Inhibition of Aminoimidazoquinoxalinc-type and Aminoimidazol-4-one-type Mutagen Formation in Liquid Reflux Models by l-Tryptophan and Other Selected Indoles

The essential amino acid l -tryptophan ( l -Trp) was found to be an effective inhibitor of the development of mutagenicity (Ames test) in liquid-reflux models known to produce identified IQ-type mutagens, such as 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQ_x) and 2-amino-3,7,8-trimethylimidazo[4,5- f ]qninoxaline (7,8-DiMeIQ_x), and in reflux models recently developed in our laboratory that have been found to produce novel IQ-"like" mutagens (aminoimidazol-4-ones), which we have identified as 2-amino-1-methyl-5-propylideneimidazol-4-one (TCP-1), and 2-amino-5-ethylidene-1-methylimidazol-4-one (TCP-2 or ACP). Selected indoles other than l -Trp were also found to be effective inhibitors of mutagen formation in these same reflux models. A mechanism of inhibition of mutagen formation based on the preferential reaction of mutagen precursor aldehydes with the indole-ring nitrogen of these inhibitors, rather than with creatinine, is indicated, and a new "concerted condensation model" for the formation of IQ-type mutagens proposed.     

Effects of glucose on the formation of PhIP in a model system.

The effect of glucose on the formation of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) was studied in a model system. When a mixture of creatine (0.9 mmol), phenylalanine (0.9 mmol) and glucose (0.45 mmol) was heated in diethylene glycol and water (3 ml, 5:1) for 10 min at 180 or 225 degrees C several mutagens were produced. Identification by HPLC, UV absorption spectroscopy and mass spectrometry revealed the presence of PhIP as well as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and minor amounts of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline. Heating the system without glucose produced PhIP as a single mutagen, but in considerably lower amount. An inhibiting effect of glucose in high concentrations was demonstrated. When glucose was added in more than or equimolar amounts of the other two reactants, the formation of mutagens was markedly reduced. Tyrosine heated under the same conditions, with creatine and glucose, showed mutagenic activity. However, no PhIP nor any other known food mutagen was identified from the tyrosine mixture.     

A prospective investigation of fish,meat and cooking-related carcinogens with endometrial cancer incidence

Background:

There are limited prospective studies of fish and meat intakes with risk of endometrial cancer and findings are inconsistent.

Methods:

We studied associations between fish and meat intakes and endometrial cancer incidence in the large, prospective National Institutes of Health-AARP Diet and Health Study. Intakes of meat mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and benzo(a)pyrene (BaP) were also calculated. We used Cox proportional hazards regression models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs).

Results:

We observed no associations with endometrial cancer risk comparing the highest to lowest intake quintiles of red (HR=0.91, 95% CI 0.77–1.08), white (0.98, 0.83–1.17), processed meats (1.02, 0.86–1.21) and fish (1.10, 95% CI 0.93–1.29). We also found no associations between meat mutagen intakes and endometrial cancer.

Conclusion:

Our findings do not support an association between meat or fish intakes or meat mutagens and endometrial cancer.     

32P-postlabelling analysis of DNA adducted with urinary mutagens from smokers of black tobacco

In order to characterize the tobacco-derived mutagens excreted in the urine of tobacco smokers, 32P-postlabelling techniques were used to examine DNA adducts formed from these mutagens with calf thymus DNA in the presence of a metabolic activation system (rat liver S9, Aroclor 1254-induced, with or without acetyl coenzyme A). Using either nuclease P1 or butanol extraction procedures, four-six and three spots, respectively, were reproducibly found on the autoradiograms in the case of the urine extract from two smokers of black tobacco. Using the urinary extract from a non-smoker, only three faint spots were detected after nuclease P1 enrichment. DNA adducts produced in smokers' urine were then compared with those formed by four N-hydroxyarylamines, N-hydroxy-2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline, N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoxaline, N-hydroxy-2-naphthylamine and N-hydroxy-4-aminobiphenyl. Visual inspection revealed that none of the reference aromatic amines contributed to the adduct pattern produced by the urinary mutagen(s). However, primary aromatic amines are mainly implicated as urinary mutagens because: (i) they produce frameshift mutations in Salmonella typhimurium strains, (ii) they are easily extractable with blue cotton and (iii) their mutagenicity is abolished by a nitrite treatment procedure for deamination.     

Isolation and identification of mutagens from a fried Norwegian meat product

Two samples of a typical Norwegian minced meat emulsion were fried at 215 degrees C. One had the regular composition while 4.2% creatine was added to the second sample prior to frying. Mutagens in both samples were purified using aqueous acid extraction, XAD-2 adsorption and a series of semipreparative and analytical high performance liquid chromatography (HPLC) purification steps monitored by the Ames/Salmonella mutagenicity test. Mutagenic activity in the creatine-fortified product was enhanced 15-fold. Mutagenicity profiles from reverse-phase and normal-phase HPLC were qualitatively similar for both samples indicating no major production of new mutagens due to the presence of additional creatine. A total of 8 distinct mutagenic peaks could be separated after three additional HPLC steps. These compounds fall into a class of compounds called amino-imidazoazaarenes (AIA). The majority of mutagenic activity is made up by the known cooking mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-3-methylimidazo[3,5-f]quinoline (IQ) and 2-amino-n,n,n-trimethylimidazopyridine (TMIP). Smaller contributions are from 2-amino-3-methylimidazo- [4,5-f]quinoxaline (IQx), 2-amino-n,n-dimethylimidazopyridine (DMIP), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and two oxygen-containing AIA. With respect to mass, MeIQx and PhIP were the dominating mutagens.     

Mutagenic metabolites in urine and feces of rats fed with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a carcinogenic mutagen present in cooked meat

To study the in vivo fate of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a carcinogenic mutagen present in cooked meat, rats were fed MeIQx in the diet and their urine and feces were analyzed for the metabolites. The isolation procedure included specific adsorption of MeIQx derivatives to blue cotton and subsequent fractionations by thin layer chromatography on silica gel and by high pressure liquid chromatography. Attention was focused on mutagenically active metabolites. Three metabolites were isolated from the urine, and their structures were elucidated on the basis of 1H nuclear magnetic resonance, ultraviolet, and mass spectra. The first metabolite characterized was 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxaline (Compound I), the second was 2-acetylamino-3,8-dimethylimidazo[4,5-f]quinoxaline (Compound II), and the third was 2-amino-8-methylimidazo[4,5-f]quinoxaline (Compound III). Compound I was isolated also from the feces. Compounds I-III were mutagenic to Salmonella typhimurium TA98 with metabolic activation. The mutagenic potency of Compounds I and II was as high as that of MeIQx, and that of Compound III was much lower than that of MeIQx.     

Disposition and metabolism of the food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rats

The disposition and metabolism of a common food mutagen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied in rats. Five rats of both sexes were given a single oral dose of 14C-labeled MeIQx (3-4 mg/kg body wt). The male rats excreted 36% of the radioactivity and 15% of the mutagenic activity of the dose given in the urine collected during the first 24 h. In the females the corresponding urine contained 41% of the radioactivity and 12% of the mutagenicity. During the next 48 h only 1-3% of the radioactive dose was excreted in urine. The remaining dose was excreted in the feces except of less than 1% that was retained by the tissues after 72 h. The liver and kidney retained more radioactivity than other organs. In a separate study the metabolites of bile, urine and feces of both sexes were investigated. After a single oral dose of 20 mg 14C-labeled MeIQx/kg body wt, three major non-mutagenic metabolites were identified. These were 2-amino-4(or 5)-(beta-D-glucuronopyranosyloxy)-3,8-dimethylimidazo[4,5-f] quinoxaline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxalin-4(or 5)-yl sulfate and N-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl) sulfamate. Another two metabolites present in bile, urine and feces were 2-(beta-D-glucuronopyranosylamino)-3,8-dimethylimidazo[4,5-f ] quinoxaline and 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxalin-4 (or 5)yl sulfate. All metabolites were essentially non-mutagenic. Most of the mutagenicity still present in bile, urine and feces could be explained by unchanged MeIQx. Unchanged MeIQx was the most abundant form excreted in urine.     

Mutagenicity of human urine caused by ingestion of fried ground beef

On ingestion of fried ground beef by humans, the urinary mutagenicity, as examined by the Ames test, increased rapidly and then decreased during a period of 12 hr to resume the original low level. The excreted mutagens were shown to differ from 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, the major mutagen in the cooked beef.     

32P Postlabelling analysis of urinary mutagens from smokers of black tobacco implicates 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as a major DNA-damaging agent

When mutagens extracted from the urine of two smokers of black tobacco were reacted with DNA in vitro in the presence of a metabolic activation system, several DNA adducts were detected by 32P-postlabelling analysis. Some of these adducts were also visible, but only faintly, on the autoradiogram for a non-smoker's urine. DNA adducts produced in vitro by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or 2-amino-1-methyl-6-phenylimidazo[3,5-b]pyridine could not account for the adduct pattern produced by the urinary mutagens. However, three or four 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-related DNA adducts were present among the five or six adducts observed for smokers in the autoradiograms of urinary mutagen-adducted nucleotides. Mutagenicity testing combined with HPLC fractionation of urinary extracts also supported the postlabelling data which implicates PhIP as a mutagen in the urine of smokers of black tobacco.     

Influence of creatine, amino acids and water on the formation of the mutagenic heterocyclic amines found in cooked meat

Creatine or one of 15 amino acids were mixed with minced pork before broiling at 200 degrees C. Total mutagenic activity and reversed-phase HPLC-separated mutagenicity profiles were determined for the crust and pan residue of all samples and also in the aerosol fraction of the smoke formed during cooking of the creatine-fortified samples. Addition of 5% (w/w) creatine increased the total mutagenicity 4-fold without changing the mutagenicity profile of either crust, pan residue or aerosol. Amino acid addition (1% w/w) increased the total mutagenicity between 1.5 (lysine) and 43 times (threonine). In most cases the mutagenicity profiles of crust and pan residues were changed by amino acid addition. Dry-heated mixtures of amino acids and creatine were all mutagenic with a 250-fold range between the amino acids. The production of known food mutagens in these mixtures was analyzed by LC-MS of HPLC-fractionated mutagenic peaks. Serine, threonine, phenylalanine, alanine, leucine and tyrosine were all shown to give rise to one of the known food mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-trimethylimidazopyridine (TMIP). Lyophilized and subsequently fried meat patties and a heated powder of lyophilized meat juice were both mutagenic, with mutagenicity profiles similar to the regular meat crust, showing that water is not a prerequisite for mutagen formation in meat. MeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-di-MeIQx) and PhIP were shown, by LC-MS, to be present in the dry-heated meat juice. It is concluded that creatine and free amino acids are the main reactants of the mutagen-forming reactions that occur during frying of meat. Creatine is probably a necessary part of all of these reactions; what specific compounds are formed in each case therefore depends upon the levels in the meat of certain free amino acids and their interactions with other, as yet unknown, compounds in the meat.     

Metabolism of 2-acetylaminofluorene and benzo(a)pyrene and activation of food-derived heterocyclic amine mutagens by human cytochromes P-450

The human P-450 CYP1A1 gene and a P450IA2 complementary DNA have been expressed in Cos-1 cells and the expressed proteins were assayed for their capacity to metabolize the carcinogens 2-acetylaminofluorene (AAF), benzo(a)pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined. The expressed human P450IA1 and P450IA2 proteins, when run on a 7.5% sodium dodecyl sulfate-polyacrylamide gel, migrated with different mobilities, with the former displaying the lower molecular weight. In human liver microsomes from 18 subjects, only a protein band corresponding to P450IA2 was detectable. Cos-1 cell-expressed P450IA1 and P450IA2 were capable of N-hydroxylating AAF and these activities were inhibited by alpha-naphthoflavone. In human liver microsomes, a correlation of r = 0.76 (P less than 0.05; n = 18) was obtained between AAF N-hydroxylase activity and P450IA2 content. AAF N-hydroxylase activity of human liver microsomes was also strongly inhibited by alpha-naphthoflavone. Except in the case of PhIP, where both proteins exhibited similar activities, P450IA2 was at least an order of magnitude more efficient than P450IA1 in activating IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline to mutagens as measured in the Ames test. Statistically significant correlations were obtained between IQ activation and P450IA2 content (r = 0.75, r2 = 0.56) and PhIP activation and P450IA2 content (r = 0.71, r2 = 0.5) in human liver microsomes. The activation of both IQ and PhIP by expressed proteins and human liver microsomes was strongly inhibited by alpha-naphthoflavone. The above data suggest a major role for P450IA2 in activation (N-hydroxylation) of aromatic amides and amines in human liver. When benzo(a)pyrene hydroxylase activity was determined, only Cos-1 cell-expressed P450IA1 exhibited appreciable activity. While alpha-naphthoflavone inhibited Cos-1 cell-expressed P450IA1 benzo(a)pyrene hydroxylase activity, it caused a marked stimulation of this activity in human liver microsomes, which lack P450IA1 protein. The lack of a role for P450IA proteins in benzo(a)pyrene metabolism is further supported by the poor correlation (r = 0.43, P greater than 0.05) between this activity and P450IA2 content of human liver microsomes. However, when P450IIIA3 content of the above human liver microsomes was determined by using the Western blot technique and correlated with benzo(a)pyrene metabolism, an r value of 0.70 (P less than 0.5) was obtained. These data suggest that human P450IIIA proteins are involved in benzo(a)pyrene metabolism.     

High levels of dietary fat: alteration of hepatic promutagen activation in the rat

Male Ola:Sprague-Dawley rats were fed diets containing either low or high levels of fats. After being fed these diets for 4 weeks, the rats were killed and individual hepatic postmitochondrial (S9) fractions were prepared. The ability of these fractions to convert the heterocyclic aromatic amines (HAAs)--2-amino-3-methylimidazo[4,5-f]quinoline; 2-amino-3,4-dimethylimidazo[4,5-f] quinoline; and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (compounds produced during the cooking of proteinaceous food)--to bacterial mutagens was studied, with the use of Salmonella typhimurium TA98 as indicator. Fractions from rats fed high-fat diets exhibited a greater ability to activate the HAAs than did those from rats fed the low-fat diet. The magnitude of the increase was dependent on the type of fat used.     

Substances in human urine that strongly inhibit bacterial mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and related heterocyclic amines.

Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.     

Meat mutagens and risk of distal colon adenoma in a cohort of U.S. men.

Cooking meats at high temperatures and for long duration produces heterocyclic amines and other mutagens. These meat-derived mutagenic compounds have been hypothesized to increase risk of colorectal neoplasia, but prospective data are unavailable. We examined the association between intakes of the heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5,-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,4,8-trimethylimidazo[4,5,-f]quinoxaline (DiMeIQx), and meat-derived mutagenicity (MDM) and risk of distal colon adenoma using a cooking method questionnaire administered in 1996 in the Health Professionals Follow-up Study cohort. Between 1996 and 2002, 581 distal colon adenoma cases were identified. Higher intake of MDM was marginally associated with increased risk of distal adenoma [fourth versus lowest quintile: odds ratio (OR), 1.39; 95% confidence interval (95% CI), 1.05-1.84; highest versus lowest quintile: OR, 1.29; 95% CI, 0.97-1.72; P(trend) = 0.08]. Adjusting for total red meat or processed meat intake did not explain those associations. Our data also suggested a positive association between higher MeIQx (highest versus lowest quintile: OR, 1.28; 95% CI, 0.95-1.71; P(trend) = 0.22) and risk of adenoma, but this association was attenuated after adjusting for processed meat intake. DiMeIQx and PhIP did not seem to be associated with risk of adenoma. In conclusion, higher consumption of mutagens from meats cooked at higher temperature and longer duration may be associated with higher risk of distal colon adenoma independent of overall meat intake. Because mutagens other than heterocyclic amines also contribute to MDM, our results suggest that mutagens other than heterocyclic amines in cooked meats may also play a role in increasing the risk of distal adenoma.     

The presence of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in smoked dry bonito (katsuobushi)

Smoked dry bonito (katsuobushi), an everyday food item for most Japanese people, was found to contain 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), the content of which was estimated at about 2 ng/g. This content is similar to the known MeIQx content of cooked beef. The katsuobushi also contained another mutagenic component, the total activity of which was 1/6-1/3 that of the MeIQx. This component was similar to 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) with respect to its behavior in high-pressure liquid chromatography and its ultraviolet absorption spectrum.     

DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds produced during the pyrolysis of creatine, amino acids and proteins. The major subclass of HCAs found in the human diet comprise the aminoimidazoazaarenes (AIAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All, except DiMeIQx, have been shown to be carcinogenic in animals. These compounds are present in cooked muscle meats at the p.p.b. level. Since the discovery of the HCAs in the late 1970s, many studies have examined the DNA adducts of these compounds. This review compiles the literature on AIA-DNA adducts including their identification and characterization, pathways of formation, mutagenesis in vitro and in vivo, and their association with carcinogenesis in animal models. It is now known that metabolic activation leading to the formation of DNA adducts is critical for mutagenicity and carcinogenicity of these compounds. All of the AIAs studied adduct to the guanine base, the major adduct being formed at the C8 position. Two AIAs, IQ and MeIQx, also form minor adducts at the N2 position of guanine. A growing body of literature has reported on the mutation spectra induced by AIA-guanine adducts. Studies of animal tumors induced by AIAs have begun to relate AIA-DNA adduct-induced mutagenic events with the mutations found in critical genes associated with oncogenesis. Several studies have demonstrated the feasibility of chemoprevention of AIA tumorigenesis. Only a few studies have reported on the detection of AIA-DNA adducts in human tissues; difficulties persist in the routine detection of AIA-DNA adducts in humans for the purpose of biomonitoring of exposure to AIAs. The AIAs are nevertheless regarded as possible human carcinogens, and future research on AIA-DNA adducts is likely to help address the role of AIAs in human cancer.     

Detoxification of carcinogenic aromatic and heterocyclic amines by enzymatic reduction of the N-hydroxy derivative.

The metabolic activation pathways associated with carcinogenic aromatic and heterocyclic amines have long been known to involve N-oxidation, catalyzed primarily by cytochrome P4501A2, and subsequent O-esterification, often catalyzed by acetyltransferases (NATs) and sulfotransferases (SULTs). We have found a new enzymatic mechanism of carcinogen detoxification: a microsomal NADH-dependent reductase that rapidly converts the N-hydroxy arylamine back to the parent amine. The following N-OH-arylamines and N-OH-heterocyclic amines were rapidly reduced by both human and rat liver microsomes: NOH-4-aminoazobenzene, N-OH-4-aminobiphenyl (N-OH-ABP), N-OH-aniline, N-OH-2-naphthylamine, N-OH-2-aminofluorene, N-OH-4,4'-methylenebis(2-chloroaniline) (N-OH-MOCA), N-OH-1-naphthyamine, N-OH-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), N-OH-2-amino-alpha-carboline (N-OH-AalphaC), N-OH-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx), and N-OH-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ). In addition, primary rat hepatocytes and human HepG2 cells efficiently reduced N-OH-PhIP to PhIP. This previously unrecognized detoxification pathway may limit the bioavailability of carcinogenic N-OH heterocyclic and aromatic amines for further activation, DNA adduct formation, and carcinogenesis.     

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline by complementary DNA-expressed human liver P-450

Eight forms of human liver microsomal P-450 were individually expressed in human hepatoma Hep G2 cells with a vaccinia virus cDNA expression system. Using the Ames test, each expressed P-450 was examined for its ability to activate to mutagenic products the compounds, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, respectively. Three forms of human P-450 significantly activated 2-amino-3-methylimidazo[4,5-f]quinoline when the latter was at high substrate concentrations, but only a single form, P-450IA2, showed very high activation of all promutagens at lower substrate concentrations. Human IA2 had extraordinarily high affinity towards four promutagens tested and is likely the predominant P-450 enzyme responsible for their mutagenic activation in human liver.     

Bioactivation of the cooked food mutagen N-hydroxy-2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine by estrogen sulfotransferase in cultured human mammary epithelial cells

Cooked food mutagens from fried meat and fish have recently been suggested to contribute to the etiology of breast cancer. Thus, the most prevalent of these compounds, i.e. 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine, or rather its more mutagenic N- hydroxylated metabolite (N-OH-PhIP), forms DNA adducts in mammary cells, including human mammary epithelial (HME) cells. The objective of this study was to determine the involvement of estrogen sulfotransferase (EST), the only sulfotransferase identified in HME cells, in the further bioactivation of N-OH-PhIP. These studies were done in vitro using human recombinant EST and in intact HME cells. Human recombinant EST increased the covalent binding of [3H]N-OH-PhIP to calf thymus DNA approximately 3.5-fold in the presence of the sulfotransferase co-substrate 3'-phosphoadenosine-5'-phosphosulfate at each N-OH-PhIP concentration (1, 10 and 100 microM) (n = 6, P < 0.001). In contrast, EST did not catalyze the DNA binding of two other cooked food mutagens, N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline and N- hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, which are mainly hepatocarcinogens. Cultured HME cells displayed high EST activity, which could be completely inhibited by 1 microM estrone. When the cells were incubated with [3H]N-OH-PhIP, binding to native DNA occurred at 60- 240 pmol/mg DNA. This binding was inhibited to 55% of control by 1 microM estrone (P < 0.01, n = 8), suggesting that EST plays a significant role in carcinogen bioactivation in human breast tissue.      

Dietary intake of heterocyclic amines, meat-derived mutagenic activity, and risk of colorectal adenomas.

Meats cooked well-done by high temperature techniques produce mutagenic compounds such as heterocyclic amines (HCAs), but the amounts of these compounds vary by cooking techniques, temperature, time, and type of meat. We investigated the role of HCAs in the etiology of colorectal adenomas and the extent to which they may explain the previously observed risk for red meat and meat-cooking methods. In a case-control study of colorectal adenomas, cases (n = 146) were diagnosed with colorectal adenomas at sigmoidoscopy or colonoscopy, and controls (n = 228) were found not to have colorectal adenomas at sigmoidoscopy. Using a meat-derived HCA and mutagen database and responses from a meat-cooking questionnaire module, we estimated intake of 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and mutagenic activity. We calculated odds ratios and 95% confidence intervals using logistic regression adjusting for several established risk factors for colorectal adenomas or cancer. The odds ratios (95% confidence interval; P for trend test) fifth versus first quintiles are: 2.2 (1.2-4.1; P = 0.02) for DiMeIQx; 2.1 (1.0-4.3; P = 0.002) for MeIQx; 2.5(1.1-5.5; P = 0.02) for PhIP; and 3.1 (1.4-6.8; P = 0.001) for mutagenic activity. When the three HCAs were adjusted for the other two, only the trend for MeIQx (P = 0.04) remained statistically significant. When we tried to disentangle the relative contribution of the three HCAs from the meat variables, we found that MeIQx remained significantly associated with risk even when adjusted for red meat but not vice versa. When MeIQx and well-done meat were analyzed in the same model, the risks were attenuated for both. Mutagenic activity from meat remained significantly associated with increased risk even when adjusted for intake of red meat or well-done red meat, whereas the red meat and well-done red meat associations were no longer significant when adjusted for total mutagenic activity. In conclusion, we found an elevated risk of colorectal adenomas associated with high intake of certain HCAS: Further, mutagenic activity from cooked meat consumption, a measure that integrates all of the classes of mutagens, was strongly associated with risk and explained the excess risk with intake of well-done red meat.