Mutagenic Heterocyclic Amines Research Articles

Inhibitory action of antimicrobial peptides against the formation of carcinogenic and mutagenic heterocyclic amines in meat

Thermal processing of meat leads to the development of Maillard's reaction intermediates, and carcinogenic toxicants. For the first time, the effectiveness of three (HX-12A, HX-12B and HX-12C) antimicrobial peptides (AMPs) against the formation of heterocyclic amines (HAs) in chemical and meat model systems. The results showed that AMPs especially 12A and 12C have strong metal chelation potential (48 and 40% at 1 mg/ml) and antioxidant activity (35 and 25% at 1 mg/ml), respectively, which were endorsed by their secondary structure (FTIR analysis) in terms of high β-sheets (1628 cm−1 and 1672 cm−1) in those AMPs. UPLC-MS analysis revealed that 12A and 12C were the most capable AMPs in MeIQx and PhIP-producing chemical models, respectively, whereas 12B promoted the HAs formation even higher than control. In particular, 12C AMP significantly (P < 0.05) decreased the most abundant carcinogenic HAs (PhIP) up to 90% at 9 mg/100 g of fresh meat, whereas 12A inhibited up to 80% of mutagenic HAs at same level compared to control and 12B. Low Field Nuclear Magnetic Resonance (LF-NMR) test showed that inhibitory effect of 12A and 12C was mediated by means of retaining water (lower T22 and T23 relaxation time) inside the macromolecules. This favorable effect was also evidenced by significantly enhanced tryptophan fluorescent intensity. Finally, based on correlation and principle component analysis, the mechanism of action has been proposed. These outcomes recommend that 12A and 12C are potential AMPs for the attenuation of HAs in thermally processed meat-based products.

First report on a series of food-derived mutagenic and carcinogenic heterocyclic amines.

Lifestyle, especially diet, significantly impacts cancer development. Sugimura, T. et al. discovered that grilled fish smoke and charred parts are highly mutagenic in Salmonella typhimurium. They identified two novel mutagenic heterocyclic amines (HCAs), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and its derivative, Trp-P-2, from tryptophan pyrolysate. Published in Proc. Jpn. Acad. (53, 58-61, 1977), their findings initiated the identification of over 10 mutagenic HCAs in cooked foods, most of them newly registered. These 10 HCAs were demonstrated to induce cancers in organs including the liver, colon, breast, and prostate in mice and rats. HCAs are metabolized primarily by CYP1A2 to hydroxyamino derivatives. Their ester forms then adduct at guanine bases, altering genes such as Apc and β-catenin. Quantification of HCAs in cooked foods and human samples, along with epidemiological observations, suggests HCAs likely contribute to human cancers.

Formation of Carcinogens in Processed Meat and Its Measurement with the Usage of Artificial Digestion-A Review.

Meat is a rich source of various nutrients. However, it needs processing before consumption, what in turn generates formation of carcinogenic compounds, i.a., polycyclic aromatic hydrocarbons (PAH), nitrosamines (NOCs), and the most mutagenic heterocyclic aromatic amines (HAAs). It was widely found that many factors affect the content of carcinogens in processed meat. However, it has recently been discovered that after digestion free HAAs are released, which are not detectable before enzymatic treatment. It was established that the highest percentage of carcinogens is released in the small intestine and that its amount can be increased up to 6.6-fold. The change in free HAAs content in analyzed samples was dependent on many factors such as meat type, doneness, particle size of meat, and the enzyme concentration used for digestion. In turn, introduction of bacteria naturally occurring in the human digestive tract into the model significantly decreases total amount of HAAs. Contrary, the addition of food ingredients rich in polyphenols, fiber, and water (pepper powder, onions, apples) increases free HAAs’ release up to 56.06%. Results suggests that in vitro digestion should be an integral step of sample preparation. Artificial digestion introduced before chromatographic analysis will allow to estimate accurately the content of carcinogens in processed meat.

Changes in Insulin Signaling and Gluconeogenic Gene Expression in Human Hepatocytes Following Exposure to Heterocyclic Amines

Humans are exposed to heterocyclic amine (HCA) mutagens produced in meats cooked at high temperatures or until well-done. A recent epidemiological study documented associations of high HCA intake with increased prevalence of insulin resistance, which is one of the hallmarks of type II diabetes mellitus and metabolic syndrome. However, it is unknown if HCAs directly induce insulin resistance. To investigate the effects of HCA exposure on insulin sensitivity, we subjected HepG2 (hepatocellular carcinoma) cells and primary human hepatocytes to 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and measured changes in insulin signaling, gluconeogenic gene expression, and glucose production. HepG2 cells were treated with varying concentrations of MeIQ (0-100 μM), then treated with insulin and analyzed for changes in insulin signaling via p-AKT induction on Western blot. MeIQ exposure decreased insulin-induced p-AKT levels in low glucose conditions, suggesting that the cells become insulin resistant in the presence of MeIQ. Additionally, the MeIQ treatment significantly upregulated the transcripts of genes involved in gluconeogenesis (e.g., G6PC and PCK1) in HepG2 cells. Similarly, primary human hepatocytes exposed to MeIQ also showed significant increases in gluconeogenic genes, compared to the vehicle treated cells. These results suggest that MeIQ impairs insulin signaling and potentially induces glucose production in hepatocytes. We then tested if the gene expression changes induced by MeIQ leads to increases in glucose production in primary hepatocytes. Despite upregulation of gluconeogenic genes, there were no significant changes in glucose production between control and MeIQ-treated cells. Future studies will examine whether MeIQ exposure alters the suppressive effects of insulin on gluconeogenic gene expression and glucose production and test additional HCAs. The alterations in insulin signaling and gluconeogenic gene expression following MeIQ exposure in hepatocytes indicate that MeIQ may directly alter insulin sensitivity.

Evaluating the effects of temperature and time on heterocyclic aromatic amine profiles in roasted pork using combined UHPLC-MS/MS and multivariate analysis

In this study, ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) combined with principal component analysis (PCA) were used to investigate the effects of process conditions on the profiles of carcinogenic and mutagenic heterocyclic aromatic amine (HAA) in the pork roasted at 175 °C, 200 °C, 225 °C and 250 °C for 10, 15, 20, 25, 30, 35 and 40 min. Twelve HAAs from four categories, including carboline (Norharman, Harman, and Phe-p-1), imidazopyridine (PhIP, 4′-OH-PhIP, DMIP, and 1,5,6-TMIP), imidazoquinoline (IQ, IQ [4,5-b], and MeIQ), and imidazoquinoxaline (MeIQx and 4,8-DiMeIQx), were detected, quantified and used to compose the HAA profiles in roasted pork. After being Analyzed by PCA, the distributions of HAA profiles from different temperature on the PCA score plot demonstrated that there are significant differences among the HAA profiles from different temperatures. The loading plot also showed that PhIP, 4′-OH-PhIP, IQ[4,5-b], and MeIQ were mainly responsible for the difference. The profiles from higher temperature distribute more scattered than the lower ones, illustrating that the time effects on the HAA profiles from higher temperature are stronger than the lower ones. Comparing the score and loading plots of different heating times, the diversities of the HAA profiles at different temperatures increased under prolonged heating because of the changingpyridines levels. The results of PCA that comparing the HAA from different categories displayed that the formation features of four categories HAAs were significantly differed because of their formation discrepancy under low temperatures and short–term roasting. Using HAA profiles as an entirety, these findings obtained in this study are more close to the real process of HAA formation in roasted pork, and make the complex effects of temperature and time on multiple HAA formations more simply to be concluded.

Takashi Sugimura: In Memoriam (1926–2020)–A Personal Perspective

![][1] Takashi Sugimura was a pioneer in cancer research and a figure recognized internationally for his seminal contributions to the understanding of mutagenicity and chemical carcinogenesis. Rather than focusing on the many accolades, honors, and influential positions that he held,

Inhibitory effects of some hydrocolloids on the formation of heterocyclic amines in roast beef

Heterocyclic amines (HAs) are food-borne mutagenic compounds formed during food preparation and processing. They are considered as significant health risk in the long term and inhibiting their formation represents one of the most effective approaches to attenuate their potential health hazards. Hydrocolloids are long-chain high-molecular-weight hydrophilic polymers. In this study, the effects of six hydrocolloids, including alginic acid, sodium carboxymethylcellulose (CMC-Na), chitosan, carrageenan, konjac glucomannan (KGM) and xanthan gum, on major HAs formation (PhIP, MeIQx and 4,8-DiMeIQx) in both chemical model systems and roast beef patties were investigated. The influence of addition method (marinating) was also evaluated. HPLC-PDA and UPLC-MS analysis showed that all the six tested hydrocolloids added in form of powders in chemical models effectively inhibited PhIP formation by 19.3–42.7% at relatively lower doses (5 and 10 mg), while they exhibited better inhibitory effects against MeIQx formation at higher dose (30 mg). Among them, CMC-Na had the strongest inhibitory effect, followed by chitosan in roast beef patties. Further analysis showed chitosan, but not CMC-Na, synergized with a polyphenol, phloridzin to inhibit the formation of HAs, particularly PhIP. Marination with CMC-Na solution led to even greater inhibitory effects on HA formation with the contents of PhIP, MeIQx and 4,8-DiMeIQx being reduced by over 90%, 80% and 70%, respectively. These findings suggest that hydrocolloids are potent HA formation inhibitors and their application in meat preparation and processing could effectively reduce human dietary exposure to mutagenic HAs.

Preventive potential and mechanism of dietary polyphenols on the formation of heterocyclic aromatic amines

AbstractThermal processing is the most important and popular domestic cooking method. More than 30 heterocyclic aromatic amines have been identified in cooked meat using various methods. This review highlights preventive potential and mechanism of dietary polyphenols on the formation of heterocyclic amines. Tea, coffee, fruits, vegetable, and spice extracts rich in polyphenols exerted significant inhibition against the formation of heterocyclic aromatic amines. Some polyphenols, such as naringenin and epigallocatechin 3‐O‐gallate, can actively participate into food chemistry reaction to trap Strecker aldehyde and lower the formation of heterocyclic aromatic amines. In addition, some polyphenols can lower the mutagenicity of heterocyclic aromatic amines. More specifically, polyphenols possessing two hydroxyl groups at the meta position of aromatic ring are the most efficient one, but the presence of carboxylic or alkyl groups as substituents in the aromatic ring slightly reduced the inhibitory effect.

Chinese chive and Mongolian leek suppress heterocyclic amine formation and enhance nutritional profile of roasted cod.

Heterocyclic amines (HAs) are potent mutagens, which can form DNA adducts in various human tissues. There is increasing evidence that mutagenic HA formation and nutrition loss can occur concurrently in fish during vigorous heat treatment. Our study investigated the effects of five Allium spp. (garlic, onion, welsh onion, Chinese chive, and Mongolian leek) on reducing HA formation and improving nutritional quality of roasted cod (Gadus morhua). The results showed that cod patties pretreated with powders of the selected Allium spp. had significantly (P < 0.05) lower levels of HAs (82–92%, except garlic, 49%) than the control. The contents of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the patties exhibited strong negative correlations with total antioxidant activity (−0.937), phenolic (−0.948), and lipophilic flavonoid (−0.933) contents, whereas the 2-amino-3,8 dimethylimidazo [4,5-f] quinoxaline (MeIQx) (made up only ∼0.7–3% of total HAs) contents exhibited significant positive correlations with these antioxidant parameters. In terms of nutrient composition change, Chinese chive and Mongolian leek were the most effective in preventing oxidative degradation of proteins and unsaturated fatty acids in roasted cod patties, which was translated into significantly higher contents of soluble proteins, essential amino acids, and polyunsaturated fatty acids. This has been the first report on the strong HA-formation inhibitory effect of Chinese chive and Mongolian leek. The dual beneficial functionality of these two Allium spp. may be utilized to reduce the intake of hazardous by-products while enhancing the nutritional and antioxidant properties of roasted cod and probably other protein-rich heat-processed foods.

Cooking with elaborate recipes can reduce the formation of mutagenic heterocyclic amines and promote co-mutagenic amines

ABSTRACTHeterocyclic amines (HCAs) are foodborne carcinogens for which their formation is highly dependent on cooking conditions. HCAs have been commonly quantified in food items prepared with simple procedures. This approach is suitable for elucidating HCAs’ formation, but it only partially reflects the contamination in consumed food. In the current investigation, the generation of HCAs has been investigated in fried beef items prepared with elaborated cooking recipes, and their occurrence has been compared with control beef fried without the addition of ingredients other than oil. The food recipes that included a variety of food ingredients had lower yields of mutagenic HCAs (≥47% reduction, with individual HCA levels ranging between 0.01 and 2.22 ng/g) with respect to the control beef. In contrast, the co-mutagens norharman and harman were formed generally at greater levels (up to three times the contamination in the control fried beef) in the items prepared including a greater variety of ingredients.

Inhibitory effect of mixture herbs/spices on formation of heterocyclic amines and mutagenic activity of grilled beef

ABSTRACTNatural antioxidants in spices and herbs have attracted considerable attention as potential inhibitors against the formation of mutagenic heterocyclic amines (HCAs) in heat-processed meat. In this study, the inhibitory activity of four spices/herbs and their mixtures on HCAs formation in grilled beef were examined. A simplex centroid mixture design with four components comprising turmeric, curry leaf, torch ginger and lemon grass in 19 different proportions were applied on beef samples before grilling at 240 ºC for 10 min. The HCAs were extracted from the samples using solid phase extraction (SPE) method and analysed using Liquid chromatography mass spectrometry LC-MS/MS. All spices/herbs in single or mixture forms were found to reduce total HCA concentrations in marinated grilled beef ranging from 21.2% for beef marinated with curry leaf to 94.7% for the combination of turmeric and lemon grass (50:50 w/w). At the optimum marinade formula (turmeric: lemon grass 52.4%: 47.6%), concentration of 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), Harman, Norharman and AαC were 2.2, 1.4, 0.5, 2.8 and 1.2 ng/g, respectively. The results of the mutagenic activity demonstrated that this optimised marinade formula significantly (p < 0.05) diminished mutagenicity of marinated grilled beef in bacterial Ames test.

Changes in the mutagenicity of heterocyclic amines, nitrite, and N-nitroso compound in pork patties during in vitro human digestion

The objective of this study was to determine the changes in mutagenicity caused by heterocyclic amines (HCAs), nitrite, and N-nitrosodiethylamine (NDEA) in pork patties during in vitro human digestion. The mutagenicity was higher in raw pork patties containing HCAs than in those containing nitrite or NDEA. In cooked pork patties too, the mutagenicity was higher in HCA-containing patties than that in nitrite- or NDEA-containing patties, both before and after in vitro human digestion. However, the mutagenicity of all pork patties decreased after in vitro digestion. In particular, their mutagenicity was drastically reduced during simulated large intestine digestion with enterobacteria, i.e., Escherichia coli and/or Lactobacillus sakei. These results indicate that high amounts of HCAs in pork patties show higher mutagenicity than nitrite and NDEA, although the mutagenicity decreases after in vitro human digestion.

UPLC-MS/MS and multivariate analysis of inhibition of heterocyclic amine profiles by black pepper and piperine in roast beef patties

In this study, UPLC-MS/MS combined with principal component analysis (PCA) with the help of scores, loadings, and contribution plots was used to investigate the inhibition of the profiles of carcinogenic and mutagenic heterocyclic amines (HAs) in roast beef patties by different levels of black pepper (0.05, 0.10, and 0.15%, g/g) or piperine (0.005, 0.010, and 0.015%, g/g). Six HAs from three categories, imidazopyridine (PhIP), imidazoquinoxaline (IQx, MeIQx, and 4,8-DiMeIQx), and β-carboline (harman and norharman), were detected and quantified in the roast beef patties. The PCA results demonstrated that both black pepper and piperine can significantly suppress the HA profiles and that lower levels of black pepper or piperine lead to better inhibition of HA formation, except the HA profiles of beef patties with 0.005% and 0.01% piperine were similar. In comparison, piperine inhibited HA formation better than did black pepper for all six HAs, as shown by the contribution plot. The total HAs were inhibited by all levels of black pepper (42%, 34%, 25%) and piperine (62%, 60%, 56%) in a dose-dependent manner, although black pepper suppressed only PhIP, IQx, MeIQx, and 4,8-DiMeIQx, whereas piperine inhibited all six HAs. The results show that UPLC-MS/MS in conjunction with PCA is a powerful tool for screening the inhibitory effects of spices and their components along with their relationship to HAs and can thus be used for safety control in food processing procedures.

Simultaneous determination of 9 heterocyclic aromatic amines in pork products by liquid chromatography coupled with triple quadrupole tandem mass spectrometry

Heterocyclic aromatic amines (HAAs) are potent mutagens that formed at high temperature in cooked, protein-rich food. Owing to their frequent intake, an accurate method is essential to access human health risk of HAAs exposure through detecting these compounds in various heat-treated meat products. In this study, a liquid chromatography-electrospray tandem mass spectrometry (LC--ESI-MS/MS) method was developed to perform the determination of 9 mutagenic heterocyclic amines (HAAs) in meat samples with multiple reaction monitoring (MRM) mode. Ultrasound assisted extraction and diatomaceous earth was employed to extract HAAs from food samples, and the analytes were purified and enriched using tandem solid phase extraction, with propyl sulfonic acid coupled to a C18 cartridge. Two parameters, extraction time and eluent, were carefully optimized to improve the extraction and purification efficiency. The LC separation was carried out using a Zorbax SB-C18 (3.5 μm particle size, 2.1 × 150 mm i.d.) column and optimized some parameters, such as pH, concentration and volume. Under the optimal experimental conditions, recoveries ranged from 52.97% to 97.11% with good quality parameters: limit of detection values between 0.02 and 0.24 ng mL-1, linearity (R2>0.998), and run-to-run and day-to-day precisions lower than 9.81% achieved. To evaluate the performance of the method in high throughput analysis of complex meat samples, the LC-MS/MS method was applied to the analysis of HAAs in three food samples, and the results demonstrated that the method can be used for the trace determination of HAAs in pork samples.

된장에서 분리된 유산균의 결합력에 의한 Heterocyclic Amines 제거

단백질이 풍부한 식품을 고온 하에서 조리하는 과정 중에 주로 발생되는 돌연변이원 heterocyclic amines (HCAs)에 대한 유산균의 결합력 및 제거능을 조사하였다. 당 발효능 및 16S rRNA 염기서열 분석을 통해 동정된 19종의 유산균 중 Lactobacillus acidophilus D11, Enterococcus faecium D12, Pediococcus acidilactici D19, L. acidophilus D38, Lactobacillus sakei D44, Enterococcus faecalis D66 및 Lactobacillus plantarum D70의 세포이나 배양 상등액은 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1)과 3-amino-1-methyl-5Hpyrido[4,3-b] indole (Trp-P-2)에 의한 Salmonella typhimurium TA98 및 TA100의 돌연변이 유발을 억제할 수 있었다. HCAs에 대한 유산균 세포의 결합력은 cell wall, exopolysaccharide 및 peptidoglycan 보다 높게 나타났다. 한편, 이들의 결합력은 단백질 분해효소, 가열, sodium metaperiodate 및 산 처리에 의해 유의하게 감소되었으므로 세포벽에 존재하는 당이나 단백질 성분이 이들 HCAs을 결합시키는데 중요한 역할을 하는 것으로 확인되었다. 또한 E. faecium D12, L. acidophilus D38 및 E. faecalis D66의 결합력은 SDS나 금속이온에 의해 감소되었으므로 이들세포와 돌연변이원 사이에는 이온 결합이나 소수성 결합이 작용하는 것으로 추정되었다. 한편, HCAs 결합력이 높은 L. acidophilus D38과 L. plantarum D70은 장관 상피세포에 대한 부착력이 낮으므로 돌연변이원을 세포에 결합시켜 체외로 배출함으로써 독성물질을 제거하는데 효과적인 것으로 확인되었다. The objective of the this study was to investigate the binding capacity and removal ability of lactic acid bacterial strains obtained from Korean soybean paste for mutagenic heterocyclic amines (HCAs) formed during cooking of protein-rich food at high temperature. Among 19 strains identified by carbohydrate fermentation and 16S rRNA sequencing, the live cell or cell-free culture supernatant of Lactobacillus acidophilus D11, Enterococcus faecium D12, Pediococcus acidilactici D19, L. acidophilus D38, Lactobacillus sakei D44, Enterococcus faecalis D66, and Lactobacillus plantarum D70 inhibited the mutagenesis caused by either 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole (Trp-P-1) or 3-amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) in Salmonella typhimurium TA98 and TA100. The bacterial cells of the isolated strains showed greater binding activity than the pure cell wall, exopolysaccharide, and pepetidoglycan. The carbohydrate moieties of the cell wall or protein molecules on the cell surface have a significant role in binding Trp-P-1 and Trp-P-2, since protease, heating, sodium metaperiodate, or acidic pH treatments significantly (P<0.05) reduced the binding efficacy of the tested bacteria. Addition of metal ions or sodium dodecyl sulfate decreased the binding ability of E. faecium D12, L. acidophilus D38, and E. faecalis D66. Therefore, the binding mechanisms of these strains may consist of ion-exchange and hydrophobic bonds. Especially, the high mutagen binding by L. acidophilus D38 and L. plantarum D70 may reduce the accumulation or absorption of Trp-P-1 and Trp-P-2 in the small intestine via increased excretion of a mutagen-bacteria complex.

Relationship between Mutagenicity of Heterocyclic Aromatic Amines and Stability of Their DNA Adducts: A Study by Semi-empirical Molecular Orbital Method

Quantitative structure-mutagenicity correlations were investigated for heterocyclic aromatic amines (HCAs) by use of a DNA model with three-base pairs. DNA adducts of thirteen HCAs were optimized by the PM3 method and energy decrease, ΔE, of each HCA due to formation of a DNA adduct was obtained as the stability of the adduct. The calculations for the HCA-DNA adducts revealed the interaction between HCA's methyl group and DNA's phosphate, which plays an important role in the stabilization of the adducts. The ΔE values plotted against the logarithm of HCA's mutagenicity, M, provided an almost straight line with the regression coefficient (R) of −0.89 (R 2= 0.79). This good correlation suggests that binding reaction between HCA's nitrenium ion and DNA is an important rate-determining step in the metabolic transformation of HCAs.

Red Meat-Derived Heterocyclic Amines Increase Risk of Colon Cancer: A Population-Based Case-Control Study

Formation of mutagenic heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) is one pathway believed to drive the association of colon cancer with meat consumption. Limited data exist on the associations of individual HCAs and PAHs in red or white meat with colon cancer. Analyzing data from a validated meat preparation questionnaire completed by 1062 incident colon cancer cases and 1645 population controls from an ongoing case-control study, risks of colon cancer were estimated using unconditional logistic regression models, comparing the fourth to the first quartile of mutagen estimates derived from a CHARRED based food frequency questionnaire. Total dietary intake of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) [adjusted odds ratio (aOR) = 1.87, 95% confidence interval (CI) = 1.44–2.44, P trend < 0.0001], 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) (aOR = 1.68, 95% CI = 1.29–2.17, P trend = 0.001) and meat-derived mutagenic activity (aOR = 1.77, 95% CI = 1.36–2.30, P trend < 0.0001) were statistically significantly associated with colon cancer risk. Meat type specific analyses revealed statistically significant associations for red meat-derived MeIQx, DiMeIQx, and mutagenic activity but not for the same mutagens derived from white meat. Our study adds evidence supporting red meat-derived, but not white-meat derived HCAs and PAHs, as an important pathway for environmental colon cancer carcinogenesis.

Trace Analysis of Mutagenic Heterocyclic Aromatic Amines in Cigarette Smoke Condensate and its Base Fractions via Silylation-GC-MS

Abstract Among the more than 5000 chemicals reported in cigarette smoke condensate (CSC), heterocyclic aromatic amines (HAAs) are considered to be a contributor to observed biological activity. HAAs are non-volatile and are reported at ppb levels in CSC. A new method for HAA analysis at the trace level is reported here. N, O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane was employed to derivatize amino groups by heating the reagent containing a sample of CSC at 80 °C for 30 min followed by analysis employing gas chromatography-mass spectroscopy (GC-MS) in the selected-ion-monitoring (SIM) mode. This derivatization method afforded symmetrical peak shapes on a ZB-50 stationary phase and achieved instrumental limits of quantification (LOQ) at 10:1 S/N from -1 ng/mL for AαC to120 ng/mL for Glu-P-1. The chemical identity of each derivative was confirmed by comparison of retention time and mass spectra of standards. The latter were characterized by the following ions: M·+ or [M-1]+, [M-15]+, and m/z 73 (i.e., trimethylsilyl). CSC and its base sub-fractions were studied using the GC-MS method. Ten HAAs were screened and five were quantified in cigarette smoke condensate, while 2-5 HAAs were quantified in each of three base sub-fractions. Values obtained with the new procedure agree well with values reported in the literature and with results obtained from a commercial laboratory via a different analytical method. The potential contribution of each HAA to the overall mutagenic activity observed for CSC and its base fractions is discussed. When considered together, HAAs account for only a small portion (-7.8%) of the observed mutagenicity of the CSC.

Physicochemical characterisation of dietary fibre components and their ability to bind some process-induced mutagenic heterocyclic amines, Trp-P-1, Trp-P-2, AαC and MeAαC

The physicochemical properties of potato fibre, wheat bran and oat samples were investigated, along with their binding capability to heterocyclic amines (HCAs). Potato fibre displayed highest total dietary fibre content (71.8/100g dry weight basis, dwb), followed by wheat bran (57.2/100gdwb) and oat sample 2 (53.0/100gdwb). Oat samples 1, 3 and 4 displayed considerably lower dietary fibre content (20.5–28.8/100g, dwb). Oat samples 3 and 4 displayed highest soluble fibre content (70–83%), and oat sample 3 also displayed highest swelling and water retention capacity (WRC). Dietary fibre samples, except samples 3 and 4, displayed improved binding to HCAs as sample weight increased. The behaviour of wheat bran and potato fibre was similar to oat samples 1 and 2. Binding of MeAαC was comparatively greater than that of other HCAs. Dietary fibre fractions with high insoluble fibre and functional groups of HCAs may significantly contribute to the binding capacity.

Anti-genotoxic activity of Vitis coignetiae Pulliat towards heterocyclic amines and isolation and identification of caftaric acid as an antimutagenic component from the juice

Our study demonstrated that the formation of DNA adducts in liver, lungs, colon and kidneys of mice given a carcinogenic heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), in the diet significantly decreased following the administration of the juice of Vitis coignetiae, purple berries from a vine tree. The juice of V. coignetiae significantly inhibited the clastogenicity and mutagenicity of heterocyclic amines in the micronucleus assay and the Ames test, and was an effective inhibitor of the activities of phase I enzymes (cytochrome P450 1A1 and cytochrome P450 1A2) and enhancer of the activities of phase II enzymes (uridine 5′-diphospho-glucuronosyltransferase and glutathione S-transferase). We investigated the purification and isolation of an active compound in the juice of V. coignetiae using antimutagenicity as a separation marker. Caftaric acid, a polyphenolic compound, was identified as a component responsible for antimutagenicity in the juice of V. coignetiae towards the carcinogenic heterocyclic amine 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2). This is the first report of antimutagenicity of caftaric acid. Caftaric acid was reported as an inhibitor of the protein–protein interactions mediated by the Src-family kinases. The impact of the juice of V. coignetiae and its constituents on tumor initiation and promotion thus warrants further study.