Activation Of Heterocyclic Amines Research Articles

Factors Determining Colorectal Cancer: The Role of the Intestinal Microbiota

The gastrointestinal tract, in particular the colon, holds a complex community of microorganisms, which are essential for maintaining homeostasis. However, in recent years, many studies have implicated microbiota in the development of colorectal cancer (CRC), with this disease considered a major cause of death in the western world. The mechanisms underlying bacterial contribution in its development are complex and are not yet fully understood. However, there is increasing evidence showing a connection between intestinal microbiota and CRC. Intestinal microorganisms cause the onset and progression of CRC using different mechanisms, such as the induction of a chronic inflammation state, the biosynthesis of genotoxins that interfere with cell cycle regulation, the production of toxic metabolites, or heterocyclic amine activation of pro-diet carcinogenic compounds. Despite these advances, additional studies in humans and animal models will further decipher the relationship between microbiota and CRC, and aid in developing alternate therapies based on microbiota manipulation.

Metabolic Activation of Heterocyclic Amines and Expression of Xenobiotic-Metabolizing Enzymes in the Gastrointestinal Tract of Rats.

Heterocyclic amines get entry into human body mainly through ingestion of pan-fried meats cooked at high temperatures. Exposure of the gastrointestinal tract (GIT) to ingested xenobiotics prior to delivery to the liver may lead to metabolic activation, which may explain the high incidence of GIT carcinogenesis. Therefore, this study investigated the mutagenic activation of 2 heterocyclic amines, 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the GIT of rats. In addition, the constitutive mRNA expression profiles of xenobiotic-metabolizing enzymes (XMEs) in the GIT of rats were examined. Metabolic activation of 2-AA was detected in all GIT tissues except the duodenum and rectum, and it was detected at high levels in the ileum and cecum. Furthermore, we revealed high metabolic activation of 2-AA and Trp-P-2 in the jejunum. The mRNA expression of phase I and II enzymes in rat GIT corresponded with their mutagenic activation ability. In conclusion, our results suggest that different expression levels of XME among GIT tissues may contribute to the tissue-specific differences in metabolic activation of xenobiotics such as heterocyclic amines in rats. This study declares mutagenic activation of 2 heterocyclic amines namely 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the gastrointestinal tract (GIT) of rats. In addition, results obtained in this study suggest that GIT tissue-specific expression of xenobiotic metabolizing enzymes may contribute to the tissue-specific mutagenesis/carcinogenesis.

Metabolite profiling of polyphenols in a Terminalia chebula Retzius ayurvedic decoction and evaluation of its chemopreventive activity

Ethnopharmacological relevanceThe decoction of Terminalia chebula fruit is an ayurvedic remedy whose prolonged oral administration is prized as a generic intestinal and hepatic detoxifying agent. Its administration is suggested also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. Aim of the studyTo evaluate the phytochemical profile and the chemopreventive potential of Terminalia chebula fruit decoction prepared according to the ayurvedic decoction recipe. Materials and methodsThe quali- and quantitative metabolite profiling of polyphenols was obtained using HPLC–UV/DAD and HPLC-ESI-MS. The crude decoction and purified compounds were tested for their capability to interact with the EphA2-ephrin-A1 system and for their antimutagenic properties against dietary and environmental mutagens (AA, 2-NF, NaN3, and heterocyclic amines IQ, MeIQ, MeIQx, Glu-P1, Glu-P2,) in the Ames–Salmonella/microsome assay, with and without enzymatic induction. ResultsThe decoction was found to contain 3,4,6-tri-O-galloyl-d-glucose (55.87mg/g), chebulic acid (54.03mg/g), β-punicalagin (41.25mg/g), corilagin (40.31mg/g), α-punicalagin (35.55mg/g), chebulagic acid (29.09mg/g), gallic acid (27.96mg/g) 1,3,4,6-tri-O-galloyl-β-d-glucose (24.25mg/g) chebulinic acid (20.23mg/g), 1,2,3,4,6-penta-O-galloyl-d-glucose (13.53mg/g), ellagic acid (8.00mg/g), 1,6-di-O-galloyl-d-glucose (4.16mg/g). An inhibitory effect was recorded in both Salmonella typhimurium TA98 and TA100 strains against the mutagenic activity of heterocyclic amines (22–61%), promutagen AA (91–97%) and directly acting mutagen 2-NF (52%) with but not against NaN3 (7%). Galloyl derivatives allowed an inhibition of mutagenicity induced by MeIQ above 80% at 0.01mol/plate. Both decoction and purified compounds were able to modulate the EphA2-ephrinA1 system, suggesting a potential multiple chemopreventive mechanism. ConclusionsThe traditional ayurvedic decoction of Terminalia chebula may harbour a potential as a safe and low-cost chemopreventive agent at the intestinal level, if administered according to the ayurvedic specifications. Moreover, its recourse may enhance the presence of some polyphenolic constituents.

Analysis of Substrate Recognition Site 2 (SRS2) in human cytochrome P450 1A2 using whole-plasmid random mutagenesis

The metabolic activation of many carcinogenic heterocyclic and aryl amines are attributed to hepatic cytochrome P450 enzymes including P450 1A2. Bioactivation mechanism analysis of P450 1A2 in the limited crystallographic data requires the generation of a large number of mutants. In this study, the SRS2 region in P450 1A2 was randomly mutated with random primers designed for the non-wild type using whole-plasmid random mutagenesis, followed the screening of the mutant library. Eight mutants in Asn222 and Glu225 were selected after Escherichia coli genotoxicity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). Each mutant E. coli membrane was used to determine k cat and K m values for phenacetin and 7-ethoxyresorufin O-deethylation. Most mutants showed considerably increased activity for phenacetin. The Glu225Asn and Asn222Lys mutants increased catalytic efficiencies (k cat/K m) by 5- and 4-fold, respectively. However, the catalytic efficiencies of 7-ethoxyresorufin O-deethylation were not increased mainly because of the increase of K m values. The Glu225Asn and Asn222Lys mutants showed increased k cat values but exhibited greater increases in K m values, which resulted in lower catalytic efficiencies (k cat/K m) for 7-ethoxyresorufin O-deethylation. These results suggested that structural changes of metabolic enzymes in the process of mutagenesis result in different effects of activity by its substrates.

The effect of green tea and olive oil on the mutagenic activity of heterocyclic amines extracted from common food consumed in Saudi Arabia

The effect of green tea (GT) and green tea with olive oil (GT+OL) as antioxidants on the formation and mutagenic activity of heterocyclic aromatic amines (HCAs) extracted from beef shawerma, grilled chicken and fried beef liver was examined. HCAs were extracted by blue rayon, analyzed as spiked and unspiked samples with high-performance liquid chromatography and its mutagenic response was assessed by Sallmonela typhimurium 100 in the Ames test. Surprisingly, GT and GT+OL augmented HCAs measured in beef shawerma and grilled chicken but total HCAs measured in GT+OL were less than GT treatment. Both treatments altered the HCA profile as imidazoquinoline type became the most abundant. In control and GT+OL fried beef liver no HCAs were detected, but Trp-P1 was detected in GT treatment. Generally, the mutagenic response of HCAs measured in GT+OL was less than GT in beef shawerma and grilled chicken. However, the mutagenic response of control and 2% GT+OL fried liver was negative. These data suggest that GT concentrations used in this study may induce free radical formation during the Millared reaction due to its pro-oxidative effect, which augmented the HCAs formed and its mutagenic response. In order to optimize both safety and quality of our diets, more need to be done to fully understand the risk of HCAs in food.

Metabolic Activation of Heterocyclic Amines and Expression of CYP1A1 in the Tongue

Xenobiotic metabolism in oral tissues, especially in the tongue, has never been reported. In the present study, the metabolic activation/detoxification ability of promutagens in the tongue and the expression levels of related enzymes were investigated. Quantitative PCR analysis of rat tongue demonstrated constitutive messenger RNA (mRNA) expression of numerous drug-metabolizing enzymes. In particular, we detected mRNA, protein expression, and enzymatic activity of cytochrome P450 (CYP)1A1 in the tongue tissue. Metabolic activation of promutagens in the tongue was estimated using benzo[a]pyrene or heterocyclic amines (HCAs), found in cooked meat and tobacco products. Metabolic activation levels of HCAs in the tongue were comparable to those in the liver. In contrast, the expression levels of glutathione-S-transferase (GST) and uridine diphosphate-glucuronosyltransferase (UGT) in the tongue were considerably lower compared with those in the liver, and as a result, the mutagenic activity in the tongue was not decreased by GST- or UGT-dependent conjugation. Treatment of rats with sudan III, a typical inducer of CYP1A1, resulted in markedly increased CYP1A1 mRNA, protein expressions, and CYP1A-dependent enzymatic and mutagenic activities. In addition, CYP1A1 mRNA expression in carcinoma cells (SAS) was induced by sudan III exposure. In conclusion, mutagenic activation of xenobiotics and an increased risk of cancer in the tongue were observed in this study. Furthermore, ingestion of drug-metabolizing enzyme inducers has the potential to increase the metabolic activation in the tongue tissue and increase the risk of biomolecular attack by promutagens.

The effect of rosemary on the mutagenic activity of heterocyclic amines extracted from common food consumed in Saudi Arabia

Meat intake may increase cancer risk as heterocyclic amines (HCAs) are one of the food mutagens produced in meat cooked at high temperature. The consumption of meat in Saudi Arabia is high compared with other developing countries and the incidence of cancer has been increasing during the past 30 years. The present study aimed to quantitatively determine the effect of rosemary on the mutagenic activity and the amount of HCAs formed in beef Shawerma, grilled chicken and fried liver as an attempt to minimize the carcinogenic risk of HCAs formed in these commonly consumed meat dishes. Surprisingly, rosemary extracts (2%, 5%, 10% and 15%) apparently enhanced the total amount of HCAs measured in beef Shawerma, whereas 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the only mutagenic amine inhibited by 2% rosemary with a reduction up to 61.6% compared with control. In grilled chicken, the total amount of HCAs measured in 2% rosemary samples was reduced seven-fold lower than the control level, whereas PhIp and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (TrpP1) were inhibited to non-detectable levels. These data demonstrate that 2% rosemary may play an important role in attenuating the production of PhIP in both Shawerma and fried chicken. In fried liver, HCAs were not detected either in the control or in 2% treated samples whereas augmented levels of TrpP1 were measured in 5%, 10% and 15% rosemary. The mutagenic activity of HCAs extracted from all beef Shawerma and grilled chicken treated samples increased over the control sample using Salmonella typhimurium TA100. In fried liver, the mutagenic activity detected in the control sample was higher than treated samples, which suggests that S. typhimurium TA100 might be less sensitive in detecting the mutagenic response of TrpP1 extracted from the real food system. We believe more research is needed to assess the role of antioxidants in the formation of HCAs in order to optimize both safety and quality of our diets.

Meat and Heterocyclic Amine Intake, Smoking, NAT1 and NAT2 Polymorphisms, and Colorectal Cancer Risk in the Multiethnic Cohort Study

N-acetyltransferases (NAT) 1 and 2 are polymorphic enzymes catalyzing the metabolic activation of heterocyclic amines. We investigated the modifying effects of NAT1 and NAT2 polymorphisms on the association of meat consumption, heterocyclic amine intake, and smoking with colorectal cancer risk. In the Multiethnic Cohort study, participants completed a smoking history and a food-frequency questionnaire at recruitment and a cooked meat module 5 years later to estimate heterocyclic amine intake (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline). Blood samples were collected from incident cases and age-, sex-, ethnicity-, frequency-matched controls to determine genotypes. For analysis of meat intake and smoking, data were available for 1,009 cases and 1,522 controls; for heterocyclic amine intake analyses, 398 cases and 1,444 controls were available. Multivariate logistic regression models were used to estimate odds ratios. Smoking was associated with an increased colorectal cancer risk (odds ratio, 1.51; 95% confidence interval, 1.17-1.95) for > or =30 pack-years compared with never smokers (P trend = 0.0004). The association was stronger with presence of the "rapid" compared with the "slow/intermediate" NAT2 genotype (P interaction = 0.003). No significant associations were observed for intakes of red meat, processed meat, and heterocyclic amine, or meat doneness preference, but a dietary pattern high in meat showed a weak positive interaction with the NAT2 genotype (P interaction = 0.05). The enhanced association between smoking and colorectal cancer risk in subjects with the NAT2 rapid genotype supports a role for NAT2 and tobacco smoke heterocyclic amines in the etiology of colorectal cancer. This study only provides weak support for a similar association with meat heterocyclic amines.

The mutagenic activity of heterocyclic amines formed in common foods in Saudi Arabia

Fried liver, spicy grilled chicken and beef Shawerma are common foods regularly consumed in Saudi Arabia. The aim of present study is measuring the mutagenic activity of heterocyclic amines (HAs) extracted from these foods using Ames test with induced rat microsome S9. All samples were prepared in our laboratory and cooked in different restaurants. HAs were extracted by blue rayon method and determined by reversed‐phase HPLC. In unmarinated foods, the highest amount of HAs was observed in beef Shawerma (15.85 ng/g) which mainly contains 7.46 ng MeIQx/g and 8.39 ng Trp‐P1/g. In marinated foods, HAs was only observed in beef Shawerma (9.2 ng /g) which mainly contains 6.59 ng MeIQx/g and 2.61 ng Trp‐P2/g. Significant levels of mutagenic activity were detected in all samples except unmarinated beef Shawerma before cooking. Our data suggested that the mutagenic activity of these foods may be due to HAs and other cooking toxicants especially polycyclic aromatic hydrocarbons (PAHs) since the cooking methods involved intense heat over direct flame. To the best of our knowledge this is the first study on determining HAs in Saudi foods. Clearly, accurate determination of human exposure to HAs and identification of the key enzyme systems involved will be required for rational assessment of its carcinogenic risk among Saudi population.

Effects of Rosemary Extracts on the Reduction of Heterocyclic Amines in Beef Patties

ABSTRACT: The effects of rosmarinic acid and a rosemary antioxidant powder were evaluated on the reduction and mutagenicity of MeIQx, PhIP, and comutagens norharman and harman in beef patties fried at 375 °F for 5 min each side and 400 °F for 7.5 min each side. Both rosemary extracts were found to effectively decrease the formation and overall mutagenic activity of heterocyclic amines (HCAs) due to their antioxidative characteristics. At the lower temperature, rosmarinic acid was able to reduce MeIQx by up to 64% and PhIP by 48%, while rosemary powder reduced the formations of MeIQx up to 69% and PhIP up to 66%. The effects of the rosemary extracts were more dramatic when cooking temperature and time were increased, as rosmarinic acid was able to reduce MeIQx formation up to 70% and PhIP up to 64%; also, rosemary powder significantly reduced MeIQx up to 57% and PhIP up to 77%. The overall mutagenic activity was evaluated by the Ames Salmonella assay and both rosemary extracts were capable of reducing mutagenicity in beef patties at the higher cooking temperature of 400 °F. At 375 °F there was an insignificant trend of the rosemary extracts decreasing the number of Salmonella revertants. There was no significant inhibiting effect by any of the rosemary extracts on the formation of either norharman and harman as measured in this study.

Antioxidant and antigenotoxic activities of Angelica keiskei, Oenanthe javanica and Brassica oleracea in the Salmonella mutagenicity assay and in HCT116 human colon cancer cells

Epidemiological studies indicate that consumption of green-yellow vegetables rich in chlorophyll, vitamin C, vitamin E, and carotenoids reduce the risk of cancer. We sought to examine the antigenotoxic and antioxidant properties of chlorophyll-rich methanol extracts of Angelica keiskei, Oenanthe javanica, and Brassica oleracea (kale). In the Salmonella mutagenicity assay, A. keiskei caused dose-dependent inhibition against three heterocyclic amine mutagens in the presence of S9, O. javanica was antimutagenic only at the highest concentration in the assay (2 mg/plate), and B. oleracea showed no consistent inhibitory activity at non-toxic levels. None of the extracts were effective against three direct-acting mutagens in the absence of S9. Extracts of A. keiskei and, to a lesser extent O. javanica, inhibited two of the major enzymes that play a role in the metabolic activation of heterocyclic amines, based on ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase assays in vitro. All three plant extracts were highly effective in assays which measured ferric reducing/antioxidant power, oxygen radical absorbance capacity, and Fe2+/H2O2-mediated DNA nicking. Finally, using the 'comet' assay, all three plant extracts protected against H2O2-induced genotoxic damage in human HCT116 colon cancer cells. These findings provide support for the antigenotoxic and antioxidant properties of chlorophyll-rich extracts of A. keiskei, O. javanica, and B. oleracea, through mechanisms that include inhibition of carcinogen activation and scavenging of reactive oxygen species.

Effects of -naphthyl isothiocyanate and a heterocyclic amine, PhIP, on cytochrome P-450, mutagenic activation of various carcinogens and glucuronidation in rat liver

To elucidate the mechanism underlying suppression by alpha-naphthyl isothiocyanate (ANIT) of mammary carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), we evaluated hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of environmental carcinogens and UDP-glucuronyltransferase (UDPGT) activities in female Sprague-Dawley rats fed a high fat diet. Immunoblot analyses revealed induction of CYP1A1, newly found 51 and 53 kDa proteins and constitutive CYP1A2 and 2B2 by intragastric treatment with 85 mg/kg PhIP eight times for 11 days. Although the extents of induction were not as high as in the case of PhIP, 3 weeks feeding of 400 p.p.m. ANIT induced CYP1A1 and the 51 and 53 kDa proteins. CP1A2 level was decreased by the feeding of ANIT. The mutagenicity in strain TA98 of PhIP, four other heterocyclic amines (HCAs) and benzo[a]pyrene was greatly enhanced in the presence of liver S9 mix prepared from rats pretreated with PhIP but not with ANIT. The mutagenicities of these five HCAs were significantly decreased in the presence of liver S9 from rats pretreated with a combination of PhIP and ANIT as compared with that pretreated with PhIP alone. The level of hepatic CYP1A2, which is known to be involved in the metabolic activation of PhIP, was consistently decreased in liver microsomes from rats administered PhIP plus ANIT as compared with that from rats administered PhIP alone. On the other hand, UDPGT activity towards 4-nitrophenol (4-NP) was enhanced using liver microsomes prepared from rats pretreated with a combination of PhIP and ANIT as compared with those pretreated with PhIP or ANIT alone. These results show that chemoprevention by ANIT against PhIP-induced rat mammary carcinogenesis can be explained by a dual action mechanism, i.e. a reduction in metabolic activation by hepatic CYP1A2 and an enhancement of detoxification by 4-NP UDPGT. The role of the newly found 51 and 53 kDa proteins in activation of HCAs is also discussed.

Expression of cytochromes P450 and glutathioneS-transferases in human prostate, and the potential for activation of heterocyclic amine carcinogensvia acetyl-coA-, PAPS- and ATP-dependent pathways

Dietary factors appear to be involved in the high incidence of prostate cancer in "Westernized" countries, implicating dietary carcinogens such as heterocyclic amines (HAs) in the initiation of prostate carcinogenesis. We examined 24 human prostate samples with respect to their potential for activation and detoxification of HAs and the presence of DNA adducts formed in vivo. Cytochromes P450 1B1, 3A4 and 3A5 were expressed at low levels (<0.1-6.2 pmol/mg microsomal protein). N-Acetyltransferase (NAT) activities, using p-aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates, were <5-5,500 and <5-43 pmol/min/mg cytosolic protein, respectively. Glutathione S-transferases (GSTs) P1, M2 and M3 were expressed at 0.038-1.284, 0.005-0.126 and 0.010-0.270 microg/mg cytosolic protein, respectively; GSTM1 was expressed in all GSTM1-positive samples (0.012-0.291 microg/mg cytosolic protein); and GSTA1 was expressed at low levels (<0.01-0.11 microg/mg cytosolic protein). Binding of N-hydroxy-PhIP to DNA in vitro occurred primarily by an AcCoA-dependent process (<1-54 pmol/mg/DNA), PAPS- and ATP-dependent binding being <1-7 pmol/mg DNA. In vivo, putative PhIP- or 4-aminobiphenyl-DNA adducts were found in 4 samples (0.4-0.8 adducts/10(8) bases); putative hydrophobic adducts were found in 6 samples (8-64 adducts/10(8) bases). Thus, the prostate appears to have low potential for N-hydroxylation of HAs but greater potential for activation of N-hydroxy HAs to genotoxic N-acetoxy esters. The prostate has potential for GSTP1-dependent detoxification of ATP-activated N-hydroxy-PhIP but little potential for detoxification of N-acetoxy-PhIP by GSTA1. However, there were no significant correlations between expression/activities and DNA adducts formed in vitro or in vivo, DNA adducts in vivo possibly reflecting carcinogen exposure.

Inhibitory Effects of Heterocyclic Amine-Induced DNA Adduct Formation in Mouse Liver and Lungs by Beer

An evaluation of the in vivo antigenotoxic potential of beer components on heterocyclic amines including 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 3-amino-1-methyl-5H-pyrido[4.3-b]indole (Trp-P-2) was determined with particular focus on the target organs of tumorigenesis, and the protective mechanisms involved were investigated. Beer-solution, consisting of a freeze-dried and dissolved sample, given as drinking water, reduced the formation of MeIQx-DNA adducts in mouse liver and lungs. Beer-solution added in the diet as a mimic of food additives also significantly reduced the amount of DNA adducts present in the liver, lung, and kidney DNA of mice fed with MeIQx compared to control mice fed with MeIQx in the absence of beer-solution. The amount of adducts present in the liver of mice with single or continuous administration of Trp-P-2 was significantly reduced when beer-solution was given as part of the diet compared to control mice given Trp-P-2 without beer-solution. Protective effects were observed both with lager- and stout-type samples. In an effort to investigate the mechanisms responsible for the observed protective effects, the effects of beer-solution on metabolizing enzymes for heterocyclic amines were examined. Beer-solutions inhibited the metabolic activation of Trp-P-2 to Trp-P-2(NHOH), as demonstrated by HPLC analysis. Considering the overall suppression of the genotoxicity of MeIQx and Trp-P-2 by beer, we have shown that beer components can inhibit the metabolic activation of heterocyclic amines and subsequently suppress the observed genotoxicity. The results of this study show that beer components are protective against the genotoxic effects of heterocyclic amines on target organs associated with tumorigenesis in vivo.

Prenylated flavanones isolated from flowers of Azadirachta indica (the neem tree) as antimutagenic constituents against heterocyclic amines.

Four prenylated flavanones were isolated from the methanol extract of the flowers of Azadirachta indica (the neem tree) as potent antimutagens against Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) in the Salmonella typhimurium TA98 assay by activity-guided fractionation. Spectroscopic properties revealed that those compounds were 5,7,4'-trihydroxy-8-prenylflavanone (1), 5,4'-dihydroxy-7-methoxy-8-prenylflavanone (2), 5,7,4'-trihydroxy-3',8-diprenylflavanone (3), and 5,7,4'-trihydroxy-3',5'-diprenylflavanone (4). All isolated compounds were found for the first time in this plant. The antimutagenic IC(50) values of compounds 1-4 were 2.7 +/- 0.1, 3.7 +/- 0.1, 11.1 +/- 0.1, and 18.6 +/- 0.1 microM in the preincubation mixture, respectively. These compounds also similarly inhibited the mutagenicity of Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine). All of the compounds 1-4 strongly inhibited ethoxyresorufin O-dealkylation activity of cytochrome P450 1A isoforms, which catalyze N-hydroxylation of heterocyclic amines. However, compounds 1-4 did not show significant inhibition against the direct-acting mutagen NaN(3). Thus, the antimutagenic effect of compounds 1-4 would be mainly based on the inhibition of the enzymatic activation of heterocyclic amines.

Modelling the metabolic action of human and rat CYP1A2 and its relationship with the carcinogenicity of heterocyclic amines

Cytochrome P450 (CYP) is a family of enzymes responsible for organism detoxification. However, some of the members of the CYP1A subfamily also catalyse the activation of heterocyclic amines (HAs), present in cooked meat, to carcinogenic compounds which have been shown to increase the risk of breast, colorectal and lung cancer. In humans, CYP1A2 is the enzyme with the most significant action in HA metabolism but in rodents CYP1A1 is also important in this biotransformation. Understanding the metabolic action of these enzymes is essential to predict the factors that enable the formation of the carcinogenic products. We have built two models of CYP1A2, one for the human enzyme and one for the rat homologue. The templates chosen include the only X-ray structure published to date for a mammal CYP, a quimeric C2A5 from rabbit, as well as CYPs belonging to Bacillus megaterium (CYPBm-3), Pseudomonas putida (CYPcam), Pseudomonas sp. (CYPterp), and Saccharopolyspora erythraea (CYPeryf). Two HAs, MeIQ (2-amino-3,4-dimethylimidazo[4,5-ƒ]quinoline) and MeIQx (2-amino-3,8-dimethylimidazo[4,5-ƒ]quinoxaline), known substrates of human and rat CYPIA2, were docked in the active site of the models, providing information regarding the different catalytic rates associated with the metabolisms in both enzymes. This is important for analysing the behaviour of animal models concerning the testing of anticancer drugs.

Modulation of heterocyclic amine-induced mutagenicity and carcinogenicity: an 'A-to-Z' guide to chemopreventive agents, promoters, and transgenic models.

A landmark report by Widmark in 1939 describing "cancer-producing substances in roasted food", and the seminal work of Sugimura and colleagues in the 1970s on the isolation of potent mutagens from cooked meat and fish stimulated a major international effort on the study of heterocyclic amines and their modulators. The latter term is used in its broadest context to mean agents or conditions that positively or negatively influence the mutagenic or carcinogenic activities of heterocyclic amines in vitro or in vivo. An 'A-to-Z' list of these modulators includes well over 150 natural or synthetic phytochemicals, micronutrients and antioxidants, as well as several large chemical classes (polyphenols, flavones, retinoids, porphyrins), food fractions, and food preparation methods. In many cases, the findings reported in the literature can be regarded as descriptive, but for a number of specific agents there is sufficient evidence to glean some understanding of the inhibitory or promotional mechanisms of action. These mechanisms can be divided into 11 separate sub-categories, arranged within a general classification scheme that encompasses such terms as 'blocking agents', 'suppressing agents', 'desmutagens', 'bioantimutagens', 'interceptor molecules' and 'tumor promoters'. In addition, new research directions, most notably during the past 2-3 years or so, have led to the use of novel dosing protocols and unique animal models (including transgenic species) that provide insight into exposure conditions and genetic background as modulators of heterocyclic amine activity in vitro and in vivo. Overall, the more than 250 citations on the subject give ample evidence of the growing interest in modulators of heterocyclic amine carcinogenesis and mutagenesis, and their possible importance in determining human cancer risk in defined populations.

N-Acetyltransferases, sulfotransferases and heterocyclic amine activation in the breast.

Heterocyclic amines are mammary carcinogens in rats and their N-hydroxy metabolites are substrates for subsequent metabolic activation by N-acetyltransferases (NAT) and sulfotransferases (SULT) in man. We investigated the expression of these enzymes in human breast tissue and the relationship between NAT genotype and NAT mRNA expression or enzyme activity. Immunohistochemical staining of sections of breast tissue identified expression of NAT1 and NAT2 protein in human mammary epithelial cells, but not in the stroma. We also measured the formation of DNA adducts of the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in calf thymus DNA after incubation of their promutagenic N-hydroxy metabolites with mammary cytosols prepared from reduction mammoplasty tissue. Experimental observations gained from use of enzyme cofactors and NAT and/or SULT inhibitors on cytosolic enzyme activity, recombinant NAT1 activity and heterocyclic amine-DNA adduct formation suggest that both NAT1 and SULT1A enzymes contribute significantly to the activation of N-hydroxylated heterocyclic amines in mammary tissue. NAT1 mRNA transcript levels were found to be two- to three-fold higher than mRNA transcripts of the NAT2 gene in reduction mammoplasty tissue and mammary epithelial cells. NAT1-specific p-aminobenzoic acid acetylation activity, but not NAT2-specific sulfamethazine acetylation activity, was detectable in mammary cytosols. There was no association apparent between NAT genotype and the levels of NAT mRNA or NAT enzyme activity, or between NAT1 genotype and IQ-DNA adduct formation mediated by mammary cytosols. Western blot analysis of mammary cytosolic protein showed detectable levels of SULT1A1 and SULT1A3.

Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.

Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium TA98. These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (panduratin A; 6). Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds. The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively. They also similarly inhibited the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All of them strongly inhibited the N-hydroxylation of Trp-P-2. Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium ( S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase ( O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC′′ lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5- f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5 H-pyrido[4,3- b]-indole and 3-amino-1-methyl-5 H-pyrido[4,3- b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B 1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. β-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs.