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The carcinogenicity of the low amounts of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and to investigate mechanisms by which IQ exerts its carcinogenic effects. A total of 1595 male F344 rats were divided into seven groups and administered with IQ at doses of 0, 0.001, 0.01, 0.1, 1, 10 and 100 p.p.m. in the diet for 16 weeks. We found that IQ doses of 1 p.p.m. and below did not induce preneoplastic lesions in either the liver or the colon, while IQ doses of 10 and 100 p.p.m. induced preneoplastic lesions in both of these organs. These results demonstrate the presence of no-effect levels of IQ for both liver and colon carcinogenicity in rats. The finding that p21Cip/WAF1 was significantly induced in the liver at doses well below those required for IQ mediated carcinogenic effects suggests that induction of p21Cip/WAF1 is one of the mechanisms responsible for the observed no-effect of low doses of IQ. Furthermore, IQ administration caused significant induction of CYP1A2 at doses of 0.01–10 p.p.m., but administration of 100 p.p.m. IQ induced CYP1A1 rather than CYP1A2. This result indicates the importance of dosage when interpreting data on the carcinogenicity and metabolic activation of IQ. Overall, our results suggest the existence of no-effect levels for the carcinogenicity of this genotoxic compound. (Cancer Sci 2011; 102: 88–94)
Exposure to environmental carcinogens is one of the most significant causes of human cancers. Determination of the dose-response relationship between carcinogen exposure and induction of cancer is one of the most important areas of chemical risk assessment. Of particularly high priority is the cancer risk assessment of dietary carcinogens.
Heterocyclic amines (HCA) are well known dietary genotoxic carcinogens derived from cooked protein-rich foods such as meat and fish,(1–3) and the carcinogenicities of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) have been widely investigated in various animal models. MeIQx induces cancers of the liver, zymbal gland, skin and clitoral gland in rats,(4) and caners of the liver and lung, and lymphoma and leukemia in mice.(5) PhIP induces colon cancers and mammary gland cancers in rats,(6) and lymphomas in mice.(7) IQ induces cancers of the liver, colon, mammary and zymbal glands in rats, caners of the liver, lung and forestomach in mice, and cancer of the liver in nonhuman primates.(8–10) MeIQx and PhIP are classified as category 2B compounds (possibly carcinogenic to humans) and IQ is classified as a category 2A compound (probably carcinogenic to humans) by the International Agency for Research on Cancer.(11) Therefore, although the concentrations of HCA in food are low, they constitute a potential hazard, and there is concern regarding the carcinogenic effects of low doses of these HCA.
Based on the view that even minute doses of a genotoxic carcinogen has the potential to produce irreversible deleterious genetic changes in the DNA of a target organ cell and the argument that if sufficient numbers of test animals are used the carcinogenic effect of a minute dose can be demonstrated, it is generally assumed that genotoxic carcinogens exert a non-threshold carcinogenic effect. However, the carcinogenicities of most genotoxic carcinogens are determined by experimental animal carcinogenicity studies using doses that are generally orders of magnitude higher than actual human exposure levels and the dose-response curves obtained are then extrapolated to zero using a non-threshold mathematical model. This approach, however, is being challenged as advancements in the understanding of the molecular mechanisms of carcinogenesis are being made and experimental evidence showing that genotoxic carcinogens do not exert mutagenic and carcinogenic effects at low doses accumulates.(12–19)
Previously, we demonstrated the existence of no-effect levels of MeIQx for both hepatocarcinogenicity and in vivo mutagenicity in various carcinogenesis models in different rat strains.(17,20–22) It has also been shown that low doses of PhIP do not exert either initiation or promotion activities in colon carcinogenesis in the rat.(23,24) However, little is known about the carcinogenic potential of low doses of IQ.
In addition, little is known about the mechanisms underlying the carcinogenicities of lower doses of HCA, but incorporation of mechanistic information is critical for quantitative cancer risk assessment. The purpose of the present study is to determine the relationship between administration of low doses of IQ and induction of preneoplastic lesions in the liver and colon in rats, and to investigate carcinogenic mechanisms of action of various doses of IQ by evaluating DNA-adduct formation, oxidative DNA damage and expression levels of genes involved in metabolic activation of IQ, cell proliferation and DNA damage repair in the liver.
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Dose-response relationships for genotoxic carcinogens have been a topic of intense scientific and public debate. High doses of the genotoxic dietary carcinogen IQ have been demonstrated to induce liver and colon cancers in rats (300 p.p.m. in diet)(8) and liver cancers in nonhuman primates (10 mg/kg b.w./day).(10) However, as the concentrations of IQ in food are generally extremely low,(11) there is uncertainty regarding the carcinogenicity of the doses of IQ to which humans are exposed. The present study shows that IQ at doses of 1 p.p.m. (0.08 mg/kg body weight [b.w.]/day) and lower did not induce either GST-P-positive foci in the liver or ACF in the colon. Only in the groups administered higher doses of IQ, 10 p.p.m. (0.76 mg/kg b.w./day) and 100 p.p.m. (7.83 mg/kg b.w./day), were increases in GST-P-positive foci and ACF observed.
GST-P-positive foci and ACF are well-established preneoplastic lesions of the liver and colon, respectively, in rats. These lesions have been accepted as useful end-point markers in the assessment of carcinogenic effects of environmentally relevant concentrations of carcinogens as they can extend the range of observable effect levels.(24,26) Therefore, the results of the present study suggest the presence of no-effect levels of IQ for both liver and colon carcinogenicity in rats and indicate that the dose-response relationship for carcinogenicity of low dose IQ is nonlinear.
Several threshold mechanisms for genotoxic carcinogens have been suggested, including induction of detoxification processes, cell cycle delay, DNA repair, apoptosis and the suppression of neoplastically transformed cells by the immune system.(12,13,15,33) However, little in vivo evidence is available. To explore mechanisms underlying the carcinogenicity of low doses of IQ, we examined the relative mRNA expression of a panel of genes involved in cell proliferation, cell cycle regulation, DNA repair and IQ metabolic activation. We found that the cell proliferation marker PCNA was significantly increased only at a dose of 100 p.p.m., a dose that is carcinogenic. The cell cycle negative regulator p21Cip/WAF1, on the other hand, was significantly induced at a dose of 0.01 p.p.m., a dose well below that which induced the formation of preneoplastic lesions. Furthermore, the finding that the levels of p21Cip/WAF1 in the groups administered 10 p.p.m. and less were much lower than that of the group administered 100 p.p.m. implies that hepatocytes have adequate capacity to cope with the type of damage that is repaired by the p21Cip/WAF1 pathway when exposed to low doses of IQ, but that the repair capacity of these hepatocytes, even in the presence of high p21Cip/WAF1 expression, can be overwhelmed when the cell is subjected to very high doses of IQ. It is reasonable to suggest that suppression of cell cycle progression by p21Cip/WAF1 followed by DNA repair is at least one of the mechanisms responsible for the observed no-effect of low doses of IQ in rats in the present model.
It is known that the vast majority of DNA damage is repaired by base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR).(34) APE-1 plays an essential role in the BER repair process by cleaving the phosphodiester backbone.(35) The activities of two different heterodimeric complexes, MSH2-MSH3 and MSH2-MSH6, belonging to the MMR system are mainly responsible for the post-replicative repair of mismatches.(36) We found that IQ significantly increased the expression levels of APE-1 but not MSH2 and MSH3 at doses of 10 and 100 p.p.m. in the liver. It has also been reported that IQ has no effect on expression of ERCC1, which is a key molecule in the NER process.(37) These findings suggest that BER rather than MMR or NER responds to IQ-induced DNA damage.
GADD45 is involved in a variety of growth regulatory mechanisms, including DNA repair, growth arrest and apoptosis.(38) It is induced by genotoxic and certain other cell stresses by p53-dependent and independent pathways.(39,40) GADD45 expression was significantly induced in the 100 p.p.m. group. The fact that significant induction of APE-1 and GADD45 was observed only at the highest doses of 10 and/or 100 p.p.m. indicate the IQ-induced DNA damage response is dose-dependent. Moreover, the fact that in the groups with low doses expression of APE-1 and GADD45 were not affected and that there was a significant but moderate induction of p21Cip/WAF1 imply that normal physiological levels of these genes are sufficient to repair the DNA damage caused by low doses of IQ. However, the expression levels of these genes are all increased by higher carcinogenic doses of IQ. A reasonable explanation of the no-effect of low doses of IQ and the carcinogenicity of high doses of IQ is that carcinogenicity is the consequence of a disruption in the balance between DNA damage and repair and between abnormal cell proliferation and apoptosis or cell cycle regulation.
Our results show that p53 gene expression is not induced by administration of IQ. Furthermore, p53-deficient mice do not show higher susceptibility to IQ-induced liver carcinogenesis than wild type mice.(41) These results suggest that p53 does not have a significant impact on the carcinogenicity of IQ.
DNA adduct formation by metabolic activation of IQ is believed to play an important role in the carcinogenicity of IQ.(42) Formation of IQ-DNA adducts in the liver showed a linear dose-dependency and proved to be one of the most sensitive end-points for the detection of exposure to IQ. Adduct formation was detectable in groups administered far lower doses of IQ compared with detection of other end-points such as cell proliferation and preneoplastic lesion induction. That IQ-DNA adduct formation was not detected in the 0.001 p.p.m. group was most likely due to the detection limit of the assay. It should be noted that DNA adduct is a premutagenic lesion and not necessarily correlated to the frequencies of mutation and cancer induced by genotoxic compounds. For example, it is known that IQ forms DNA adducts in the kidneys and stomach of both rats and monkeys, but does not induce tumors in these organs.(43,44) Our present findings of a linear dose-response of IQ-DNA adduct formation and a nonlinear carcinogenic dose-response to IQ administration support the idea that IQ-DNA adducts do not necessarily lead to mutation and formation of cancerous lesions. Our results are also in line with previous results on HCA including MeIQx(1,18,45) and PhIP.(24) These results can be explained, at least in part, by the actions of gene products such as p21Cip/WAF1, GADD45 and APE-1 and the other repair genes for DNA damage. Moreover, in the case of MeIQx, it has been suggested that formation of DNA adducts alone might not be sufficient to produce cancers and that the MeIQx-induced genetic alterations in the liver are enhanced by liver regeneration induced by high doses of MeIQx itself.(1) Therefore, while IQ-DNA adduct formation is important in IQ carcinogenicity, high levels of adduct formation are likely required and other factors such as cell proliferation can affect the balance between DNA damage and repair and lead to fixation of DNA mutations into the cell’s genome.
It has been demonstrated in vitro that IQ is more efficiently metabolized and activated by CYP1A2 than by CYP1A1 or CYP1B1.(46) However, limited in vivo data are available. In a study by McPherson et al.(47), no significant induction in mRNA expression level or activity of either CYP1A1 or CYP1A2 were reported in the livers of rats receiving 300 p.p.m. IQ in the diet for 52 weeks, but these enzymes were significantly increased after daily administration of 20 mg/kg b.w. IQ by oral gavage for 3 days; in the average adult rat, a dose of 300 p.p.m. IQ in the diet is approximately equivalent to administration of 20 mg/kg b.w. IQ by oral gavage. The results of the present study revealed that IQ significantly induced CYP1A2 expression at doses from 0.01 to 10 p.p.m., but CYP1A2 was not induced in the 100 p.p.m. group. The lack of effect of 100 p.p.m. IQ on CYP1A2 expression is consistent with the results in rats receiving 300 p.p.m. IQ in the diet for 52 weeks.(47) Significant increases in CYP1A1 expression in the 100 p.p.m. group provide an alternative mechanism that can compensate for decreased CYP1A2 activity. However, as noted above, in apparent contrast to our results, in the study by McPherson et al.,(47) administration of 300 p.p.m. IQ over the course of 52 weeks did not induce CYP1A1. Therefore, it is reasonable to postulate that the dose-relationship between IQ and induction of CYP1A1 is not a simple dose-response. CYP1B1 does not appear to be involved in the metabolism of IQ at doses up to 100 p.p.m. in rats. The findings described above demonstrate the importance of taking into account dosage, duration and route of exposure in interpretation of the data on metabolic activation of IQ. Further studies on the dose-response relationships between chronic IQ exposure and the protein expression levels and activities of detoxifying enzymes, especially at doses relevant to human exposure, would provide further insight into the role of metabolic activation in IQ carcinogenicity.
Oxidative DNA damage does not appear to play a role in IQ-induced carcinogenesis. In the present study, no significant changes in 8-OHdG levels or Ogg1 expression levels in the livers of IQ-treated rats were observed. Our results are consistent with the recent findings in IQ-treated Big Blue rats that oxidative stress was not responsible for the initiation of IQ-induced carcinogenesis in the liver and colon.(37) In this respect, IQ is different from MeIQx, in which oxidative DNA damage plays an important role in liver carcinogenesis.(48)
In summary, the present study provides the first experimental data on the carcinogenicity of low doses of IQ in both the liver and colon of the test animal and compares the effect of IQ at the cellular level with its carcinogenic effect. Our findings support the idea that there is a practical threshold that should be considered when evaluating the risk of genotoxic carcinogens. To this end, further accumulation of data, especially mechanistic data, should be promoted to facilitate not only an understanding of the carcinogenic effects of low doses of genotoxic carcinogens but also to establish an accurate means of quantitative risk assessment.