Carcinogenic risk of heterocyclic amines in combination – Assessment with a liver initiation model

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Abstract

Carcinogenic potential of heterocyclic amines (HCAs) was investigated using an in vivo 5-week initiation assay with quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver. Numbers of GST-P positive foci were significantly increased with individual administration of six different HCAs, indicating utility of the assay. It was therefore applied to investigate risk with multiple HCAs in combination. Unexpectedly, concomitant treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) did not result in any additive carcinogenicity. In the rats taking MeIQx prior to PhIP the value was almost equal to the sum total of individual data, indicating additive initiation activities. In contrast, simultaneous or prior administration of PhIP rather exerted inhibitory effects on the carcinogenic potential of MeIQx. Moreover, microarray and quantitative RT-PCR assessment revealed that PhIP induced cytochrome P450 1A1, responsible for both activation and detoxification of HCAs, more strongly than MeIQx. It is noteworthy that complex exposure to multiple HCAs is not necessarily associated with increased risk of carcinogenesis because they are simultaneously and continuously ingested under normal circumstances.

Introduction

More than 10 mutagenic and carcinogenic heterocyclic amines (HCAs) are produced by cooking or heating of meat or fish and these are now generally considered as important environmental risk factors for human carcinogenesis (Sugimura et al., 2004). Since humans are presumed to be simultaneously and continuously exposed to several HCAs in their diets, assessment of risks of these agents in combination is particularly important. In animal experiments, additive or synergistic effects of 5 or 10 different HCAs were indicated in the post-initiation phase using a medium-term liver bioassay (Hasegawa et al., 1991, Hasegawa et al., 1994). On the other hand, Tsuda et al. indicated that mixtures of HCAs do not always act additively and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) rather tended to inhibit the carcinogenic potential of other HCAs (Tsuda et al., 1999).

Difficulties may be experienced in conducting long-term animal experiments for assessment of combined risks since they require large numbers of animals and massive investment in facilities. One way to overcome the problems is to adopt an in vivo 5-week initiation assay system. This is based on the two stage hypothesis of carcinogenesis and has already been employed for assessment of initiation activities of chemicals by quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver (Sakai et al., 2002b). These preneoplastic lesions are considered reliable surrogates for liver tumors (Tatematsu et al., 1987). The 5-week initiation assay consists of induction of cell proliferation by two-thirds partial hepatectomy (PH) and subsequent administration of test chemicals to rats. If initiated cells are present in a liver, they rapidly grow to form GST-P positive foci with use of selection pressure, achieved with exposure to 2-acethylaminofluorene (2-AAF) and carbon tetrachloride (CCl4). In this assay non-hepatocarcinogens can also induce GST-P positive foci regardless of their normal target organs (Sakai et al., 2002b), presumably due to the proliferating status of hepatocytes during exposure. Importantly, we earlier found that initiation activities of different carcinogens were summated with multiple chemical administration in this assay (Sakai et al., 2002a).

In the present study, the same in vivo 5-week initiation assay model was employed to evaluate the initiation activities of six HCAs individually and in combination, with a special focus on PhIP and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Moreover, the expression of metabolic enzymes after administration of PhIP and/or MeIQx was investigated with cDNA microarrays and quantitative RT-PCR analysis to cast light on mechanisms of interaction of these two carcinogens.

Section snippets

Animals

Six weeks old male F344 rats were purchased from Charles River Japan Inc. (Atsugi, Japan) and housed in plastic cages on wood chips for bedding under constant conditions (12 h light/dark cycle, 60% humidity at 22 ± 2 °C). They were fed Oriental NMF diet (Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum and allowed to acclimatize for 1 week before the start of experiment, at which time they were 7 weeks old. All animals were handled in accordance with the guidelines for animal

Initiation activities of HCAs

Data for the numbers of GST-P positive foci induced by the six kinds of HCAs are summarized in Table 1. First, we individually administered each HCA at 50 mg/kg b.w. Induction of GST-P positive foci was significant with MeIQx, IQ, Trp-P-1, and Glu-P-1. There were no significant differences from the control with low doses of PhIP (50 and 100 mg/kg) and Glu-P-2 (50 mg/kg). However, on increasing the administration doses for PhIP to 200 mg/kg and Glu-P-2 to 100 mg/kg, significant development of GST-P

Discussion

Induction of GST-P positive foci was significant with each HCA, including the non-hepatocarcinogen PhIP, using the present in vivo 5-week initiation assay (Table 1), indicating its utility for analysis of the carcinogenic potential of this important group of environmental agents. HCAs are N-hydroxylated mainly by CYP 1A2 at the first step of their metabolic activation (Sugimura et al., 2004) and it has been reported that the metabolic activity of CYP 1A2 is maintained after PH (Trautwein et

Conflict of interest statement

The authors declare that there are no conflict of interest.

Acknowledgements

We thank Dr. Malcolm A. Moore for revision of the scientific English language. This work was supported in part by Grants-in-Aid from the Ministry of Health, Labour and Welfare and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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