Dietary Heterocyclic Aromatic Amines Research Articles

Inhibition of the CYP Enzymatic System Responsible of Heterocyclic Amines Bioactivation by an Asclepias subulata Extract.

Asclepias subulata plant extract has previously demonstrated antiproliferative activity and antimutagenicity against heterocyclic aromatic amines (HAAs) commonly found in cooked meat. The objective of this work was to evaluate the in vitro ability of an ethanolic extract from the medicinal plant Asclepias subulata extract (ASE), non-heated and heated (180 °C), to inhibit the activity of CYP1A1 and CYP1A2, which are largely responsible for HAAs bioactivation. Ethoxyresorufin and methoxyresorufin O-dealkylation assays were performed in rat liver microsomes exposed to ASE (0.002-960 µg/mL). ASE exerted an inhibitory effect in a dose-dependent manner. The half inhibitory concentration (IC50) for unheated ASE was 353.6 µg/mL and 75.9 µg/mL for heated ASE in EROD assay. An IC40 value of 288.4 ± 5.8 µg/mL was calculated for non-heated ASE in MROD assay. However, after heat treatment, the IC50 value was 232.1 ± 7.4 µg/mL. Molecular docking of corotoxigenin-3-O-glucopyranoside, one of the main components of ASE, with CYP1A1/2 structure, was performed. Results show that the interaction of corotoxigenin-3-O-glucopyranoside with CYP1A1/2s' α-helices, which are related with the active site and the heme cofactor, may explain the plant extract's inhibitory properties. Results showed that ASE inhibits CYP1A enzymatic subfamily and may potentially act as a chemopreventive agent by inhibiting bioactivation of promutagenic dietary HAAs.

PhIP exposure in rodents produces neuropathology potentially relevant to Alzheimer’s disease

Alzheimer’s disease (AD) is a public health crisis due to debilitating cognitive symptoms and lack of curative treatments, in the context of increasing prevalence. Thus, it is critical to identify modifiable risk factors. High levels of meat consumption may increase AD risk. Many toxins are formed during meat cooking such as heterocyclic aromatic amines (HAAs). Our prior studies have shown that HAAs produce dopaminergic neurotoxicity. Given the mechanistic and pathological overlap between AD and dopaminergic disorders we investigated whether exposure to 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a prevalent dietary HAA formed during high-temperature meat cooking, may produce AD-relevant neurotoxicity. Here, C57BL/6 mice were treated with 100 or 200 mg/kg PhIP for 8 h or 75 mg/kg for 4 weeks and 16 weeks. PhIP exposure for 8 h produced oxidative damage, and AD-relevant alterations in hippocampal synaptic proteins, Amyloid-beta precursor protein (APP), and β-Site amyloid precursor protein cleaving enzyme 1 (BACE1). PhIP exposure for 4 weeks resulted in an increase in BACE1. PhIP exposure for 16 weeks resulted in increased hippocampal oxidative damage, APP, BACE1, Aβ aggregation, and tau phosphorylation. Quantification of intracellular nitrotyrosine revealed oxidative damage in cholinergic neurons after 8 h, 4 weeks and 16 weeks of PhIP exposure. Our study demonstrates that increase in oxidative damage, APP and BACE1 might be a possible mechanism by which PhIP promotes Aβ aggregation. Given many patients with AD or PD exhibit neuropathological overlap, our study suggests that HAA exposure should be further studied for roles in mediating pathogenic overlap.

Acid-labile protein-adducted heterocyclic aromatic amines in human blood are not viable biomarkers of dietary exposure: A systematic study

Heterocyclic aromatic amines (HCA) are carcinogenic mutagens formed during cooking of protein-rich foods. HCA residues adducted to blood proteins have been postulated as biomarkers of HCA exposure. However, the viability of quantifying HCAs following hydrolytic release from adducts in vivo and correlation with dietary intake are unproven. To definitively assess the potential of labile HCA-protein adducts as biomarkers, a highly sensitive UPLC-MS/MS method was validated for four major HCAs: 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 (4,8-DiMeIQx) and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx). Limits of detection were 1–5 pg/ml plasma and recoveries 91–115%. Efficacy of hydrolysis was demonstrated by HCA-protein adducts synthesised in vitro. Plasma and 7-day food diaries were collected from 122 fasting adults consuming their habitual diets. Estimated HCA intakes ranged from 0 to 2.5 mg/day. An extensive range of hydrolysis conditions was examined for release of adducted HCAs in plasma. HCA was detected in only one sample (PhIP, 9.7 pg/ml), demonstrating conclusively for the first time that acid-labile HCA adducts do not reflect dietary HCA intake and are present at such low concentrations that they are not feasible biomarkers of exposure. Identification of biomarkers remains important. The search should concentrate on stabilised HCA-peptide markers and use of untargeted proteomic and metabolomic approaches.

Abstract A10: Exposure to heterocyclic aromatic amines, genetic susceptibility and bulky DNA adduct levels in blood leukocytes

Abstract Background: There is compelling evidence to suggest that aspects of diet influence cancer risk; specifically, high meat consumption is associated with elevated risks. Exposure to heterocyclic aromatic amines (HAAs), carcinogens produced in meat when cooked at high temperatures, is a hypothesized explanation for the meat-cancer relationship. Reactive HAA metabolites form adducts with DNA; left unrepaired, adducts can induce mutations which may initiate/promote carcinogenesis. Genetic differences in the ability to biotransform HAAs (as conferred by polymorphisms in CYP1A1, CYP1A2, CYP1B1, NAT1 and NAT2) and repair DNA adducts (as conferred by polymorphisms in XPA, XPD and XRCC1) is postulated to modify the dietary HAAs-DNA adduct relationship. Methods: In a cross-sectional study of 99 healthy volunteers recruited from Kingston, Ontario, Canada, dietary exposure to HAAs and bulky DNA adduct levels in blood leukocytes was investigated. A detailed questionnaire was used in combination with a mutagen database to estimate average intake of dietary HAAs. Specifically, the detailed questionnaire obtained the average frequency of consumption, usual level of doneness and usual serving size of nine commonly consumed meats items with high HAA content. Bulky DNA adduct levels were measured in blood collected after an overnight fast using 32P-postlabelling. Least squares regression was used to examine the relationship between dietary HAA exposure and bulky DNA adduct levels in blood. Gene-diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair were also examined. Results: No main effects of dietary exposure to HAAs on bulky DNA adduct levels were observed. However, polymorphisms in NAT1 were found to associate with bulky DNA adduct levels. Specifically, those with the putative NAT1*10 rapid acetylator phenotype had a lower adduct level than those with the slow acetylator phenotype (p=0.02). Furthermore, having five or more ‘at-risk’ genotypes was associated with higher bulky DNA adduct levels (p=0.03). Gene-diet interactions were also observed between the NAT1*10 allele and dietary HAAs (p<0.05); specifically, among the slow acetylator phenotype, higher intakes of dietary HAAs were associated with an increase in DNA adduct levels compared to lower intakes. Conclusions: This study provides evidence of a biologic relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct formation. The lack of a strong independent association between dietary HAAs and DNA adducts suggests that dietary HAAs are not a large contributor to bulky DNA adducts in this Canadian population; future studies should consider relevant gene-diet interactions to clarify the potential role of HAAs in carcinogenesis. Citation Format: Vikki Ho, Sarah Peacock, Thomas E. Massey, Roger W. L. Godschalk, Frederik-Jan van Schooten, Jian Chen, Will D. King. Exposure to heterocyclic aromatic amines, genetic susceptibility and bulky DNA adduct levels in blood leukocytes. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A10.

Gene-diet interactions in exposure to heterocyclic aromatic amines and bulky DNA adduct levels in blood leukocytes.

Heterocyclic aromatic amines (HAAs), carcinogens produced in meat when cooked at high temperatures, are an emerging biologic explanation for the meat-colorectal cancer relationship. HAAs form DNA adducts; left unrepaired, adducts can induce mutations, which may initiate/promote carcinogenesis. The purpose of this research was to investigate the relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct levels. Least squares regression was used to examine the relationship between dietary HAA exposure and bulky DNA adduct levels in blood measured using (32)P-postlabeling among 99 healthy volunteers. Gene-diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair were also examined. No main effects of dietary HAAs on bulky DNA adduct levels was found. However, those with the putative NAT1 rapid acetylator phenotype had lower adduct levels than those with the slow acetylator phenotype (P = 0.02). Furthermore, having five or more 'at-risk' genotypes was associated with higher bulky DNA adduct levels (P = 0.03). Gene-diet interactions were observed between NAT1 polymorphisms and dietary HAAs (P < 0.05); among the slow acetylator phenotype, higher intakes of HAAs were associated with an increase in DNA adduct levels compared to lower intakes. This study provides evidence of a biologic relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct formation. However, the lack of a strong main effect of HAAs suggests that dietary HAAs are not a large contributor to bulky DNA adducts in this population; future studies should consider relevant gene-diet interactions to clarify the role of HAAs in carcinogenesis.

Meat-derived carcinogens, genetic susceptibility and colorectal adenoma risk.

Exposure to heterocyclic aromatic amines (HAAs), carcinogens produced when meat is cooked at high temperatures, is an emerging risk factor for colorectal cancer (CRC). In a cross-sectional study of 342 patients undergoing a screening colonoscopy, the role of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), the three most abundant HAAs found in cooked meats, and total mutagenic activity in cooked meats were examined in relation to colorectal adenoma risk. Given that genetic differences in the ability to biotransform HAAs and repair DNA are postulated to modify the HAA-CRC relationship, gene-diet interactions were also examined. Among the total study population, no relationships were observed between dietary HAAs or meat mutagenicity, and colorectal adenoma risk; however, in males, positive associations between dietary HAAs/meat mutagenicity exposures and adenoma risk were suggestive of a relationship. In a separate analysis, polymorphisms in CYP1B1 were found to be associated with colorectal adenoma risk. Additionally, gene-diet interactions were observed for dietary PhIP and polymorphisms in CYP1B1 and XPD, dietary DiMeIQx and XPD polymorphisms, and meat mutagenicity exposure and CYP1B1 polymorphisms. Overall, increased colorectal adenoma risk was observed with higher HAA/meat mutagenicity exposures among those with polymorphisms which confer greater activity to biotransform HAAs and/or lower ability to repair DNA. This research supports the link between dietary HAAs and genetic susceptibility in colorectal adenoma etiology. The vast majority of CRCs arise from colorectal adenomas; thus, the results of this study suggest that changes in meat preparation practices limiting the production of HAAs may be beneficial for CRC prevention.

Validity of a self-administered food frequency questionnaire in the estimation of heterocyclic aromatic amines.

Clarification of the putative etiologic role of heterocyclic aromatic amines (HAAs) in the development of cancer requires a validated assessment tool for dietary HAAs. This study primarily aimed to evaluate the validity of a food frequency questionnaire (FFQ) in estimating HAA intake, using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in human hair as the reference method. We first updated analytical methods of PhIP using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) and measured 44 fur samples from nine rats from a feeding study as part-verification of the quantitative performance of LC-ESI/MS/MS. We next measured PhIP level in human hair samples from a validation study of the FFQ (n = 65). HAA intake from the FFQ was estimated using information on intake from six fish items and seven meat items and data on HAA content in each food item. Correlation coefficients between PhIP level in human hair and HAA intake from the FFQ were calculated. The animal feeding study of PhIP found a significant dose-response relationship between dosage and PhIP in rat fur. Mean level was 53.8 pg/g hair among subjects with values over the limit of detection (LOD) (n = 57). We found significant positive correlation coefficients between PhIP in human hair and HAA intake from the FFQ, with Spearman rank correlation coefficients of 0.35 for all subjects, 0.21 for subjects with over LOD values, and 0.34 for subjects with over limit of quantification. Findings from the validation study suggest that the FFQ is reasonably valid for the assessment of HAA intake.

Dietary intake of meat and meat-derived heterocyclic aromatic amines and their correlation with DNA adducts in female breast tissue

It was the aim of this study to examine the association of the consumption of meat in general, meat prepared by different cooking methods and the dietary intake of heterocyclic aromatic amines (HCA) with the level of DNA adducts in the breast tissue of women undergoing reduction mammoplasty. Dietary intake of meat and HCA were assessed via questionnaire in 44 women undergoing reduction mammoplasty. DNA adduct analysis in breast tissue was performed by (32)P-postlabelling analysis. Spearman rank correlation coefficients (r) were calculated to examine the association of meat consumption and dietary HCA intake with tissue DNA adduct levels. A median DNA adduct level of 18.45 (interquartile range 12.81-25.65) per 10(9) nucleotides in breast tissue was observed; median HCA intake was 40.43 ng/day (interquartile range 19.55-102.33 ng/day). Total HCA intake (r = 0.33, P = 0.03), consumption of fried meat (r = 0.39, P = 0.01), beef (r = 0.32, P = 0.03) and processed meat (r = 0.51, P = 0.0004) were statistically significantly correlated with the level of DNA adducts in breast tissue. The detected DNA adducts could not be confirmed to be specific HCA-derived DNA adducts by comparison with external standards, using the (32)P-postlabelling assay. We observed strong correlations of dietary HCA intake and consumption of fried and processed meat with DNA adduct levels in breast tissue of 44 women. Since the detected DNA adducts were not necessarily specific only for HCA, it is possible that HCA intake is a surrogate of other genotoxic substances, such as polycyclic aromatic hydrocarbons, in meat prepared at high temperatures.

Effects of polyunsaturated fatty acids on prostaglandin synthesis and cyclooxygenase-mediated DNA adduct formation by heterocyclic aromatic amines in human adenocarcinoma colon cells.

Dietary heterocyclic aromatic amines (HCA) and polyunsaturated fatty acids (PUFA) are both believed to play a role in colon carcinogenesis, and are both substrate for the enzyme cyclooxygenase (COX). In HCA-7 cells, highly expressing isoform COX-2, we investigated the effects of PUFA on prostaglandin synthesis and DNA adduct formation by the HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Furthermore, we studied the role of COX, COX-2 in particular, and cytochrome P4501A2 (CYP1A2) by using the enzyme inhibitors indomethacin (IM), NS-398, and phenethyl isothiocyanate (PEITC), respectively. COX-mediated formation of prostaglandin E2 (PGE2) from linoleic acid (LA) showed that HCA-7 cells can convert LA into arachidonic acid (AA). Alternatively, eicosapentaenoic acid (EPA) was found to compete with AA for COX. Strongly decreased PGE2 levels by addition of IM demonstrated involvement of COX in PUFA metabolism. Both IM and NS-398 inhibited adduct formation by HCA to nearly the same extent, indicating involvement of COX-2 rather than COX-1, while CYP1A2 activity in HCA-7 cells was demonstrated by addition of PEITC. Overall, inhibiting effects were stronger for PhIP than for IQ. HCA-DNA adduct formation was stimulated by addition of PUFA, although high PUFA concentrations partly reduced this stimulating effect. Finally, similar effects for n-3 and n-6 fatty acids suggested that adduct formation may not be the crucial mechanism behind the differential effects of PUFA on colon carcinogenesis that have been described. These results show that COX, and COX-2 in particular, can play a substantial role in HCA activation, especially in extrahepatic tissues like the colon. Furthermore, the obvious interactions between PUFA and HCA in COX-2 expressing cancer cells may be important in modulating colorectal cancer risk.

Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites

People are commonly exposed to organophosphorus ester (OP) insecticides through the treatment of pets, homes, lawns, gardens, workplaces and in commercial agriculture. Aromatic amines are another chemical class with wide human exposure particularly dietary heterocyclic aromatic amines (HAAs). Previously, we reported that specific aromatic amines and ethyl paraoxon (the metabolite of the insecticide ethyl parathion) induced enhanced mutagenic responses in Salmonella typhimurium. In the present study, we demonstrated that the mutagenicity of 2-acetoxyacetylaminofluorene (2AAAF) and the heterocyclic dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5- b)pyridine (PhIP) was enhanced in the presence of the OP insecticides, ethyl parathion or methyl parathion or a metabolite (methyl paraoxon). The mutagenicity of 2-amino-3-methylimidazo-(4,5- f)quinoline (IQ) was increased by methyl parathion and methyl paraoxon but not by ethyl parathion. This mutagenic synergy was expressed in S. typhimurium strain YG1024. Mammalian microsomal activation was required for PhIP and IQ to express mutagenic synergy. Synergistic responses are rarely incorporated in risk assessment models, yet such responses are important in establishing accurate toxicological characteristics of agents. Under real world conditions where people are exposed to a multitude of agents, the results of this study raise a concern about the environmental and public health impacts of OP insecticides.

Dietary heterocyclic amines and microsatellite instability in colon adenocarcinomas.

Microsatellite instability (MSI) is now an accepted and important pathway in colon tumorigenesis, occurring in 10-15% of sporadic colon cancers and almost all hereditary nonpolyposis colon cancers. Little is known about possible environmental influences on MSI status in colon cancer. We conducted an epidemiological study of 276 colon cancer cases in Los Angeles County that was designed to determine the population prevalence of MSI(+) colon cancer and to identify environmental influences in the development of MSI(+) tumors. Our results support the cigarette smoking and MSI(+) association recently reported [Slattery,M.L., Curtin,K., Anderson,K. et al. (2000) J. Natl Cancer Inst., 92, 1831-1836]. Risk of MSI(+) colon cancers increased with increasing dose (number of cigarettes per day) and increasing duration (years of smoking) of smoking. Compared with never-smokers, those who smoked 1-20 pack-years and >20 pack-years showed odds ratios of 1.39 and 1.64, respectively (P for trend = 0.03). In addition, our results show, for the first time, that after adjustment for cigarette smoking habits and red meat intake, patients with MSI(+) colon cancers had significantly higher dietary exposure to heterocyclic aromatic amines (HAA) as determined by two surrogates of high dietary HAA exposure: preference for well-done red meat and high frequencies of certain cooking practices (frying, barbecuing, broiling and using meat drippings). The risk of MSI(+) colon cancer was increased 3-fold (smoking/red meat intake adjusted OR = 3.03, 95% CI = 1.06, 8.63) among patients who preferred to eat red meat that was very well-done and was increased >2-fold (smoking/red meat adjusted OR = 2.33, 95% CI = 1.00, 5.25) among those who frequently fried/barbecued/broiled their meats or used meat drippings. The risk of MSI(+) colon cancer associated with cigarette smoking remained statistically significant after adjustment for high dietary HAA exposure. The significant association between cigarette smoking and dietary HAA with a specific subset of colon cancers may explain, at least in part, inconsistencies in results linking these two exposures to colon cancers. These results provide a potential mechanism of linking HAA exposure and cigarette smoking to a specific subset of colon cancers.

N-Acetyltransferase 2 (NAT2) Genotype and Colorectal Carcinoma: Risk Variability According to Tumour Site?

Background: Dietary heterocyclic aromatic amines (HAAs) are members of a family of chemicals that comprise highly mutagenic compounds related to colon cancer. The polymorphic N-acetyltransferase 2 enzyme (NAT2, E.C. 2.3.1.5) plays a key role in the transformation of HAAs to ultimate carcinogens. NAT2 enzyme activity is expressed in a genotype-dependent manner in colon epithelium. Therefore local activation of HAAs in colon, and hence increased risk to develop colon cancer, is likely to be related to high NAT2 enzyme activity. This study is aimed at analysing the association between genotypes leading to high NAT2 activity and colorectal cancer risk. Methods: Genomic DNA from 120 colorectal cancer patients and 258 healthy individuals were analysed for enzyme-inactivating mutations at the coding region of the NAT2 gene by means of a mutation-specific polymerase chain reaction. Results: Among patients with sigmoid colon cancer, a significant excess of individuals with genotypes leading to high NAT2 activity was observed as compared both to controls and to the rest of patients with colorectal cancer (P < 0.05). Conclusions: Our findings, which require independent confirmation, suggest that the NAT2 genotype constitutes a secondary risk factor to develop sigmoid colon cancer.