Extended quantitative structure-activity relationships for 80 aromatic and heterocyclic amines: structural, electronic, and hydropathic factors affecting mutagenic potency.

The mutagenic/carcinogenic heterocyclic amines formed during the cooking of protein foods have been determined to be probable or possible human carcinogens. As part of a comprehensive study of the food mutagens, our laboratory has produced a series of quantitative structure-activity relationships (QSARs) of aromatic and heterocyclic amines, to attempt to elucidate the mechanisms of mutagenesis/carcinogenesis. Amines are genotoxically active only after activation by a series of reactions converting the parent compound to an electrophilic derivative, which is postulated to be a nitrenium ion that covalently binds to and damages DNA. An important agent in this conversion is cytochrome P450. In this report we develop a QSAR for 80 amines of diverse structure and a range of 10 orders of magnitude in mutagenic potency. New structural factors and quantum chemical ab initio and Hückel calculations are included. The results are interpreted to show that a main determinant of mutagenic potency is the extent of the aromatic pi-electron system. Small contributions are made by both the dipole moment and the calculated stability of the nitrenium ion. Multiple linear regression models account for nearly two-thirds of the variance in potency, leaving room for additional unknown factors. The role of cytochrome P450 1A in amine toxification is supported, and further theoretical and experimental research on its reaction mechanisms and modeling of its active site are proposed.     

Separation of heteroaromatic amines in food products

In recent years, many studies have dealt with the role of certain heteroaromatic amines (HAs) as mutagenic compounds, and their occurrence in foodstuffs. Here we examine the determination of HAs, focusing on the analytical strategies for their extraction and preconcentration from several matrices. We summarise the properties of heteroaromatic amines and the main drawbacks involved in their analysis, and then concentrate on the separation procedures, sorbents and solvents used in the sample treatment. We discuss the requirements of the analytical techniques and the strategies most frequently followed to achieve accurate results.     

Quantitative structure-activity relationship of flavonoids for inhibition of heterocyclic amine mutagenicity

The mutagenic/carcinogenic heterocyclic amines formed during the cooking of protein foods have been determined to be a potential risk to human health. Therefore, mitigation measures are beginning to be studied. A recent finding is that the induction of mutation in Salmonella by these amines can be inhibited by the addition of flavonoids to the assay. This study combines data on the inhibitory process with structural, ab initio quantum chemical, hydropathic, and antioxidant factors to develop a quantitative structure-activity relationship (QSAR) database and statistical analysis. For 39 diverse flavonoids the inhibitory potency varied approximately 100-fold. Three predictive variables, in order of decreasing contribution to variance, are: (1) a large dipole moment; (2) after geometric minimization of energy, a small departure from planarity (i.e., small dihedral angle between the benzopyran nucleus and the attached phenyl ring), and a low rotational energy barrier to achieving planarity; and (3) fewer hydroxyl groups on the phenyl ring. However, these variables account for less than half of the variance in inhibitory potency of the flavonoids. Frontier orbital energies and antioxidant or radical scavenging properties showed little or no relationship to potency. We conclude that interference by the flavonoids with cytochrome P450 activation of the promutagens is the probable mechanism for inhibition of mutagenesis, and suggest avenues for further research. Environ. Mol. Mutagen. 35:279-299, 2000 Published 2000 Wiley-Liss, Inc.     

Detection of carcinogenic glutamic acid pyrolysis products in Worcestershire sauce by high-performance liquid chromatography

Commercially available Worcestershire sauce was analyzed for mutagenic and carcinogenic glutamic acid pyrolysis products using high-performance liquid chromatography. These carcinogenic heterocyclic amines were found to be present in all brands of Worcestershire sauce analyzed. The identity of the carcinogens was confirmed by spectrometric analyses and the SOS umu-test. The concentrations of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) in the Worcestershire sauce were 695 +/- 329 pmol/liter (mean +/- SD, n = 5) and 1,839 +/- 1,321 pmol/liter (n = 5), respectively.     

Mammary gland carcinogenesis by food-derived heterocyclic amines: metabolism and additional factors influencing carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

The heterocyclic amines (HCAs) are a family of mutagenic/carcinogenic compounds found in cooked meats. Several HCAs are mammary gland carcinogens in rats. Of these compounds, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the major one present in the human diet. This report reviews the studies on rat mammary gland carcinogenesis by HCAs; discusses what is currently known regarding mechanisms of mammary gland carcinogenesis of PhIP, especially the significance of metabolic processing; and further highlights the evidence for the possible role of PhIP in human breast cancer.     

Review: Putative mutagens and carcinogens in foods. VI. Protein pyrolysate products

Diet and nutrition may be responsible for 60% of the total cancer incidence for women and greater than 40% for men. Fat, animal protein, and meat consumption are highly correlated with colon cancer incidence. The charcoal broiling of meat and fish yield mutagenic substances. Many findings support the hypothesis that the predominant mutagens are formed by the Maillard reaction. A number of mutagenic compounds have been identified both from cooked foods and from protein pyrolysates. The identified compounds are N-heterocyclic primary amine derivatives of either carbolines, imidazoquinolines, or imidazoquinoxalines. The carboline-type mutagens are structurally related to the known carcinogens 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF), while the imidazoquinoline and imidazoquinoxaline types are believed to resemble 3,2′-dimethyl-4-aminobiphenyl (DMAB). Studies support the theory that these compounds require metabolic activation and are carcinogenic. The major metabolites of several compounds have been identified as the N-hydroxy derivatives. DNA binding was found to be a necessary but not a sufficient condition for mutagenesis. The modified base products have been identified as C-8-guanyl derivatives, resembling adducts formed by the carcinogenic aromatic amines.     

Protein pyrolysate products

Diet and nutrition may be responsible for 60% of the total cancer incidence for women and greater than 40% for men. Fat, animal protein, and meat consumption are highly correlated with colon cancer incidence. The charcoal broiling of meat and fish yield mutagenic substances. Many findings support the hypothesis that the predominant mutagens are formed by the Maillard reaction. A number of mutagenic compounds have been identified both from cooked foods and from protein pyrolysates. The identified compounds are N-heterocyclic primary amine derivatives of either carbolines, imidazoquinolines, or imidazoquinoxalines. The carboline-type mutagens are structurally related to the known carcinogens 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF), while the imidazoquinoline and imidazoquinoxaline types are believed to resemble 3,2'-dimethyl-4-aminobiphenyl (DMAB). Studies support the theory that these compounds require metabolic activation and are carcinogenic. The major metabolites of several compounds have been identified as the N-hydroxy derivatives. DNA binding was found to be a necessary but not a sufficient condition for mutagenesis. The modified base products have been identified as C-8-guanyl derivatives, resembling adducts formed by the carcinogenic aromatic amines.     

Characterization of mutagenic activity in instant hot beverage powders

Extracts of several grain-based coffee-substitute blends and instant coffees were mutagenic in the Ames/Salmonella test using TA98, YG1024, and YG1029 with metabolic activation. The beverage powders induced 150 to 500 TA98 and 1,150 to 4,050 YG1024 revertant colonies/g, respectively. Increased sensitivity was achieved using strain YG1024. No mutagenic activity was found in instant hot cocoa products. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Differential bacterial strain specificity, and a requirement for metabolic activation suggest that aromatic amines are present. Characterization of the mutagenic activity, using HPLC and the Ames test of the collected fractions, showed the coffee-substitute blends and instant coffees contain several mutagenic compounds. Known heterocyclic amines are not responsible for the major part of the mutagenic activity. The main mutagenic activity in grain-based coffee-substitute blends and instant coffees is due to several unidentified compounds, which are most likely aromatic amines. © 1995 Wiley-Liss, Inc.     

Uptake of heterocyclic amines, Trp-P-1 and Trp-P-2, into clonal rat pheochromocytoma PC12h cells by dopamine uptake system

Heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), are known to be produced in food by cooking and are carcinogenic. These amines were found to be accumulated in clonal rat pheochromocytoma PC12h cells and to reduce enzyme activity related to catecholamine synthesis. The mechanism of uptake of these heterocyclic amines into PC12h cells was studied. The uptake was dependent on the incubation time, the amount of the cells, and the concentrations of Trp-P-1 and Trp-P-2 in the incubation mixture. The uptake of these amines was saturable with their concentrations, and the uptake velocity followed the Michaelis-Menten equation, indicating that the uptake was mediated by a transporting protein. The uptake was inhibited by dopamine and serotonin, but not by noradrenaline. Involvement of the dopamine uptake system in uptake of the heterocyclic amines was further indicated by the fact that nomifensine and mazindol, specific inhibitors of dopamine uptake, reduced the uptake, but sulpiride, an antagonist of D2 receptor, did not. The significance of the uptake of the carcinogenic heterocyclic amines was discussed in relation to their possible neurotoxicity in the human brain.     

Mutagenicity of products generated by the reaction between several antiparasitic drugs and nitrite

Drugs containing secondary aliphatic amines, heterocyclic nitrogen, or secondary aliphatic amido groups (chloroquine, dehydroemetine, mebendazole, and piperazine) and pyrimidine derivatives such as pyrantel pamoate were reacted in vitro with sodium nitrite at pH 3.7 and became mutagenic for Salmonella typhimurium strain TA1535. The products derived from the nitrosation of chloroquine and dehydroemetine required metabolic activation by mammalian hepatic S9 to be mutagenic. The N-nitroso derivatives of mebendazole, piperazine, and pyrantel pamoate were mutagenic with and without S9, although more activity was noted in the presence of S9 with the nitrosated compounds formed from mebendazole and piperazine. Under identical conditions, no mutagenic products were detected from quaternary ammonium salts such as bephenium hydroxynaphthoate or drugs containing tertiary heterocyclic amino groups, such as iodochlorhydroxyquin.     

Pathways of heterocyclic amine activation in the breast: DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) formed by peroxidases and in human mammary epithelial cells and fibroblasts

Most human mammary carcinomas originate in the epithelial cells of the breast ducts. A potential role of heterocyclic amines (HAs) in the aetiology of this disease has led us to investigate peroxidase-catalysed and stromal (non-epithelial) activation of the HA 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which may subsequently lead to DNA damage in the adjacent human mammary epithelial cells (HMECs). HAs are formed when proteinaceous foods are cooked at high temperature and some, but not all, can cause mammary tumours in rats. Myeloperoxidase (MPO) and lactoperoxidase (LPO) are peroxidase enzymes present in breast secretions. (32)P-post-labelling analysis showed that IQ-DNA adducts were formed after co-incubation of IQ (500 microM) with calf thymus DNA, hydrogen peroxide and either bovine LPO or horseradish peroxidase (HRP). The major HRP-mediated IQ-DNA adduct co-migrated on TLC and HPLC with the major adduct formed in HMECs, suggesting a common reactive intermediate (nitrenium ion). IQ-DNA adducts were also formed in extracellular DNA when phorbol myristate acetate-stimulated neutrophils (which activate IQ via MPO) were co-incubated with IQ (500 microM) and extracellular plasmid (4 +/- 1 adducts/10(8) nucleotides) or calf thymus DNA (6 +/- 2). Mean adduct formation was five to seven times greater in neutrophil DNA (31 +/- 20). Primary cultures of human mammary fibroblasts or epithelial cells isolated from reduction mammoplasty tissues (n = 4 individuals) were incubated with IQ (500 microM) and formed 2.5 and 14.8 adducts/10(8) nucleotides (mean values), respectively. Our results indicate the possible contribution of stromal cells and breast peroxidases to the metabolic activation of carcinogens in the mammary gland.     

Structural basis of the mutagenicity of heterocyclic amines formed during the cooking processes

A data base consisting of 61 heterocyclic amines formed during food preparation and their desamino analogs were subjected to structure-activity analysis using the CASE method, a structural activity relational expert system. The program identified the major structural determinants associated with mutagenic activity or lack thereof. The structures identified as contributing to the probability of activity as well as those associated with mutagenic potency were highly predictive of molecules not in the learning set. The major structural determinant, the aromatic amino moiety, and quantum mechanical calculations revealed that the mutagenic potency associated with this functionality derived from their contribution to the energy of the Lowest Unoccupied Molecular Orbital (LUMO.) © 1993 Wiley-Liss, Inc.     

Species and sex differences in genotoxicity of heterocyclic amine pyrolysis and cooking products in the hepatocyte primary culture/DNA repair test using rat, mouse, and hamster hepatocytes

Eleven mutagenic heterocyclic amines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3]indole (Trp-P-2), 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C), 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]quinoline (MeIQX), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-diMeIQX), and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-diMeIQX), were studied for genotoxicity in the hepatocyte/DNA repair test employing hepatocytes of male rats, male and female mice, and male hamsters. In these four assay systems, all compounds elicited DNA repair in at least three systems, except Trp-P-2, which was uniformly inactive. However, there were several significant differences in the responses of different systems. Rat and hamster hepatocytes responded to nine of the ten genotoxic compounds with the exception of Glu-P-2. Male and female mouse hepatocytes responded to Glu-P-2, whereas female, but not male, mouse hepatocytes responded to MeIQX and 4,8-diMeIQX. These results illustrate species and sex differences in response to these heterocyclic amines and suggest that a number of these compounds are carcinogenic in hamsters, as they have been in rats and mice.     

Computational insight into anti-mutagenic properties of CYP1A flavonoid ligands

Cytochrome P450 1A (CYP1A) is a subclass of enzymes involved in the biotransformation of heterocyclic amines present in cooked red meat to carcinogenic compounds. Anti-cancer properties have long been associated with flavonoids, and some compounds of this class have been shown to interact directly with CYP1A2. The understanding of this interaction is the purpose of this work. As the number of experimentally tested molecules is limited, two complementary methods in terms of information provided, are proposed for the study of protein-inhibitor interaction as alternatives to a QSAR analysis, using quantum mechanics as well as molecular mechanics.     

Mechanistic QSAR of aromatic amines: new models for discriminating between homocyclic mutagens and nonmutagens, and validation of models for carcinogens

Because of its environmental and industrial importance, the aromatic amines are the single chemical class most studied for its ability to induce mutations and cancer. The large database of mutagenicity and carcinogenicity results has been studied with Quantitative Structure-Activity Relationship (QSAR) approaches by several authors, leading to models for the following: (a) the mutagenic potency in Salmonella thyphimurium; (b) the carcinogenic potency in rodents; and (c) the discrimination between rodent carcinogens and noncarcinogens. However, satisfactory models for the discrimination between mutagens and nonmutagens are lacking. The present work provides new QSARs for mutagenic/nonmutagenic homocyclic aromatic amines in S. typhimurium strains TA98 and TA100. The two new models are validated by checking their ability to predict the mutagenicity of further aromatic amines not included in the training set, and not used to generate the QSAR models. In addition, we also validated previous QSAR models for the carcinogenicity/noncarcinogenicity of the aromatic amines with external data. The mechanistic implications of the models are discussed in light of the other QSARs for the aromatic amines. The results of the analysis point to two QSAR models (one for mutagenicity and one for rodent carcinogenicity) as reliable tools for the in silico characterization of the risk posed by the aromatic amines.     

A QSAR for the mutagenic potencies of twelve 2-amino-trimethylimidazopyridine isomers: structural, quantum chemical, and hydropathic factors

An isomeric series of heterocyclic amines related to one found in heated muscle meats was investigated for properties that predict their measured mutagenic potency. Eleven of the 12 possible 2-amino-trimethylimidazopyridine (TMIP) isomers were tested for mutagenic potency in the Ames/Salmonella test with bacterial strain TA98, and resulted in a 600-fold range in potency. Structural, quantum chemical, and hydropathic data were calculated on the parent molecules and the corresponding nitrenium ions of all of the tested isomers to establish models for predicting the potency of the unknown isomer. The principal determinants of higher mutagenic potency in these amines are: (1) a small dipole moment, (2) the combination of b-face ring fusion and N3-methyl group, (3) a lower calculated energy of the pi electron system, (4) a smaller energy gap between the amine HOMO and LUMO orbitals (Pearson "softness"), and (5) a more stable nitrenium ion. Based on predicted potency from the average of six regression models, the isomer not yet synthesized and tested is expected to have a mutagenic potency of 0.77 revertants/microg in tester strain TA98, which is near the low end of the potency range of the isomers.     

Inhibition of human brain aromatic L-amino acid decarboxylase by cooked food-derived 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other heterocyclic amines

A carcinogenic, food-derived heterocyclic amine, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), was found to inhibit aromatic L-amino acid decarboxylase isolated from human brainstem. Trp-P-2 inhibited the enzyme activity toward L-DOPA more markedly than that toward 5-hydroxytryptophan. The inhibition was competitive to a cofactor of the enzyme, pyridoxal-5-phosphate, and the Ki value of Trp-P-2 was 163 microM. The enzyme activity could be fully recovered after removal of Trp-P-2 by gel filtration, which indicates that the inhibition was reversible. Among a series of heterocyclic amines examined for their effects on the activity toward L-DOPA, Trp-P-2 was the most potent inhibitor, followed by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, then Trp-P-1. Another heterocyclic amine, 2-amino-3-methyl-9H-pyrido[2,3-b]indole also inhibited the enzyme. The inhibition of the decarboxylase activity by these heterocyclic amines may affect the catecholamine metabolism in human brain.     

Cytogenetic biomonitoring in traffic police workers: Micronucleus test in peripheral blood lymphocytes

Atmospheric pollution represents a relevant environmental hazard which has been associated with considerable excess mortality, morbidity, and increased rates of respiratory diseases in humans. To date, more than 3,000 environmental chemical compounds have been identified in the ambient atmosphere, including a variety of mutagenic and/or carcinogenic agents, such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and heterocyclic compounds. Positive associations between cytogenetic markers and airborne levels of PAHs have been reported by experimental and human studies. Traffic has been implicated as the major determinant for the concentration of PAHs and, therefore, for the genotoxic activity of urban air. A biomonitoring study has been conducted in 82 Italian traffic police workers exposed to air pollutants and 34 control subjects (matched by age, gender, and smoking habits) not exposed to traffic pollutants. The aim of this study was to assess the cytogenetic effects, such as micronucleus frequency in peripheral blood lymphocytes, and to estimate the association with individual exposure to PAH. Statistical analysis of the frequency of micronuclei in binucleated cells showed higher mean levels in referent subjects (4.03%) than in traffic police officers (3.73%). Smoking showed no effect on the frequency of micronuclei. The study failed to detect any association between micronucleus frequency and individual level of benzo(a)pyrene, considered a marker of exposure to PAHs. These findings indicate that exposure to urban air pollutants does not result in increased levels of micronuclei in peripheral white blood cells. Environ. Mol. Mutagen. 30:396–402, 1997 © 1997 Wiley-Liss, Inc.     

Molecular interactions between human cytochrome P450 1A2 and flavone derivatives

Activation by human cytochrome P450 1A2 (hCYP1A2) of heterocyclic amines is assumed to trigger of a number of carcinogenic processes. In this work, a group of natural inhibitors of human cytochrome P450 1A2 reported in literature has been theoretically analysed. These consist of flavone hydroxylated derivatives, natural compounds that exist in plants and associated products. Different theoretical/computational tools were used to describe the specific molecular interactions between these compounds and hCYP1A2. Based on this analysis, a method is proposed for helping the selection of specific molecular features that enhance protein-inhibitor interaction.     

Antimutagens, anticarcinogens, and effective worldwide cancer prevention.

Cancer is the eventual outcome of the transformation of normal cells by DNA-reactive, genotoxic carcinogens and the growth promotion of mutated cells by enhancing factors. It is important to discriminate between genotoxic carcinogens and nongenotoxic chemicals because their mechanisms of action are quite distinct. Their dose-response curves, reversibility, and organ- and species-specificity are also quite distinct. Thus, the mode of action of agents involved in cancer causation and development needs careful analysis. Genotoxic carcinogens are mutagenic, form DNA adducts, and lead to the formation of hydroxy radicals and inappropriate peroxidation reactions that the antioxidants in vegetables, fruits, and tea can effectively decrease. About 35% of known cancers are associated with tobacco use and about 55% with inappropriate nutritional habits. Cancer induction can be decreased by (1) avoiding the formation of carcinogens, (2) reducing their metabolic activation, or (3) increasing their detoxification. Nutritional factors play a major role in cancer prevention. Vegetables, fruits, and the beverage, tea, provide select means to prevent activation of carcinogens or to increase their detoxification. Salting and pickling of certain foods generate direct-acting mutagens and carcinogens that affect the stomach and probably the esophagus. Thus, controlling salt use and increasing vegetable and fruit intake can prevent these diseases. Frying and broiling protein foods generate heterocyclic amines that affect the colon, breast, prostate, and pancreas, and their formation is decreased by antioxidants. Heterocyclic amines are converted to reactive components by specific cytochrome P450 enzymes and N-acetyltransferases. The tobacco-specific nitrosamines and polycyclic aromatic hydrocarbons undergo specific activation and detoxification processes. These reactions are controlled by antioxidants such as quercetin in vegetables, polyphenols in tea, genistein and daidzin in soy, sulforaphane in broccoli, and 3-methylindole and isothiocyanates in such protective foods. In the Western world, the type and amount of fat play a critical role that operates through specific promoting mechanisms to modify the action of genotoxic carcinogens. In most Western countries where mixed fats and oils are consumed, the total amount of fat at 35 to 40% of calories acts as a powerful promoter. On the other hand, in Italy and Greece, where the monounsaturated oil, olive oil, is used almost exclusively without promoting effect, little enhancement by fat is observed. Also, in the Mediterranean region, meats, mainly veal, are not browned to the point of generating heterocyclic amines. Wheat bran fiber increases stool bulk and eliminates carcinogens and the promoters of colon and breast cancers. Thus, the current base of knowledge on the mechanisms of cancer causation provides effective ways of preventing most types of cancer, the definitive means of cancer control.