No-Observed Effect Levels for Carcinogenicity and for in vivo Mutagenicity of a Genotoxic Carcinogen

Manabu Hoshi*,{dagger}, Keiichirou Morimura*, Hideki Wanibuchi*, Min Wei*, Eriko Okochi{ddagger}, Toshikazu Ushijima{ddagger}, Kunio Takaoka{dagger} and Shoji Fukushima*,1

* Department of Pathology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan; {dagger} Department of Orthopaedic Surgery, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan; and {ddagger} Carcinogenesis Division, National Cancer Center Research Institute, 1-1, Tsukiji, 5 chome, Chuo-ku, Tokyo 104-0045, Japan

Received April 14, 2004; accepted June 10, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To elucidate the relationship between in vivo carcinogenic and mutagenic potentials of genotoxic carcinogens, low doses were tested in the livers of Big Blue transgenic rats with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Male Big Blue rats were fed a diet containing 0.001, 0.01, 0.1, 1, 10, or 100 ppm of MeIQx for 16 weeks, and the frequencies of lacI mutants and glutathione S-transferase placental form (GST-P) positive foci in the liver were determined. The mutation frequencies significantly increased at doses of 10 and 100 ppm, and GST-P positive foci significantly increased at a dose of 100 ppm. However, no statistical increases in both frequencies were observed at lower doses. MeIQx most frequently induced G frameshifts, followed by G to T transversions. Thus, no observed effect level (NOEL) was demonstrated for both carcinogenicity in terms of preneoplastic lesion induction and in vivo mutagenicity of MeIQx, and the NOEL for in vivo mutagenicity was lower than that for carcinogenicity.

Key Words: MeIQx; no-observed effect level; in vivo mutagenicity; carcinogenicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
More than 80% of human cancers may be related to carcinogenic environmental chemicals (Doll and Peto, 1981Go). Carcinogenicity is generally detected by long-term carcinogenicity testing in rodents using doses considerably in excess of human exposure levels. The situation is complicated by the fact that carcinogens can be classified as either genotoxic or non-genotoxic according to results of in vitro genotoxicity tests. The currently accepted view is that no threshold exists for the carcinogenic potential of genotoxic carcinogens, with the response curve approaching zero in a linear fashion with extrapolation from the doses used in carcinogenicity testing (Preussmann, 1980Go). Actual human cancer risk of genotoxic carcinogens is very difficult to assess, because few directly obtained data are available from the carcinogenicity testing at low doses such as human daily exposure levels (Truhaut, 1979Go). Therefore, practical information concerning cancer risk remains inadequate, and the need for investigation in this field is urgent.

A heterocyclic amine produced in the cooking of meat and fish, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), is a potent genotoxic carcinogen that shows strong mutagenicty in the Ames assay (Johansson and Jagerstad, 1994Go; Sugimura, 1986Go). MeIQx is metabolized in vivo to N-hydroxyamino derivatives by cytochrome P450 and then activated by the esterification enzymes, acetyltransferase and sulfotransferase. The activated form produces DNA adducts, particularly involving guanine bases, which can lead to mutations (Langouet et al., 2001Go). MeIQx induces hepatocellular carcinomas in male rats with exposure at high doses (Kato et al., 1988Go; Kushida et al., 1994Go) and also glutathione S-transferase placental form (GST-P)-positive foci, well-established preneoplastic lesions in the livers of rats which have been accepted as end-points for assessing carcinogenic potential in the liver in medium-term bioassay (Ito et al., 1988Go).

Somatic mutation is considered responsible for carcinogenesis with stepwise accumulation of alterations in cancer-related genes leading to malignant neoplasia (Vogelstein and Kinzler, 1993Go). DNA damage and irreversible DNA base change appear to play an important role in this multiple-step carcinogenesis. The Big Blue transgenic rodent mutagenesis assay is used widely for in vivo mutagenicity assays, and the reliability of this system has been established (Kohler et al., 1991Go). The system originally was designed to assess DNA damage in vivo in a tissue-specific manner. We have used the rats in this system to determine both mutagenic and carcinogenic potentials of test substances in the same individual animal.

Our group recently showed no increase in quantitative data for GST-P-positive foci in liver on treatment of rats with very low doses of MeIQx, indicating the existence of a threshold for carcinogenicity (Fukushima, et al., 2002Go, 2003Go). Mutagenicity remained at issue, however (Solomon et al., 1996Go) and in vitro mutagenicity tests of genotoxic carcinogens generally show DNA damage to be dose-dependent in a truly linear manner (Maier and Schawalder, 1988Go). Therefore, a discrepancy may exist between in vivo carcinogenic and in vitro mutagenic potential. However, little information is available concerning in vivo mutagenicity of genotoxic carcinogens, especially at low doses.

As the first step toward to elucidating the mechanism of MeIQx-induced mutagenicity, it is necessary to establish suitable target genes as biomarkers in the field of genotoxicology. Most genotoxic carcinogens may exert not only carcinogenicity but also in vivo mutagenicity in an organ specific manner but this aspect has not been hitherto explored in detail. Few background data on susceptibility of non-target organs to induction of mutations by chemical carcinogens have been generated (Nishikawa et al., 1997Go). Therefore, there is further need for investigation at the whole body level, including target and non-target organs after treatment with genotoxic carcinogens and for identification of the tissue-specificity of in vivo mutagenicity, compared to its carcinogenicity.

In the present study, to determine in vivo carcinogenicity and mutagenicity from treatment with MeIQx at low doses representing human exposure levels, we used a rodent mutagenesis system together with a medium-term liver carcinogenicity test, which we employed previously for low dose carcinogenicity of rats (Fukushima et al., 2002Go). At high doses, in vivo mutagenicity was examined for multiple organ systems.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. The carcinogen, MeIQx (purity, 99.9%), was obtained from Nard Institute (Nishinomiya, Japan).

Animals and treatments. A total of 40 Big Blue male rats with genetic background of Fisher 344 were purchased from Stratagene (La Jolla, CA) at the age of 4–5 weeks. The animals were divided into seven groups, receiving MeIQx at doses of 0 ppm (group 1, control), 0.001 ppm (group 2), 0.01 ppm (group 3), 0.1 ppm (group 4), 1 ppm (group 5), 10 ppm (group 6), and 100 ppm (group 7) in powdered basal diet (Oriental MF, Oriental Yeast, Co., Ltd., Tokyo, Japan) continuously for 16 weeks after an acclimation period of one week. The lowest level, 0.001 ppm of MeIQx, was established as equivalent to the daily intake of this carcinogen in humans (IARC, 1993Go). MeIQx diets were prepared by Oriental Yeast Co., Ltd, and the MeIQx concentration in each was confirmed by high-performance liquid chromatography (HPLC). Group 1 comprised 10 rats, while groups 2 to 7 included 5 rats each. The animals were housed two or three to a plastic cage with paper chips for bedding under constant conditions (room temperature, 22 ± 2°C; relative humidity, 55.5%; light/dark cycle, 12:12 h). Body weights, food consumption, and water intake were recorded weekly.

Collection of tissue samples. Upon completion of MeIQx treatment or basal diet alone, the rats were killed under ether anesthesia for removal of livers for mutation analysis and immunohistochemical examination to detect GST-P-positive foci and proliferating cell nuclear antigen (PCNA). Formalin-fixed, paraffin-embedded liver tissue (three sections from each of left lateral lobe, medial lobe, and the right lateral lobe) was either routinely stained with hematoxylin and eosin (H&E) or immunohistochemically stained for GST-P-positive foci or PCNA positive cells. Remaining unfixed liver tissues were quickly frozen in liquid nitrogen and kept frozen at –80°C until DNA isolation.

For multiple organ analysis, Big Blue rats at the age of 4–5 weeks were employed. They were divided into two groups of five animals each receiving MeIQx at doses of 0 and 100 ppm continuously for 16 weeks. The rats were sacrificed and liver, colon, Zymbal gland, kidney, spleen, lung, testis, heart, brain, fat tissue, and skeletal muscle of quadriceps femoris were collected. Formalin-fixed samples of skeletal muscle of brain, liver, lung, testis, kidney, spleen, and heart, were routinely embedded in paraffin wax for staining of sections with hematoxylin and eosin. The remaining tissues were quickly frozen in liquid nitrogen and kept frozen –80°C until DNA isolation.

Plaque color screening assay. High-molecular-weight genomic DNA was extracted by the phenol/chloroform extraction methods, and phages were recovered using a Transpack Packaging Extract (Stratagene, CA) (Rogers et al., 1995Go). Mutation frequency in lacI gene was analyzed by the method recommended by Stratagene (Okochi et al., 1999Go; Suzuki et al., 1996aGo). Phages from each fully blue plaque were stored in 500 µl of SM buffer (0.1 M NaCl, 8 mM MgSO4, 50 mM Tris-HCl at pH 7.5, and 0.01% gelatin) with 50 µl of chloroform at 4°C. For analysis of the lacI gene, blue plaques were isolated and replated at low density on X-gal containing NZYM agar plates to confirm the mutant plaque. The mutant phages were independently collected in SM buffer containing 10% chloroform for direct sequencing.

DNA sequencing of mutant lacI. Direct sequencing was performed with an ABI PRISM 3100 Genetic Analyzer (PE Applied Biosystems. Chiba, Japan). The DNA sequences of all mutants isolated from the livers of control and MeIQx-treated rats were analyzed with designed primers (Ushijima et al., 1995Go). Mutations were classified into three categories: single base substitution, frameshift, and others. When a base pair deletion mutation occurred in the same nucleotide, the nucleotide with the lowest number was assigned as the mutation site.

Immunohistochemical detection of GST-P-positive foci and PCNA. Immunohistochemical detection of GST-P-positive foci was performed by the avidin-biotin-peroxidase complex (ABC) method described previously (Kitano et al., 1998Go). Foci comprising two or more positive hepatocytes were counted under a light microscope. Total areas of sections were measured using a color image processor (IPAP Sumika Technos, Osaka, Japan), and numbers of foci per square centimeter of liver tissue were calculated by a well-trained pathologist. Proliferating epithelial cells ratio were detected with anti-PCNA antibody staining (DAKO Japan, Kyoto, Japan), also using the ABC method.

Statistical analysis. Statistical analysis of the observed values was performed using the Student's t-test. All calculations were performed with the aid of the Stat View statistical package (Abacus Concepts, Inc., Berkeley, CA). Significance of differences between the expected and the observed values of mutation frequencies and GST-P positive foci were analyzed using Super ANOVA Duncan New Multiple Range analysis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Final Body Weights, Liver Weights, Total Intakes of MeIQx, and Histological Examination
Final body weights, liver weights, and total intakes of MeIQx are shown in Table 1. All rats survived in good condition until the scheduled time, 16 weeks, for liver examination, and no adverse effects were observed in rats treated with MeIQx at various doses. No statistically significant differences were found between groups with regard to body or liver weights. Likewise, food consumption and water intake did not differ (data not shown). Average total MeIQx intakes in each group were dose-dependent. No obvious macroscopic or microscopic changes, including tumors, were detected in the liver, colon, Zymbal gland, kidney, spleen, lung, testis, heart, brain, fat tissue, and skeletal muscle of quadriceps femoris of any rat.


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TABLE 1 Final Body Weights, Liver Weights, and Total MeIQx Intakes

 
In vivo Mutagenicity at Low-Dose Treatment of MeIQx
For the in vivo mutagenesis assay at low doses, data for total plaque forming units, mutation-containing-plaque forming units, and calculated mutation frequencies are shown in Table 2. Mutation frequency in the liver of the control group treated without MeIQx was 14.8 ± 7.6, and mutation frequencies in groups treated with 1 ppm and less were not significantly different from that of the control group. Values for 10 and 100 ppm MeIQx groups were significantly increased compared with the control group frequency. The in vivo mutagenic dose-response curve of mutation frequency at low doses did not parallel the MeIQx dose (Fig. 1A).


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TABLE 2 Mutation Frequency of LacI and GST-P Positive Foci in Liver

 


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FIG. 1. Mutation frequencies (A) and GST-P-positive foci in the liver (B). Mutation frequencies of groups treated with 1 ppm and less MeIQx demonstrate no significant difference from the control group. In contrast, the values for 10 and 100 ppm MeIQx groups are significantly increased. The numbers of GST-P positive foci of groups treated with 10 ppm and less similarly demonstrate no statistically significant difference from the controls, only the value for the 100 ppm-treated group being significantly increased. *p < 0.001 vs. control. Broken lines (A, B) represent dose-response linear lines, which are predicted from two points at doses of 0 and 100 ppm.

 
Carcinogenicity at Low-Dose Treatment of MeIQx
Numbers of GST-P-positive foci in the liver did not differ significantly from those in the control group at 10 ppm and less (Table 2). Only foci in animals receiving the 100 ppm treatment were significantly increased beyond control-group numbers. The in vivo carcinogenic dose-response curve in terms of numbers of GST-P-positive foci did not parallel MeIQx doses (Fig. 1B).

PCNA-Positive Cells
PCNA-positive cells were counted to quantify cell proliferative ratio in the liver. PCNA-positive liver cells per 1500 hepatocytes were expressed as percentages. No significant differences were observed between MeIQx treatments and control groups (Fig. 2). Thus, the hepatocyte-proliferation rate was similar in all groups. Furthermore, with the highest dose no significant differences were detected between MeIQx-treated and control groups in the various organs, Zymbal gland, kidney, spleen, lung, testis, heart, brain, and skeletal muscle (data not shown).



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FIG. 2. PCNA indices in the liver. The cell proliferation indices do not differ significantly between the groups. Columns show means; Bars show SD.

 
In vivo Mutagenicity at High-Dose Treatment of MeIQx
Data for total plaque forming units (pfu), mutated pfu, and calculated mutation frequency for the multiple organs in the high dose experiment are shown in Table 3. The administration of MeIQx (five mice) significantly increased lacI mutation frequency in the liver (p < 0.0001), Zymbal gland (p = 0.0078), and colon (p = 0.006), compared to the control (five rats). Especially in the liver, mutation frequency was extremely elevated. On the other hand, no statistically significant elevation of the lacI mutation frequency was observed in the kidney, spleen, lung, testis, heart, brain, fat tissue, and skeletal muscle, lung, kidney, spleen, testis, brain, heart and fat tissue.


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TABLE 3 LacI Mutation Frequency in Big Blue Rat

 
LacI Sequence Analysis
The results of sequence analysis of 150 MeIQx-treated group and two control group liver mutated blue plaques are shown in Table 4. The most frequent mutation type in the MeIQx-treated group was the frameshift (60.0%), followed by base substitution (32.0%). The most frequent mutation was 1 bp GC deletions (48.0%), followed by GC to TA transversion (26.7%) and then 2 bp GC deletions (9.3%). The two spontaneous mutations were a GC to AT transition and a T insertion. CpG sites were involved in 55.3% of the liver mutations.


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TABLE 4 Mutation Spectra of LacI in Liver

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study disclosed new aspects of carcinogenic and in vivo mutagenic properties of the genotoxic carcinogen, MeIQx. Dose-response curves for induction of GST-P positive foci and in vivo mutagenicity in liver were nonlinear in the region near zero. Thus, NOEL was demonstrated not only for carcinogenicity but also for in vivo mutagenicity at low doses. The NOEL for in vivo mutagenicity was lower than that for carcinogenicity.

Absence of a threshold for carcinogenicity of genotoxic carcinogens has long been assumed because, even when low doses of carcinogen do not induce tumors in rodents, genotoxic carcinogens are considered capable of damaging DNA at any exposure level, ultimately resulting in a tumor causing (Lutz, 1998Go). However, the present in vivo data disagree with the assumption.

To support non-linearity of dose-response curve induced by MeIQx treatment, we compared our non-linear curve of mutation frequency and GST-P positive foci to presumptive dose-response linear curve. Assuming linearity of dose-response for at low dose extrapolating from high dose, we could represent the dose-response linear line of mutation frequency from two points at dose of 0 and 100 ppm in this report. Herein, cancer risk per ppm (6.26/ppm) could be calculated from 641.5 ± 14.8 per 100 ppm (Fig. 1A). At 10 ppm one would expect a frequency of 14.8 + 6.26 x 10 = 77.4, compared to the observed value of 51.4 ± 14.2; at 1 ppm 14.8 + 6.26 x 1 = 21.1, compared to the observed value of 29.4 ± 21.7, samely, at 0.1 ppm 14.8 + 0.626 = 15.4, compared to the observed value of 19.9 ± 17.5, at 0.01 ppm 14.8 + 0.06 = 14.9, compared to the observed value of 15.6 ± 9.6, at 0.001 ppm 14.8 + 0.006 = 14.8, compared to the observed value of 14.9 ± 12.2. This assumption of the dose–response linear line was also applied to the data of GST-P positive foci (Fig. 1B). The expected numbers were 1.13 at 10 ppm, 0.50 at 1 ppm, 0.44 at 0.1 ppm, 0.43 at 0.01 ppm, 0.43 at 0.001 ppm, compared to the observed value of 0.78 ± 0.49, 0.19 ± 0.26, 0.33 ± 0.38, 0.31 ± 0.68, 0.18 ± 0.20, respectively, at each dose. For analysis of relationship between the expected and our observed value of mutation frequencies and GST-P positive foci, these data was statistically calculated. Concerning the mutation frequencies, the significance (p < 0.05) was found at dose of 10 ppm, and the others were not. Concerning the value of GST-P positive foci, no significance was found at any dose.

Genotoxic carcinogens including MeIQx can induce DNA damage and fixation to result in potentially carcinogenic mutations (Langouet et al., 2001Go). While specific cancer-related genes targeted by MeIQx have not been identified, if the 30 to 40 copies of lacI genes inserted into chromosome 4 in Big Blue rats are randomly affected by genotoxic carcinogens, they can serve as a marker of overall genetic instability in the presence of a mutagen. Thus, we studied mutation frequency of the lacI gene as a surrogate for cancer-related genes.

According to our results, NOEL of genetoxic carcinogens may exist at low doses, at which many adaptive and/or defensive responses within the organism may work to maintain biologic homeostasis. Metabolic pathways for detoxification, immune responses, cytokines, scavenger systems, hormones, and up-regulation of suppressor genes may play important roles (Lutz, 2001Go). Apoptosis, as well as DNA repair mechanisms by which altered bases are removed from DNA and replaced with the correct base, could counteract low level damage to DNA (Oesch et al., 2000Go). In radiation carcinogenesis, especially at low doses, such biologic adaptive responses have been reported (Wolff, 1998Go), and defenses against low doses of chemical carcinogens may be similar.

In estimation of the carcinogenic influence of genotoxic carcinogens, DNA adducts are useful markers for examination for exposure assessment (Bailey et al., 1994Go; Troxel et al., 1997Go). However, this method does not represent demonstration of a carcinogenic effect, since DNA adduct formation does not always result in actual mutations. Moreover, MeIQx-DNA adducts can be detected in rat liver in a dose-dependent manner (Yamashita et al., 1990Go), with small but apparent effects at low doses which are not in agreement with the exhibited threshold for carcinogenic potential at low doses (Fukushima et al., 2003Go).

Cell proliferation must be considered for understanding mechanisms of chemical carcinogenesis (Cohen and Ellwein, 1990Go), since DNA alterations induced in cancer-related genes can influence cell growth. We presently used immunohistochemistry for PCNA to estimate cell proliferative activity. If cell proliferation is activated, mutation frequency also may be enhanced with cell cycle acceleration, since gene mutations such as those of lacI gene can be transmitted to daughter cells by mitosis. However, our PCNA analysis showed no significant difference between controls, low doses, and high doses of MeIQx. So, the increase of mutation frequency at high-dose MeIQx treatment was not caused by clonal expansion.

Human cancer risk assessment is presently estimated based on extrapolation to low doses of results from animals receiving much higher doses than with normal human exposure. Other researchers have described curves in carcinogenic potential tests at high doses of MeIQx as showing a dose-dependent response (Yamashita et al., 1990Go) but dose-response curves for carcinogenicity and initiation activity actually differ between high and low exposure (Fukushima et al., 2002Go, 2003Go), and Williams et al. (2000)Go also demonstrated the mechanistic basis for nonlinearities and thresholds in rat liver carcinogenesis by genetoxic carcinogens. Taking together, we would argue that genotoxic carcinogens might show threshold, at least practical threshold.

Transgenic animal mutagenesis systems like the Big Blue have been developed to allow measurement of in vivo mutagenicity of any organ of interest, although the validity of the lacI gene as a human cancer risk marker remains to be confirmed (Suzuki et al., 1996bGo). Our present study showed in vivo mutagenicity to be elevated in the liver, Zymbal gland, and colon, but not in any of the other tissues examined. It should be noted that high-dose treatment with MeIQx was reported to induce hepatocellular carcinomas and squamous cell carcinomas of the Zymbal gland in male F344 rats in long-term animal experiment (Kato et al., 1988Go). No report has demonstrated tumor occurrence in the colon in animal long-term bioassays as far as we know. However, macroscopically, aberrant crypt foci (ACF), considered to be possible preneoplastic lesions in this organ, have actually been detected in MeIQx-treated F344 rats (Tanakamaru et al., 2001Go). So our experiments showed in vivo mutagenicity and carcinogenicity were completely collaborated at organ level, differing from the previous article showing no direct correlation between mutation frequencies and cancer incidences in mice (Nagao et al., 2001Go). This discrepancy might be probably due to the differences of species. Further studies were required to disclose these points.

Most genotoxic carcinogens in our surroundings are known to leave specific DNA changes at the individual level, which accumulate to ultimately induce tumors. In order to understand the influence of a genotoxic carcinogen, it is very important to determine the spectrum of DNA damage, as well as the quantity in terms of mutation frequency. Some authors have reported that in the liver of transgenic mice treated with MeIQx, the main mutation is the GC to TA transversion (Ryu et al., 1999Go). High-dose MeIQx induces c-k-ras mutations in codon 12 in the colon of rats, featuring GGT to GAT single base substitutions (Kudo et al., 1991Go). In zebrafish embryos treated with MeIQx, about 60% of mutations were found to be deletions (Amanuma et al., 2002Go). In our present study, DNA samples from some of hepatic mutated blue plaque (150 plaques from exposed animals and 2 plaques from controls) were sequenced to examine the nature of the mutations. The mutational spectrum in the liver showed MeIQx to mainly cause mutations at GC pair sites, in accordance with occurrence of bulky adduct at guanine in DNA, such as C8-guanine adduct, N2-guanine adduct and oxidative stress marker, 8-hydroxyguanosine, although the contribution of these adducts due to MeIQx in the mutation spectrum is not yet known. Furthermore, most mutations involved CpG sites (55.3%), where MeIQx may also induce destablization, producing a bulky intermediate that finally causes mismatch. Our mutational spectrum for in vivo mutagenicity, especially involving guanine bases, is completely compatible with in vitro mutagenicity in the lacZ gene as a reporter of mutations of E. coli (Solomon et al., 1996Go). We concluded that MeIQx produces a very characteristic mutation spectrum, characterized by G deletion in vivo.

In conclusion, low doses of the genotoxic carcinogen, MeIQx may show a practical threshold, below which there are no effects, not only for carcinogenic potential but also for in vivo mutagenesis. The assumption that the carcinogenic response curve linearly approaches zero at very low doses is not reasonable for assessing human risk.


    ACKNOWLEDGMENTS
 
The author thanks Miss C. Imazato, Miss K. Touma, Miss M. Imanaka, Miss M. Dokoh, and Miss Y. Onishi for expert technical assistance. This research was supported by a grant from the Japan Science and Technology Corporation, included into the Project of Core Research for Evolution Science and Technology (CREST) in Japan.


    NOTES
 

1 To whom correspondence should be addressed. Fax: +81-6-6646-3093. E-mail: fukuchan{at}med.osaka-cu.ac.jp.


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