 rats with phenobarbital as promoter of hepatocarcinogenesis, the GST-P positive foci in the liver were signiˆcantly increased in the 10 and 100 ppm dose groups, while no diŠerence compared to non-treated controls was found at a dose of 1 ppm or less (5). A summary of the results obtained in our experiments is presented in Fig. 2. Through the investigation of the eŠects of MeIQx application in rat hepatocarcinogenesis, in particular, the activity of the carcinogen at diŠerent doses, the formation of DNA-MeIQx adducts was observed at very low doses, thereafter with increasing the doses, elevation of 8-OHdG formation level occurred, followed by the rise in lacI gene mutation, and at-last, due to the strengthening of initiation activity, increased rat liver preneoplastic lesions (GST-P positive foci) formation was found. From these data, the existence of the broad range for no-observed eŠect levels of the examined markers has been proven. Thus, from the investigation of the markers, which are indicators of hepatocarcinogenesis, the no-observed eŠect level was detected for each of them, and according to the analysis on the basis of carcinogenicity mechanisms, it has been concluded that MeIQx has a threshold, at least a practical threshold, for its hepatocarcinogenicity in the rat.

the two-stage chemical carcinogenesis model. Moreover, induction of cell proliferation in‰uences the preneoplastic lesions to develop quickly from mutated cells and to give rise to neoplasms, which are induced from the preneoplastic lesions in the promotion stage of this model. In this paper, we report on the low-dose carcinogenicities of genotoxic carcinogens studied in a medium-term bioassay from the view point of the carcinogenic mechanism.

Low Dose Carcinogenicity of 2-Amino-3,8dimethylimidazo[4,5-f ]quinoxaline (MeIQx) in the Rat Liver MeIQx is a heterocyclic amine contained in fried meat and ˆsh, and 100 to 400 ppm doses of MeIQx were found to be carcinogenic for the rat liver (2). To investigate the eŠect of MeIQx exposure at low doses 1145, 21-day-old male F344 rats were administered MeIQx in the diet at a dose of 0, 0.001, 0.01, 0.1, 10 ppm (low dose groups) or 100 ppm (high dose group) for 16 and 32 weeks (3). The data on the induction of GST-P positive foci, a preneoplastic marker in rat hepatocarcinogenesis and the endpoint marker in the rat liver mediumterm carcinogenicity bioassay are presented in Fig. 1 (3). The numbers and areas of GST-P positive foci were not changed in the 0.001¿1 ppm MeIQx groups, however, at 10 ppm there was an indication for an increase and at 100 ppm a signiˆcant elevation was detected as compared to the non-treated controls, in a 16week experiment. The same result was observed when treatment with MeIQx was continued for 32 weeks. Furthermore, the formation of MeIQx-DNA adducts was dose-dependently induced after 4 weeks of treatment (Fig. 1). The marker level for the oxidative DNA damage, 8-hydroxy-2?-deoxyguanosine (8-OHdG) formation, was elevated after treatment with MeIQx at doses higher than 1 ppm (Fig. 1). Furthermore, the mutation level of H-ras gene, the role of which in rat hepatocarcinogenesis is still unclear, was signiˆcantly increased in the liver of rats treated with MeIQx at doses

Fig. 1. Rat hepatocarcinogenicity of MeIQx at low doses. Male F344 rats were treated with MeIQx at wide range of doses for 4, 16 and 32 weeks for the examination of GST-P positive foci development, formation of MeIQx adducts and DNA 8-OHdG levels. 34

Threshold in carcinogenicity of environmental carcinogens

Low Dose Carcinogenicity of 2-Amino-1methyl-6-phenylimidazo[5,6-b ]pyridine (PhIP) in the Rat Colon

Indeed, our 2-year carcinogenicity test with MeIQx in rats showed no hepatocarcinogenicity at low doses (6).

A heterocyclic amine, PhIP, a carcinogen predominantly contained in seared ˆsh and meat, has been shown to exert carcinogenicity in the rat colon. For risk assessment of carcinogens, we have investigated the carcinogenicity of PhIP in the rat colon applied at doses of 0.001 to 400 ppm (9). 1920, 6-week-old F344 male rats were administered PhIP in the diet for 16 weeks. The development of aberrant crypt foci (ACF), considered to be a surrogate marker of the preneoplastic lesions in the colon, was not altered by PhIP application doses of 0.001 to 10 ppm, however, at the doses of 50 to 400 ppm a signiˆcant increase was observed. Furthermore, signiˆcant elevation of PhIP-DNA adduct level was detected in the groups treated doses of 0.01 ppm and higher.

Low Dose Hepatocarcinogenicity of N -Nitrosocompounds

Low Dose Carcinogenicity of Potassium Bromate, KBrO3, in the Rat Kidney

N-Nitrosocompounds, such as diethylnitroamine (DEN) and dimethylnitrosamine (DMN), have been shown to be synthesized in the stomach through the reaction of secondary amines and nitrites. Furthermore, they are contained in diŠerent life-substances and known as contaminants of diŠerent natural and manufactured food products. The carcinogenicity of DEN with respect to the relationship between the applied dose and reactivity was investigated by Peto and colleagues using 4080 male rats (7). DEN at doses of 0.033 to 13.896 ppm was administered to rats in drinking water, and induction of liver cancer was found to be dependent on the applied dose of DEN. Therefore, from the existence of the relationship between the treatment dose and tumorigenicity, it was concluded that DEN had no threshold for its carcinogenicity in the rat liver. For clariˆcation, we have examined the in‰uence of DEN applied at lower doses than used in the experiment of Peto et al. (7). Two thousand 21-day-old male F344 rats were administered DEN over a wide range of doses from 0.0001 to 1 ppm in their drinking water for 16 weeks (2). No induction of GST-P positive foci was observed at DEN doses of 0 to 0.01 ppm, however, their values were signiˆcantly elevated at doses of 0.1 ppm or higher. When the same experiment was performed with DMN, and the carcinogen was applied to F344 rats at doses of 0.0001 to 10 ppm in their drinking water for 16 weeks, no induction of GST-P positive foci was found at doses of 0.001 to 0.1 ppm, however, the signiˆcant increase of their number and area was observed at 1 and 10 ppm (8).

Potassium bromate, known as a pollutant of tap water and as a food additive, was shown to exert carcinogenicity in the rat kidney (10). Furthermore, potassium bromate was reported to belong to the class of the DNA non-reactive genotoxic carcinogens (10). To investigate the mutagenicity of potassium bromate in the rat liver, it was administered to Big Blue rats in the drinking water at doses of 0.02 to 500 ppm for 16 weeks (11). The signiˆcant increase of lacI gene mutations was found at a dose of 500 ppm, however, no mutagenicity was observed for doses lower than 125 ppm. Furthermore, potassium bromate at a dose of 500 ppm was found to induce a speciˆc mutation (GC to TA transversion), which was previously shown to be induced by oxidative DNA damage. Moreover, we have measured the formation of kidney DNA 8-OHdG, one of the oxidative DNA modiˆcations, and a signiˆcant increase was found only at a dose of 500 ppm. From these results a no-observed eŠect level for the lacI gene mutation and oxidative DNA damage levels induced by potassium bromate appeared to continue from very low doses, with a detectable eŠect at the dose of 500 ppm. In line with these data, in the present experiment, no preneoplastic or neoplastic lesions were histopathologically detected in the kidney. Next, for examination of the carcinogenicity of potassium bromate in the kidney, male Wistar rats were treated with N-ethyl-N-hydroxyethylnitrosamine for initiation of kidney carcinogenesis, and thereafter administered potassium bromate over wide dose range (unpublished data). Enhancement of kidney tumors induction was observed in the highest dose group (the dose was changed during the experiment from 500 to 250 ppm due to the observed toxicity), but not for the

Fig. 2. Risk of liver cancer: Reaction curves for the carcinogenicity markers dependent on the dose of MeIQx. This is an illustration for MeIQx eŠects in log-log scale.

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Shoji Fukushima et al.

dose of 125 ppm or at lower doses. 2

Conclusions Our results indicate the existence of a threshold, at least a practical threshold, for the carcinogenicities of genotoxic carcinogens. Carcinogens can be divided into those that directly interact with DNA (DNA reactive genotoxic carcinogens) and those that indirectly aŠect DNA, for example by induction of reactive oxygen species as results of biological responses of cells to the reactions of the chemical with cellular components other than DNA (DNA non-reactive genotoxic carcinogens). The DNA reactive genotoxic carcinogens included those whose metabolites interact with DNA, e.g., heterocyclic amines and N-nitrosocompounds. There appears to be the existence of a practical or apparent threshold for the DNA reactive genotoxic carcinogens. On the other hand, threshold for carcinogenicity of the DNA nonreactive genotoxic chemicals as well as non-genotoxic carcinogens can be called a perfect or true threshold. Examples of direct and indirect genotoxic carcinogens are the heterocyclic amines and N-nitrosocompounds (DNA reactive genotoxic carcinogens), and potassium bromate (DNA non-reactive genotoxic carcinogens).

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Acknowledgements: The authors would like to acknowledge the help of Masao Hirose (Div. Pathology, National Institute of Health Sciences), Yoichi Konishi (Dept. of Oncological Pathology, Cancer Center, Nara Medical University), Dai Nakae (Tokyo Metropolitan Institute of Public Health), Shuzo Otani (Dept. Biochemistry, Osaka City Univ. Grad. Sch. Med.), Tomoyuki Shirai (Dept. Pathology, Nagoya City Univ. Grad. Sch. Med.), Michihito Takahashi (Div. Pathology, National Institute of Health Sciences), Masae Tatematsu (Div. Oncological Pathology, Aichi Cancer Center Research Institute), Hiroyuki Tsuda (Dept. Mol. Toxicol., Nagoya City Univ. Grad. Sch. Med.) and Keiji Wakabayashi (Cancer Prevention Research Div., National Cancer Center Research Institute). These studies were supported by a grant from the Japan Science and Technology Corporation, included in the Project of Core Research for Evolutional Science and Technology (CREST) and by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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