1. Introduction

Cancer is a growing health problem around the world particularly with the steady rise in life expectancy, increasing urbanization and the subsequent changes in environmental conditions, including lifestyle. According to a recent report by the World Health Organization, Until now more than ten million cases of cancer are reported per year worldwide. Much effort has gone into investigating the factors affecting the formation, yield and structures of such compounds ^{[1, 2]}. The three major causes of human carcinogenesis are nicotine from cigarette smoking, infection and inflammation and nutrition and dietary factors ^{[3, 4, 5, 6, 7]}. The search for carcinogenic agents in foods is directed towards explaining the occurrence of cancer in different groups of humans ^{[8, 9, 10]}. Heterocyclic aromatic amines (HAAs) are mutagens/carcinogens formed during the cooking of muscle meat and fish as byproducts of the Maillard reaction between free amino acids, creatine/creatinine and hexoses. HAAs are considered as important factors in a lifestyle related carcinogenesis, according to the International Agency for Research on Cancer classified as 2A/B carcinogens ^{[11, 12]} therefore being extensively investigated worldwide with a particular interest in finding methods to diminish their genotoxic potential. More than twenty four different type of food mutagens have been identified from cooked meat products. Studies have shown that PhIP (2-amino – 1methyl 6 phenylimidazo [4,5b] pyridine) which induces tumor of the prostate and colon in male rats and those of the colon and breast in females is the most abundant HAA formed, followed by MeIQx (2-amino 3,8-dimethylimidazo [4,5f ] quinoxaline ^{[11, 12, 13]} which induces liver and lung tumors in mouse models. As a result, numerous studies concerning protective effects of several dietary components against the mutagenic and carcinogenic activity of HAAs have been performed. Exposure to HAAs has been implicated in the etiology of certain human cancers including colon, prostate and breast cancer. However, much remains to be investigated to determine the mechanisms by which HAAs exert their effects and to discover which HAAs have the greatest relevance to human cancer incidence ^[14].

Our laboratory has been engaged in standardizing methods for detection of dietary mutagens ^[15], We wanted to explore new methods to validate earlier findings. Therefore, in the present study, we targeted two of the most commonly occurring carcinogens in food, which were assessed employing Electrospray ionization mass spectrometry (ESI MS).

2. Materials and Methods

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, CAS No. 105650-23-5) and 2-amino-3,8 dimethylimidazo[4,5-f]quinoxaline (MeIQx, CAS No. 77500-04-0) were purchased from Toronto Research Chemicals (North York, Ontario, Canada). Amberlite XAD-2 resin was from Supelco (Bellefonte, Pennsylvania, USA). Hydrochloric acid, sodium hydroxide and acetone were purchased from Sisco Research Laboratories (Mumbai, India). All reagents were analytical grade. Methanol obtained from Merck (Mumbai, India) was of HPLC grade. The extracts were filtered before injecting through 0.45 µm syringe filter from Axiva Sichem Biotech (New Delhi, India).

Three types of sample products namely Chicken Salami, Peppered Mackerel and Bacon Grill were purchased from local grocery stores in New Delhi, India. All the samples were stored at -20^oC until further processing. 50g of each product was weighed and used for processing. Each sample was replicated twice

Mutagens were extracted according to the method described by ^{[16, 17]} with certain modifications consisting of a liquid-liquid extraction procedure at different pH followed by a solid-liquid extraction on Amberlite XAD-2 resin column. 50g of boneless meat sample was taken each time and uniformly homogenized in a double volume of distilled water in a mixer-grinder. The homogenate was acidified with 0.1M HCl to pH 2.0 and centrifuged at 6000×g for 15 minutes. The supernatant was collected and pellet resuspended in distilled water, acidified and centrifuged again. The supernatants were combined and neutralized with 1M NaOH to pH 7.0. The cloudy supernatant obtained was filtered through Whatman filter paper no1 and the clear filtrate applied to a column of Amberlite XAD-2 resin (1.5cm×10cm) at a flow rate of 2ml/min. Subsequently, 10ml of distilled water (pH 7.0) was introduced to the column and the adsorbed compounds finally eluted with 25ml of acetone followed by 25ml of methanol. Extracts were completely evaporated to dryness in a vacuum rotary evaporator and resuspended in a volume of 1ml methanol for further analysis.

The mass of a protein can be precisely determined by ESI MS. This is an established analytical technique ^[18] which is also applicable to small molecular weight compounds. Such molecules are separated according to the ratio of their mass to charge (m/z). The method used in this study was developed and validated by us. Detection of food mutagens validate the earlier reports as indicated in ^{[19, 20]} Mass spectrometric graphs were recorded as intensity versus mass to charge (m/z) ratio. Technical details for the equipment used for our study were a WATERS SYNAPT G2 (HDMS), Mode employed was ESI +ve, Capillary Voltage = 3k V, Sampling Cone = - 40 k V, Extraction Cone = -120 V. Temperature of the Source = 120°C, Temperature of Desolvation = 350⁰C. Gas flow of Cone Gas (Litres/hour) = 50 and Desolvation Gas (Litres/hour) = 600

A stock solution of MeIQx and PhIP (1.0 mg/ml) each was prepared by dissolving the appropriate amount in methanol. Working standards were prepared fresh by dilution of stocks in methanol so that the final concentration was (1.0mg/5ml).

3. Results

Figure 1a represents the structure of PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine ) and Figure 1b shows MeIQx (2-amino-3,8 dimethylimidazo[4,5-f] quinoxaline). Both, PhIP and MeIQx were employed as standard reference mutagenic compounds.

Figure 1a. Chemical Structure of PhIP

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View next figure

Figure 1b. Chemical Structure of MeIQx

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

View next figure

Table 1 represents the ingredients of each of the sample (Peppered Mackerel, Bacon Grill and Chicken Salami) subjected to analysis. Sample energy content, protein, carbohydrate, fat (PUFA) and other parameters are illustrated in Table 2.

Table 1. Description of samples and ingredients in selected ready to eat meat products

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View next table

Figure 2. ESIMS of standard mutagen: a, PhIP; b,MeIQx

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

View next figure

Table 2. The contents of fat, protein, carbohydrates, energy, in the samples analyzed

Download as

PowerPoint Slide

Larger image(png format)

Tables index

Veiw figure

View current table in a new window

View previous table

Figure 2 represents the ESI MS chromatograms of the two standard known reference mutagens employed for this study namely MeIQx and PhIP. Results obtained for Figure 2a indicates a peak at an (m/z = 214.0972), which corresponds to MeIQx for which (m/z = 213), an additional peak is obtained at an (m/z =236.08),because of the formation of a quasimolecular ion [M+Na]+, Figure 2 b indicates a prominent peak which was obtained at an (m/z = 225.0991) which corresponds to PhIP for which (m/z = 224). An additional peak at an (m/z = 247.084) was observed, which we interpret to be that of [M+Na]+ as an added sodium ion.

Commercially available, sample meat products contain a number of additives, preservatives, binders and ingredients. Peppered Mackerel, Bacon Grill and Chicken Salami are ready to eat packed food products. The main ingredients and nutritional information of the experimental food items which we employed are enlisted in Table 1. Peppered Mackerel does not contain artificial flavours and colour, whereas Bacon Grill has smoke flavour as has been emphasized in the nutritional information. In order to investigate the presence of the representative mutagens in our samples under study: Bacon Grill, Peppered Mackerel and Chicken Salami, when subjected to ESI MS analysis, a distinct peak was observed for one of the samples (Bacon Grill) at an (m/z = 214.0908) which confirms the presence of MeIQx (m/z = 214.0972). We can safely conclude that Bacon Grill has a substantial amount of MeIQx, represented in Figure 3.

Figure 3. ESIMS chromatogram of Bacon Grill

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

View next figure

However, when Chicken Salami (Figure 4) and Peppered Mackerel (Figure 5) were subjected to analysis through the same process, we were unable to detect the presence of any such carcinogenic compound. Further, it was of interest that in Peppered Mackerel and Chicken Salami, we were able to identify a well characterized (unassigned) chromatographic peak at an (m/z = 413), which we presume to be that of a representative of a suspected polymer species being formed at the mass represented.

Figure 4. ESIMS chromatogram of Chicken Salami

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

View next figure

Figure 5. ESIMS chromatogram of Peppered Mackerel

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

4. Discussion

Carcinogenic and mutagenic heterocyclic aromatic amines are natural products often present at ng/g levels in muscle meats when cooked at temperatures above 150^OC. Many heterocyclic aromatic amines have been isolated and identified from cooked meats or model systems, but four compounds are frequently reported in food surveys. These are DiMeIQx (2 amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline), IQ (2- amino-3-methylimidazo [4,5-f]quinoline), MeIQx (2-amino-3,8-dimethylimidazo[4,5f]quinoxaline) and PhIP (2-amino-1-methyl-6phenylimidazo[4,5-b] pyridine ^[8]. PhIP has been shown to have biological properties that appear to be relevant to risk assessment, more importantly tissue specificity with respect to carcinogenicity of colon in male rats and mammary gland and colon in females. Similar cancer targets have been observed in humans exposed to a diet rich in meat, suggesting a role of these compounds in human cancer etiology which necessitates an understanding of the levels of consumption of PhIP and related compounds in our diet. Human populations are exposed to mutagenic/carcinogenic HAAs mostly through consumption of meat and meat products ^{[21, 22]}. Further, it was observed that MeIQx and PhIP were reported to occur more frequently ^[23].

To summarize this study, HAAs were determined in three different types of samples obtained from local grocery stores. The validated method provided accurate results, constituting a simple and easy method to identify the presence of such carcinogenic/mutagenic compounds in foods commonly consumed by humans. Among the three samples analyzed by us, a positive result was obtained for one of the samples, namely Bacon Grill. We propose that a higher fat content (24.6%) in pork may facilitate the formation of HAAs through Maillard reaction products such as pyridine from lipid degradation. Although pepperd mackerel also has a high fat content, it has black pepper as one of the ingredients to which may presumably be attributed the inhibition of generation of Heterocyclic Aromatic Amines as has been shown by previous workers ^[24].

Acknowledgements

The authors duly acknowledge financial assistance from the University Grants Commission, New Delhi, India for funding the Major Research Project- UGC MRP (F. No 33-221/2007) Plabon Borah is acknowledged for technical assistance in ESI MS analysis.

Statement of Competing Interests

The authors disclose there is no conflict of interest. No competing financial interests exist.

References

[1]	Sugimura T, Sato S, “Mutagens-carcinogens in foods,” Cancer Res, 43(5 Suppl) 2415s-2421s, 1983.
	In article
[2]	Pfau W, Martin F. L, Cole K. J, Venitt S, Phillips D. H, Grover P. L, Hans Marquardt. H, “Heterocyclic aromatic amines induce DNA Strand breaks and cell transformation,” Carcinogenesis, 20(4): 545-551, 1999.
	In article		CrossRef
[3]	Heeschen C, Jang J. J, Weis M, Pathak A, Kaji S, Hu R. S, Tsao P. S, Johnson F. L, Cooke J. P, “Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis,” Nat Med, 7(7): 833-839, 2001.
	In article		CrossRef
[4]	Argentin G, Cicchetti R, “Genotoxic and antiapototic effect of nicotine on human gingival fibroblasts,” Toxicol Sci, 79(1): 75-81, 2004.
	In article		CrossRef
[5]	Guo J, Ibaragi S, Zhu T, Luo L, Hu G, Huppi P. S, Chen C. Y, “Nicotine promotes mammary tumor migration via a signaling cascade involving protein kinase C and cdc42,” Cancer Res, 68(20): 8473-8481, 2008.
	In article		CrossRef
[6]	Sugimura T, “Nutrition and dietary carcinogens,” Carcinogenesis, 21 (3): 387-395, 2000.
	In article		CrossRef
[7]	Doll R, Peto R, “The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today,” J Nat Cancer Inst, 66 (6): 1191-1308, 1981.
	In article
[8]	Knize M. G, Salmon C. P, Hopmans E. C, Felton J. S, “Analysis of foods for heterocyclic aromatic amine carcinogens by solid- phase extraction and high –performance liquid chromatography,” J. Chromatogr A, 763 (1-2): 179-185,1997.
	In article		CrossRef
[9]	Felton J. S, Knize M. G, “Occurrence, identification and bacterial mutagenicity of heterocyclic amines in cooked food,” Mutat Res, 259 (3-4): 205-217, 1991.
	In article		CrossRef
[10]	IARC, “Some Naturally Occurring and Synthetic Food Components, Furocoumarins and Ultraviolet Radiation” (vol. 40 of IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, IARC, Lyon, France) 40:123-159, 1986.
	In article
[11]	IARC, “Some Naturally Occurring Substances: Food Items and Constituents: Heterocyclic Aromatic Amines and Mycotoxins,” (vol. 56 of IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, IARC, Lyon, France) 165-231, 1993.
	In article
[12]	Keating G. A, Bogen K. T, “Methods for estimating heterocyclic amine concentrations in cooked meats in the US diet,” Food Chem. Toxicol, 39 (1): 29-43, 2001.
	In article		CrossRef
[13]	Messner C, Murkovich M, “Evaluation of a new model system for studying the formation of heterocyclic amines,” J. Chromatogr B Analyt Technol Biomed Life Sci, 802 (1): 19-26, 2004.
	In article		CrossRef
[14]	Ni W, McNaughton L, LeMaster D M, Sinha R, Turesky R. J, “Quantitation of 13 heterocyclic aromatic amines in cooked beef, pork and chicken by liquid chromatography electrospray ionization/tandem mass spectrometry, “J. Agric. Food Chem, 56 (1): 68-78,2008.
	In article		CrossRef
[15]	Bjeldanes L. F, Grosse K. R, Davis P. H, Stuermer D. H, Healy S. K, Felton J. S, “An XAD-2 resin method for efficient extraction of mutagens from fried ground beef,” Mutat Res, 105 (1-2): 43-49, 1982.
	In article		CrossRef
[16]	Zaidi R, Kumar S, Rawat P. R, “ Rapid detection and quantification of dietary mutagens in food using mass spectrometry and ultra performance liquid chromatography,” Food Chem, 135 (4): 2897-2903, 2012.
	In article		CrossRef
[17]	Perez C, Lopez De Cerain A, Bello J, “Modulation of mutagenic activity in meat samples after deep-frying in vegetable oils,” Mutagenesis, 17 (1): 63-66, 2002.
	In article		CrossRef
[18]	Stryer L, Biochemistry, W. H. Freeman and Company, New York, 2000, 52-53.
	In article
[19]	Zaidi R, Rawat PR, “Evaluation of cytotoxicioty of Food in Human Hepatoma HepG2 cells: Comet Assay Coupled to the MTT Assay”, J.Nutr. Food Sciences, 2(6): 1-5, 2012.
	In article		CrossRef
[20]	Zaidi R, Rawat PR, “Identification of Heterocyclic Amines in home cooked and commercially available meat foods,” J.Nutr.Food Sciences, 1(3): 1-7, 2011.
	In article		CrossRef
[21]	Toribio F, Busquets R, Puignou L, Galceran M. T, “Heterocyclic amines in griddled beaf steak analysed using a single extract clean-up procedure,” Food Chem Toxicol, 45(4): 667-675, 2007.
	In article		CrossRef
[22]	Johansson M, Jägerstad M, “Influence of oxidised deep- frying fat and iron on the formation of food mutagens in a model system,” Food Chem. Toxicol, 31 (12): 971-979, 1993.
	In article		CrossRef
[23]	Abdulkarim B. G, Smith J. S, “Heterocyclic amines in fresh and processed meat products,” J. Agric. Food. Chem, 46, 4680-4687, 1998.
	In article		CrossRef
[24]	Fatih Oz,Mukerrem Kaya, “The inhibitory effect of black pepper on formation of heterocyclic aromatic amines in highfat meatball,” Food Control, 22 (3-4): 596-600, 2011.
	In article		CrossRef