Urinary excretion of N-OH-2-amino-3-methylimidazo[4,5-f] quinoline-N-glucuronide in F344 rats is enhanced by green tea

Carl W. Embola, John H. Weisburger1,,3 and Michael C. Weisburger2,

Department of Pathology, New York Medical College and
1 American Health Foundation, Valhalla, NY 10595 and
2 Case Western Reserve University, Cleveland, OH 44106, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
The effects of green tea on the metabolism of the food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) with emphasis on the formation of the detoxified glucuronides was studied. Two groups of 20 adult male and female Fischer 344 rats consumed 2% green tea or water for 6 weeks before being administered a single dose of 40 mg/kg body weight of [2-14C]IQ by oral gavage. Major metabolites in 24 h urine samples were separated by high-performance liquid chromatography (HPLC), including N-OH-IQ-N-glucuronide, 5-OH-IQ glucuronide and sulfate, IQ sulfamate and IQ itself. The structures of the main metabolites were established by mobility on the HPLC and by mass spectrometry. Sulfate esters and sulfamate were hydrolyzed by 0.1 N HCl for 15 min at 100°C, yielding 5-OH-IQ and high levels of IQ. HPLC of the resulting product showed the N-OH-IQ-N-glucuronide and the 5-OH-IQ glucuronide, as well as IQ. The male and female rats drinking tea displayed a significantly higher (P < 0.05) excretion of the two major glucuronides. We conclude that intake of green tea increases the excretion of N-OH-IQ-N-glucuronide, a detoxified metabolite of the proximate carcinogen N-OH-IQ.

Abbreviations: d.p.m., disintegrations per minute; HCAs, heterocyclic amines; HPLC, high performance liquid chromatography; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; N-OH-IQ, N-hydroxy-IQ; 5-OH-IQ, 5-hydroxy-IQ.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
2-Amino-3-methylamidazo[4,5-f]quinoline (IQ) belongs to a class of carcinogens known as heterocyclic arylamines formed during the cooking of meat, poultry and fish (1). Bioassays of IQ demonstrated a multi-target organotropism with specific cancer induction in the mammary gland, colon, liver and pancreas of rodents (13). Because many people in the world regularly consume foods that have been fried, grilled, barbecued or broiled, there is extensive exposure to this novel type of carcinogen. It is, therefore, important to understand its mechanism of action and also to develop ways of preventing its formation, or changing its metabolism towards more detoxification end products (14).

Heterocyclic amine (HCA) formation can be lowered appreciably by the action of tea and tea polyphenols (5,6). Tea is the most highly consumed beverage in the world, other than water. However, unlike water, tea contains many organic constituents, some of which appear to have medicinal and health benefits that were known to early Chinese civilizations. Tea contains substantial amounts of polyphenols (7). It is this broad class of compounds with its unique biological activities that may be responsible for many of the health benefits of tea that are currently undergoing intensive scientific investigation. The current research is based on our finding that UDP-glucuronosyltransferases are increased by green and black tea intake (8). Previously, we observed that tea enhanced the excretion of 5-OH-IQ glucuronide (6). Also relevant is our report that phenobarbital increased urinary glucuronides of hydroxylated metabolites of the carcinogen 2-acetylaminofluorene (N-2-fluorenylacetamide), accounting for its decreased carcinogenicity and lower formation of DNA adducts (9).

This paper explores the effectiveness of green tea in enhancing the detoxification of IQ in the F344 rat. The results show that intake of green tea increases the excretion of N-OH-IQ-N-glucuronide, a detoxified metabolite of the proximate carcinogen, N-OH-IQ, thereby most likely reducing DNA adduct formation in particular and carcinogenesis in general.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
Chemicals and reagents
[2-14C]IQ (10 mCi/mmol) and unlabeled IQ were purchased from Toronto Research Chemicals (Toronto, Canada). DMSO and unlabeled IQ metabolite standards were available from previous research (26,1012). Purity of all IQ standards were checked by HPLC and found to be over 95%. All other chemicals were purchased from laboratory supply firms at the highest available grade.

Tea
Fresh Lipton Research World Blend of green tea was donated by the Lipton Company (Dr William Franke), Englewood Cliffs, New Jersey, through the courtesy of the Tea Council of the USA. Green tea (20 g) was dissolved in 1000 ml boiling water and stirred for 5 min. The mixture was filtered to separate the liquid from the leaves. This gave a 2% (w/v) concentration of green tea.

Animals
Adult male and female Fischer 344 rats were purchased from Charles River (Kingston, New York) and kept in the specialized metabolism room in the Research Animal Facility of the American Health Foundation, accredited by the AAALAC. This project was approved by the IACUC of this Institute. When the study started, the rats were divided into four groups of 10 animals each and transferred to stainless steel metabolism cages that allowed for separate collection of urine and feces. The first two groups, designated as the control groups continued drinking water, while the other two experimental groups consumed 2% (w/v) green tea beverage, prepared freshly three times a week for 6 weeks.

Gavage, urine collection and analysis of metabolites
After 6 weeks, both groups of rats received by oral gavage single doses of 40 mg/kg body weight of [2-14C]IQ dissolved in 1.0 ml dimethyl sulfoxide (DMSO). The mean body weights ± standard deviation just before gavage were 406.3 ± 20.2 gm for males and 176.5 ± 4.8 gm for females on tea, and 404.0 ± 18.1 gm for males and 173.0±3.5 gm for females on water. Twenty-four hour urine samples were collected and metabolites separated by HPLC. Aliquots of urine were refluxed in 0.1 N HCl at 100°C for 15 min to hydrolyze the sulfate esters and sulfamates, and the resultant mixture, adjusted to pH 5.8, was resolved by HPLC. The HPLC conditions, a modification of the method by Snyderwine et al. (13), consisted of running 100% 50 mM CH3COONH4 (pH 5.8) and 0% methanol isocratically for 20 min, then increasing the percentage of methanol linearly to 10% at 40 min, to 40% at 50 min, to 80% at 60 min, and to 100% at 70 min, at a flow rate of 1.0 ml/min. Quantitation of metabolite fractions was done in triplicate by scintillation counting in an LKB-Wallac instrument, Model 1217. Mass spectra were obtained on a Hewlett Packard Model 5987A by electron spray mass chromatography, by staff of the Core Analytical Chemistry Facility of American Health Foundation.

Statistics
Statistical significance was calculated by a Student's t-test for two-sample, assuming equal variances. A value of P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
Methods for the purification and identification of IQ urinary metabolites in rats and non-human primates have been described by Inamasu et al. (10), Luks et al. (11), Vavrek and Weisburger (12) and Snyderwine et al. (13). The identities of isolated metabolites were established by a comparison of the characteristic metabolite retention times, or by co-chromatography with unlabeled known standards, where the radioactive peak coincides with that of each known standard. One minor and four major metabolites were isolated in both the male and female rats (Figure 1Go). The minor metabolite was identified as IQ-N-glucuronide, while the major metabolites included N-OH-IQ-N-glucuronide, 5-OH-IQ-glucuronide and sulfate, IQ sulfamate and the parent compound, IQ. The sulfate esters and sulfamates yielded 5-OH-IQ and IQ upon acid hydrolysis. The 5-OH-IQ was not visualized on the standard HPLC chromatogram since it is a chelating agent, as reported previously (12). The major metabolites observed after acid hydrolysis were the N-OH-IQ-N-glucuronide and the 5-OH-IQ-glucuronide, as well as high levels of IQ, stemming from the hydrolysis of the sulfamate (Figure 2Go). There was an effect of green tea on the percentage of various metabolites recovered (Table IGo). Thus, the important finding was made that the rats on tea showed a significantly higher (P < 0.05) excretion in urine of N-OH-IQ-N-glucuronide, compared with the water controls (Figure 3Go). The structure of N-OH-IQ-N-glucuronide was confirmed by electron spray mass spectrometry (Figure 4Go). Also, the HPLC mobilities were identical to those observed in our laboratory (26,1012) and by Snyderwine et al. (13).



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Fig. 1. Typical HPLC profile showing IQ metabolites in male rats (see Materials and methods). Similar elution profiles were observed in females.

 


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Fig. 2. HPLC elution pattern after acid treatment.

 

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Table I. Effect of green tea on percent of the total urinary radioactivity as metabolites of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in male and female F344 rats
 


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Fig. 3. Amounts of radioactivity recovered in urine from male and female rats on tea, or controls on water in peak 3, representing N-OH-IQ-N-glucuronide, from Figures 1 and 2GoGo.

 


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Fig. 4. Electrospray mass spectrum of N-OH-IQ-N-glucuronide (m+H)+, m.w. 391.

 

    Discussion and conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 
The food carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a typical HCA formed in protein-rich foods using the usual household cooking practices (14). Most of the HCAs have been shown to be strong mutagens and potent inducers of neoplasm in animals (1,3,11,15,16). Epidemiological studies show meat consumption and HCAs as risk factors for colon and breast cancer (17). IQ requires metabolic activation to exert its genotoxicity and form DNA adducts (1822). It is important to understand the mechanism of action and to develop detoxification routes of IQ, since there is extensive exposure of this class of carcinogen to the general non-vegetarian population (13,17).

The metabolism of IQ involves both phase I and phase II enzyme systems for bioactivation and/or detoxification. The potent carcinogenicity of IQ is mainly associated with N-hydroxylation via cytochrome P450 1A2 to the proximate carcinogen (N-OH-IQ), followed by subsequent O-esterification, catalyzed by acetyltransferases and sulfotransferases. With the carcinogen 2-acetylaminofluorene, a model arylamine, undergoing N-hydroxylation by CYP1A2, it was found that the phase II enzyme glucuronosyltransferase, induced by phenobarbital, accounted for a decreased carcinogenic effect, even though CYP1A2 was also higher. The formation of glucuronides excreted in urine outweighed a higher level of the proximate carcinogen, the N-hydroxy-2-acetylaminofluorene (9). Enzymes that contribute to the metabolic activation of HCAs, namely CYP1A2, microsomal NADPH-cytochrome P450 reductase and N,O-acetyltransferase, were inhibited by tea in vitro (23). The mutagenicity of heterocyclic amines and of other genotoxic carcinogens was inhibited by green or black tea and the tea polyphenols (2325). However, of great relevance are studies in vivo that have established that tea also induces specific cytochrome P450 and Phase II enzymes upon subchronic intake (8). Indeed, previous research demonstrated that black or green tea increased the activities of CYP1A1, -1A2, -2B1 and UDP-glucuronosyl transferase in the liver of male F344 rats. Thus, the significantly higher levels of excretion in the urine of N-OH-IQ-N-glucuronide in tea-drinking rats is accounted for by the induction of UDP-glucuronosyltransferase, the Phase II enzyme performing the glucuronidation. Increased excretion of the glucuronides was noted in male and female rats. In contrast, because of lower availability of IQ, sulfamate formation was decreased.

Glucuronidation is a major pathway in the detoxification and subsequent excretion of xenobiotics and endobiotics (26). This phase II enzyme detoxification may be a general mechanism that we observed in the detoxification of the carcinogen 2-acetylaminofluorene (N-2-fluorenylacetamide) many years ago (9). We conclude that intake of green tea increases the excretion of N-OH-IQ-N-glucuronide, a detoxified metabolite of the proximate carcinogen, N-OH-IQ, thereby most likely reducing DNA adduct formation and carcinogenesis by this class of hazardous chemical in the human food chain.


    Notes
 
3 To whom correspondence should be addressedEmail: john_weisburger{at}nymc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion and conclusions
 References
 

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Received January 8, 2001; revised March 12, 2001; accepted March 12, 2001.