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The proteolytic release of genotoxins from cooked beef
BBRC Biochemical and Biophysical Research Communications 293 (2002) 1497–1501 www.academicpress.com
The proteolytic release of genotoxins from cooked beefq Francis L. Martin,* Kathleen J. Cole, David H. Phillips, and Philip L. Grover Haddow Laboratories, Institute of Cancer Research, Cotswold Rd., Sutton, Surrey SM2 5NG, UK Received 22 April 2002
Abstract Dietary factors are important in the aetiology of human cancer and carcinogens, mostly heterocyclic aromatic amines, have been isolated from cooked proteinaceous foodstuffs. Whilst such carcinogens have induced tumours in rodent bioassays, the dosages required were much higher than estimates of human exposure levels. We have examined the possibility that genotoxins, which were not extractable prior to enzymic digestion, may be released from cooked beef by proteolysis. Dichloromethane and/or a solid-phase tandem extraction procedure were used with aqueous homogenates of pan-fried or uncooked beef, both before and after proteolysis (proteinase K). Genotoxicity was measured using the alkaline single cell–gel electrophoresis (‘Comet’) assay in MCL-5 cells and mutagenicity in Salmonella typhimurium strains TA1538 or YG1019. Proteolysis released significant amounts of DNA-damaging material that was not extractible prior to enzymic digestion, suggesting that human exposures to diet-derived genotoxins may have been underestimated. Ó 2002 Elsevier Science (USA). All rights reserved.
Heterocyclic aromatic amines (HAAs) are formed when proteinaceous foods, such as meat or fish, are cooked [1,2] and when mixtures of amino acids and creatine or creatinine are heated in modelling reactions [3,4]. More than 20 individual HAAs have been isolated and characterised and their biological properties include mutagenicity and clastogenicity in a variety of test systems [5], the transformation of mammalian cells in culture [6] and carcinogenicity, albeit at high doses, in rodents [7–9]. Because they are widespread in the human diet, HAAs are also suspected of involvement in the initiation of, for example, colon, breast, and prostate cancer [10] but this remains unproven. Prior to quantitation, HAAs have been isolated using solid phase, ion exchange resin or solvent extraction procedures [11–13], and have been found to be present at ng/g concentrations in a wide variety of cooked foods
[2]. Using the same methods, typical total human intake values for HAAs have been estimated at 3 lg=day [10]. What does not appear to have been considered previously is the possibility that HAA-like compounds could be formed, through condensation reactions between amino acid residues in proteins and creatine or creatinine, when protein-rich foods are heated but which remain covalently bound to the protein until proteolytic digestion occurs in the gastro-intestinal tract. Such compounds would not be extractible by any of the commonly used procedures for HAA isolation and would not have been included, therefore, in total daily intake estimations. In this paper we describe preliminary experiments which show that solvent- soluble genotoxins, which were not extractible prior to treatment with proteinase K, can be released from aqueous homogenates of cooked beef by proteolysis.
q Abbreviations: HAA, heterocyclic aromatic amine; SPTE, solidphase tandem extraction; DMSO, dimethylsulfoxide; SSB, singlestrand break; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; DCM, dichloromethane. * Corresponding author. Present address: Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK. Fax: +44-1524-843854. E-mail address: [email protected] (F.L. Martin).
Materials and methods Chemicals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (>97% pure) was obtained from Toronto Research Chemicals (Toronto, Canada). All other chemicals were obtained from Sigma Chemical (Poole, UK) unless otherwise stated. Beef samples. Freshly prepared hamburger patties (4 175 g, lean Aberdeen Angus beef) were purchased from a local supermarket. Two
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patties were fried, without any additional oil or fat, in a previously unused, non-stick pan until well done: the other two were used as a raw beef control. Cooked and uncooked beef samples were homogenised in de-ionised water (2 volumes) in a domestic blender (30 s) and were stored frozen at )20 °C. Prior to extraction, thawed samples (100 g beef equivalent) were re-homogenised using an Ultra Turrax homogeniser. Extracts. Extractions of the cooked and of the uncooked samples were carried out using the sequence of procedures outlined in Fig. 1. Portions of aqueous homogenate (8 g beef equivalent) were saponified by treatment with NaOH (1 M, 20 ml) and further homogenised (Ultra Turrax) for 1 min. The mixtures were then extracted using the solidphase tandem extraction (SPTE) procedure, exactly as described previously [11] and designated SPTE-1. Other portions of the aqueous homogenates (24 g beef equivalent) were extracted five times with dichloromethane (DCM; 5 2 volume). Portions of the aqueous phase (8 g beef equivalent) were then saponified by treatment with NaOH (1 M, 20 ml), homogenised (Ultra Turrax), extracted using the SPTE method, and designated SPTE-2. Other portions of a solvent-extracted aqueous phase (16 g beef equivalent) were subjected to proteolytic digestion by incubation (37 °C, 48 h) with proteinase K (EC 3.4.21.64; 1 mg/ml) in buffer (10 mM EDTA/50 mM Tris, pH 8.0) containing SDS 1% w/v), extracted with DCM (1 2 volume), and the solvent extract divided into two. One half was evaporated, the residue re-dissolved in DMSO and designated PK. The other half was evaporated, the residue saponified in NaOH (1 M, 20 ml), extracted using SPTE, and designated PK + SPTE. In each case residues (8 g beef equivalent) remaining after the evaporation of solvent were re-suspended in DMSO prior to incorporation into assays at a maximum concentration of 1% (v/v). Single-cell gel electrophoresis (‘Comet’) assay. MCL-5 cells (obtained under licence from the Gentest, Woburn, MA) suspended in PBS (1 105 cells=75 ll) were incubated in the presence of the DNArepair inhibitors, hydroxyurea (HU, 10 mM) and cytosine arabinoside (ara-C, 1.8 mM), at 37 °C for 30 min [15,16] with or without the addition of beef extracts. Cell lysis and alkaline nuclear electrophoresis were performed exactly as described [15,16]. Briefly, frosted microscope slides (Curtin Matheson Scientific, Houston, TX), on which cells were embedded in an agarose sandwich, were submerged in cold alkaline lysis solution (2.5 M NaCl, 100 mM EDTA disodium salt,
Fig. 1. Scheme showing sequence of steps carried out to yield extracts of homogenates of cooked or uncooked beef that were tested for genotoxic activity as described in the text (SPTE, solid-phase tandem extraction; DCM, dichloromethane).
10 mM Tris, 1% N-lauroyl sarcosine, adjusted to pH 10 with NaOH; then made up to 1% Triton X-100 and 10% DMSO before use), protected from light, and stored at 4 °C for at least 1 h. Under red light, slides were transferred to a horizontal electrophoresis tank, covered in alkaline electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH > 13:0), and stored in a chilled incubator at 10 °C for 40 min to allow unwinding of the DNA before electrophoresis at 0.8 V/cm and 300 mA for 36 min. Slides were neutralised (Tris, 0.5 M, pH 7.5), stained with an aqueous solution of ethidium bromide (20 ng/ml) and nuclear material was visualised by epifluorescence using a Leitz Laborlux S microscope. Images were digitized, and DNA damage was expressed as comet tail length (CTL, lm) [15,16]. A total of 50 nuclei/ data point from two slides was scored. Increases in CTL were assessed for significance using the Mann–Whitney test. Bacterial mutagenicity. Extracts were assessed for mutagenicity towards Salmonella typhimurium TA1538 and YG1019 by the plateincorporation assay. Assays, in triplicate, were performed in the presence of Aroclor 1254-induced rat-liver S9 prepared from male Wistar rats with the final S9 mix (30% v/v) containing 33 mM KCl, 8 mM MgCl2 , 5 mM glucose-6-phosphate, 3.3 mM NADPþ , 0.1 M phosphate buffer, pH 7.4 [14]. Revertants/plate were recorded following 72 h incubation at 37 °C, using a calibrated Biotran II Automatic Colony Counter. Cell viability. Trypan blue exclusion was used. Individual cell suspensions were gently mixed 1:1 with trypan blue (0.4% solution in 0.85% saline, Flow Laboratories, Scotland), allowed to stand for 5–10 min and placed in a haemocytometer. The percentage of cells that excluded trypan blue was used as an indicator of cell viability and was estimated both before and after treatment of cells.
Results Comet formation in MCL-5 cells Fig. 2 shows that comet-formation induced in MCL-5 cells by cooked beef extracts (SPTE-1) was reduced if a series of solvent extractions with DCM are performed prior to the preparation of a second set of SPTE extracts (SPTE-2). In the presence of HU/ara-C, SPTE-1 induced increases in CTL (median ¼ 49:5 lm, P < 0:0001) as opposed to control CTL ðmedian ¼ 14:0 lmÞ. However, following solvent extraction SPTE-2 was not comet-forming ðmedian ¼ 10:0 lmÞ. This suggests that ‘‘free’’ genotoxins were extracted by the solvent from cooked beef samples. However, proteinase K digestion of these previously extracted cooked beef samples generated DCM extracts (PK) that were significantly cometforming (median ¼ 81:5 lm, P < 0:0001). The level of comet-forming activity released by enzymic digestion was similar to, if not greater than, the levels of activity in the original extracts (SPTE-1). A significant proportion of this activity was removed from PK extracts by SPTE (extracts designated PK + SPTE; median ¼ 35:0 lm). These results were reproducible in subsequent experiments using other freshly cooked beef patties (data not shown). Parallel extracts (SPTE-1, SPTE-2, PK, and PK + SPTE) of homogenates of uncooked beef samples were all inactive (Fig. 3), suggesting that it is the cooking process that leads to the production of both ‘‘free’’ and ‘‘bound’’ genotoxic principles. Neither extracts of
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Fig. 2. Comet-forming activities of extracts of cooked beef (8 g equivalent), measured in MCL-5 cells. Cell suspensions in PBS (1 105 cells=75 ll) were incubated in the presence of the DNArepair inhibitors, hydroxyurea, and cytosine arabinoside (HU/ara-C) (10 mM/1.8 mM final concentration), at 37 °C for 30 min. Comet tail length ðlmÞ was used as a measure of DNA damage. MCL-5 cells were treated with vehicle control (DMSO): A, in the absence of HU/ara-C and B, in the presence of HU/ara-C. Cells were also treated, in the presence of HU/ara-C, with extracts of cooked beef, added as solutions in DMSO as follows: C, SPTE-1; D, SPTE-2; E, PK 3; F, PK+SPTE. Incubations and the Comet assay were carried out as described in Materials and methods. P , as compared with HU/ara-C control; P , PK + SPTE compared with PK.
cooked nor uncooked beef samples were observed to induce cytotoxicity, as measured by trypan blue exclusion (data not shown). Bacterial mutagenicity An exogenous metabolic activation system (Aroclor 1254-induced rat-liver S9) was found to be a requirement for the detection of bacterial mutagenicity in cooked beef extracts, since no mutagenic activity was observed in its absence (data not shown). Results for each extraction procedure were normalised (revertants/ plate/g-beef equivalent) by subtracting the mean number of background revertant colonies and then dividing the mean number of induced-revertant colonies by the g-meat equivalent for that extract. Levels of spontaneous revertants were 35 6 and 52 6 (n ¼ 12) for the strains TA1538 and YG1019, respectively. A positive control (PhIP, 0:5 lg=plate) was employed and levels of induced revertants were 844 125 and 1231 217
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Fig. 3. Comet-forming activities of extracts (8 g equivalent) of uncooked beef, measured in MCL-5 cells. Cell suspensions in PBS (1 105 cells=75 ll) were incubated in the presence of the DNArepair inhibitors, hydroxyurea, and cytosine arabinoside (HU/ara-C) (10 mM/1.8 mM final concentration), at 37 °C for 30 min. Comet tail length ðlmÞ was used as a measure of DNA damage. MCL-5 cells were treated with vehicle control (DMSO): A, in the absence of HU/ara-C and B, in the presence of HU/ara-C. Cells were also treated in the presence of HU/ara-C with extracts of uncooked beef, added as solutions in DMSO as follows: C, SPTE-1; D, SPTE-2; E, PK 3; F, PK + SPTE. Incubations and the Comet assay were carried out as described in Materials and methods.
(n ¼ 12) for the strains TA1538 and YG1019, respectively. Fig. 4 shows that levels of bacterial mutagenicity induced by a cooked beef extract (SPTE-1) are reduced, but not eliminated, if a series of solvent extractions with DCM are performed prior to the preparation of the second set of extracts (SPTE-2), suggesting that free mutagens are extractible from the aqueous homogenates of cooked beef samples. However, following a proteinase K digestion of these previously solvent-extracted beef samples, the subsequent DCM extracts (PK) induce levels of bacterial mutagenicity in strains TA1538 and YG1019 similar to, if not greater than, the levels induced by the original extracts (SPTE-1). Not all of this activity is extractable by SPTE as demonstrated by the levels of bacterial mutagenicity remaining in the PK + SPTE extracts. These results were reproducible in subsequent experiments using other freshly cooked beef patties: no significant increases above background mutagenicity were seen with any of the extracts prepared from uncooked beef (data not shown).
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Fig. 4. Normalised levels of bacterial mutagenicity induced by extracts (8 g equivalent) of cooked beef in S. typhimurium TA1538 or YG1019. Bacterial mutagenicity assays were performed in the presence of S9 as described in Materials and methods. Each point represents normalised mean revertants per plate (SD < 20%) from three plates.
Discussion Dietary and/or environmental factors are important determinants in the aetiology of both non-hereditary and hereditary cancers [17]. The best evidence for this observation comes from studies in which incidence rates or mortality have been determined for migrants moving from lower-risk to higher-risk countries [18]. For example, data from 1962–1971 showed that, although breast cancer mortality in recently arrived Italian migrants to Australia was low, it rose over time to match that of the host population [19]. Similarly, breast cancer risk in females of Chinese, Japanese, or Filipino origin migrating to the United States increased by 80% after 10 years of residence [20]. Cancers which occur with higher frequency in Western societies have been associated with the consumption of higher levels of cooked meats, especially cooked red meat [2,7,10,17]. Well-done meat contains protein-derived mutagens [21], many of which have been identified as HAAs. These compounds are formed at parts-per-billion levels as the products of protein pyrolysis or Maillard reactions [2]. They have also been produced in modelling reactions in which amino acids such as glycine or alanine are heated, either dry or in solution, in the presence of creatinine or creatine [3,4]. HAAs have induced tumours
in rodent bioassays [9] and are strongly suspected of contributing to human cancer incidence [7,10]. Their characteristic structure includes one or two heterocyclic rings fused to an aminoimidazo ring. These imidazole mutagens include the aminoimidazo-quinoxaline derivatives, the aminoimidazo-quinoline derivatives, and the pyrido-indole derivatives. The quinolines and quinoxalines are strongly mutagenic to bacteria whereas the pyridine derivatives are much less potent [5,6]. However, the pyridines seem to possess greater genotoxicity in test systems involving mammalian cells [5,6]. Although some 24 HAAs have either been identified or partially identified [10], more than 70% of the mutagenic activity extractable from grilled, fried, or broiled meat samples remains unaccounted for [22]. The present study has investigated the possibility that there are dietary genotoxins that have so far avoided detection and quantitation. When proteinaceous foods are heated, ‘‘free’’ HAAs are formed that are extractable by well-documented methods [11–13]. Higher yields can be produced if the protein is hydrolysed to its amino acid constituents prior to heating [23,24], in agreement with the results obtained in modelling reactions [25]. We have now examined the notion that other genotoxins are formed in heated proteins that remain integral with, and covalently bound to, the protein until hydrolysis occurs in the gastro-intestinal tract. Reactions between, for example, C- or N-terminal aromatic amino acid residues and creatine or creatinine might be expected to yield HAA-like compounds. The products would not be extractable prior to proteolysis and would therefore have remained undetected to date: they would not necessarily be identical to known HAAs since either the carboxylic acid or the amino group will have been part of a peptide bond when, or if, condensation reactions occurred (with ‘internal’ amino acid residues both the –COOH and the –NH2 groups would be unavailable for reactions). Results from the preliminary experiments described here have lent support to this idea. When aqueous homogenates of well-done beef were proteinase K-digested, materials that were genotoxic in the Comet assay employing MCL-5 cells became extractable that were not apparently solvent soluble before proteolysis. Quantitatively, the results obtained using the Comet assay (Fig. 2) showed that less than half the total genotoxic activity present in a cooked beef homogenate was extractible prior to proteolysis and similar but less marked results were obtained using the mutagenicity assays (Fig. 4). Some of the solvent-soluble genotoxic activities released by proteolysis were also extractible using SPTE (Figs. 2 and 4, PK + SPTE) suggesting that it may include heterocyclic or aromatic amino compounds. In addition, although no more comet-forming activity could be extracted using SPTE after five sequential DCM extractions (Fig. 2C and D), some mutagenic
F.L. Martin et al. / Biochemical and Biophysical Research Communications 293 (2002) 1497–1501
activity was present in the SPTE-2 extract (Fig. 4). The reasons for the differences between the results obtained with the two biological test systems are not known. It may be worth noting that, for some HAAs, genotoxicity in the Comet assay appears to be more closely correlated with their ability to induce morphological transformation of cells in culture than with their mutagenicity in S. typhimurium [5]. Further work is clearly required to confirm the findings reported here, to identify the genotoxins released by the enzymic digestion of heattreated proteins and to assess the contribution that such putative ‘‘closet carcinogens’’ may make to the levels of genotoxins that are ingested in the diet. Acknowledgments This study was supported by research grants from the Association for International Cancer Research, the Cancer Research Campaign, and the Department of Health. We wish to thank Professor Nitya Kundu and Drs. Wolfgang Pfau and Terry Jones for helpful discussions.
References [1] M. Nagao, M. Honda, Y. Seino, T. Yahagi, T. Sugimura, Cancer Lett. 2 (1977) 221–226. [2] J.S. Felton, M.G. Knize, Mutat. Res. 259 (1991) 205–217. [3] S. Grivas, T. Nyhammar, K. Olsson, M. Jagerstad, Mutat. Res. 151 (1985) 177–183. [4] S. Grivas, T. Nyhammar, K. Olsson, M. Jagerstad, Food Chem. 20 (1986) 127–136. [5] W. Pfau, F.L. Martin, K.J. Cole, S. Venitt, D.H. Phillips, P.L. Grover, H. Marquardt, Carcinogenesis 20 (1999) 545–551.
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[6] W. Pfau, H. Marquardt, Toxicology 166 (2001) 25–30. [7] E.G. Snyderwine, Cancer 74 (Suppl.) (1994) 1070–1077. [8] M. Nagao, T. Ushijima, K. Wakabayashi, M. Ochiai, H. Kushida, T. Sugimura, R. Hasegawa, T. Shirai, N. Ito, Cancer 74 (1984) 1063–1069. [9] T. Shirai, M. Sano, S. Tamano, S. Takahashi, M. Hirose, M. Futakuchi, R. Hasegawa, K. Imaida, K. Matsumoto, K. Wakabayashi, T. Sugimura, N. Ito, Cancer Res. 57 (1997) 195–198. [10] T. Sugimura, Princess Takamatsu Symp. 23 (1995) 214–231. [11] G.A. Gross, Carcinogenesis 11 (1990) 1597–1603. [12] G. Becher, M.G. Knize, I.F. Nes, J.S. Felton, Carcinogenesis 9 (1988) 247–253. [13] P.A. Nielsen, Mutat. Res. 276 (1992) 117–123. [14] S. Venitt, C. Crofton-Sleigh, R. Forster, in: S. Venitt, J.M. Parry (Eds.), Mutagenicity Testing: A Practical Approach, IRL Press, Oxford, 1984, pp. 45–98. [15] F.L. Martin, S. Venitt, P.L. Carmichael, C. Crofton-Sleigh, E.M. Stone, K.J. Cole, B.A. Gusterson, P.L. Grover, D.H. Phillips, Carcinogenesis 18 (1997) 2299–2305. [16] F.L. Martin, K.J. Cole, M.H. Orme, P.L. Grover, D.H. Phillips, S. Venitt, Mutat. Res. 445 (1999) 21–43. [17] R. Doll, R. Peto, J. Natl. Cancer Inst. 66 (1981) 1191–1308. [18] J. Peto, Nature 411 (2001) 390–395. [19] A.J. McMichael, G.G. Giles, Cancer Res. 48 (1988) 751–756. [20] R.G. Ziegler, R.N. Hoover, M.C. Pike, A. Hildesheim, A.M. Nomura, D.W. West, A.H. Wu-Williams, L.N. Kolonel, P.L. Horn-Ross, J.F. Rosenthal, M.B. Hyer, J. Natl. Cancer Inst. 85 (1993) 1819–1827. [21] B. Commoner, A.J. Vithayathil, P. Dolara, S. Nair, P. Madyastha, C.C. Cuca, Science 201 (1978) 913–916. [22] J.S. Felton, M.G. Knize, F.T. Hatch, M.J. Tanga, M.E. Colvin, Cancer Lett. 143 (1999) 127–134. [23] R.T. Taylor, E. Fultz, V. Shore, J. Environ. Sci. Health A19 (1984) 791–817. [24] R.T. Taylor, V. Shore, E. Fultz, J. Environ. Sci. Health A19 (1984) 819–845. [25] K. Skog, Food Chem. Toxicol. 31 (1993) 655–675.
The proteolytic release of genotoxins from cooked beefq Francis L. Martin,* Kathleen J. Cole, David H. Phillips, and Philip L. Grover Haddow Laboratories, Institute of Cancer Research, Cotswold Rd., Sutton, Surrey SM2 5NG, UK Received 22 April 2002
Abstract Dietary factors are important in the aetiology of human cancer and carcinogens, mostly heterocyclic aromatic amines, have been isolated from cooked proteinaceous foodstuffs. Whilst such carcinogens have induced tumours in rodent bioassays, the dosages required were much higher than estimates of human exposure levels. We have examined the possibility that genotoxins, which were not extractable prior to enzymic digestion, may be released from cooked beef by proteolysis. Dichloromethane and/or a solid-phase tandem extraction procedure were used with aqueous homogenates of pan-fried or uncooked beef, both before and after proteolysis (proteinase K). Genotoxicity was measured using the alkaline single cell–gel electrophoresis (‘Comet’) assay in MCL-5 cells and mutagenicity in Salmonella typhimurium strains TA1538 or YG1019. Proteolysis released significant amounts of DNA-damaging material that was not extractible prior to enzymic digestion, suggesting that human exposures to diet-derived genotoxins may have been underestimated. Ó 2002 Elsevier Science (USA). All rights reserved.
Heterocyclic aromatic amines (HAAs) are formed when proteinaceous foods, such as meat or fish, are cooked [1,2] and when mixtures of amino acids and creatine or creatinine are heated in modelling reactions [3,4]. More than 20 individual HAAs have been isolated and characterised and their biological properties include mutagenicity and clastogenicity in a variety of test systems [5], the transformation of mammalian cells in culture [6] and carcinogenicity, albeit at high doses, in rodents [7–9]. Because they are widespread in the human diet, HAAs are also suspected of involvement in the initiation of, for example, colon, breast, and prostate cancer [10] but this remains unproven. Prior to quantitation, HAAs have been isolated using solid phase, ion exchange resin or solvent extraction procedures [11–13], and have been found to be present at ng/g concentrations in a wide variety of cooked foods
[2]. Using the same methods, typical total human intake values for HAAs have been estimated at 3 lg=day [10]. What does not appear to have been considered previously is the possibility that HAA-like compounds could be formed, through condensation reactions between amino acid residues in proteins and creatine or creatinine, when protein-rich foods are heated but which remain covalently bound to the protein until proteolytic digestion occurs in the gastro-intestinal tract. Such compounds would not be extractible by any of the commonly used procedures for HAA isolation and would not have been included, therefore, in total daily intake estimations. In this paper we describe preliminary experiments which show that solvent- soluble genotoxins, which were not extractible prior to treatment with proteinase K, can be released from aqueous homogenates of cooked beef by proteolysis.
q Abbreviations: HAA, heterocyclic aromatic amine; SPTE, solidphase tandem extraction; DMSO, dimethylsulfoxide; SSB, singlestrand break; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; DCM, dichloromethane. * Corresponding author. Present address: Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK. Fax: +44-1524-843854. E-mail address: [email protected] (F.L. Martin).
Materials and methods Chemicals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (>97% pure) was obtained from Toronto Research Chemicals (Toronto, Canada). All other chemicals were obtained from Sigma Chemical (Poole, UK) unless otherwise stated. Beef samples. Freshly prepared hamburger patties (4 175 g, lean Aberdeen Angus beef) were purchased from a local supermarket. Two
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patties were fried, without any additional oil or fat, in a previously unused, non-stick pan until well done: the other two were used as a raw beef control. Cooked and uncooked beef samples were homogenised in de-ionised water (2 volumes) in a domestic blender (30 s) and were stored frozen at )20 °C. Prior to extraction, thawed samples (100 g beef equivalent) were re-homogenised using an Ultra Turrax homogeniser. Extracts. Extractions of the cooked and of the uncooked samples were carried out using the sequence of procedures outlined in Fig. 1. Portions of aqueous homogenate (8 g beef equivalent) were saponified by treatment with NaOH (1 M, 20 ml) and further homogenised (Ultra Turrax) for 1 min. The mixtures were then extracted using the solidphase tandem extraction (SPTE) procedure, exactly as described previously [11] and designated SPTE-1. Other portions of the aqueous homogenates (24 g beef equivalent) were extracted five times with dichloromethane (DCM; 5 2 volume). Portions of the aqueous phase (8 g beef equivalent) were then saponified by treatment with NaOH (1 M, 20 ml), homogenised (Ultra Turrax), extracted using the SPTE method, and designated SPTE-2. Other portions of a solvent-extracted aqueous phase (16 g beef equivalent) were subjected to proteolytic digestion by incubation (37 °C, 48 h) with proteinase K (EC 3.4.21.64; 1 mg/ml) in buffer (10 mM EDTA/50 mM Tris, pH 8.0) containing SDS 1% w/v), extracted with DCM (1 2 volume), and the solvent extract divided into two. One half was evaporated, the residue re-dissolved in DMSO and designated PK. The other half was evaporated, the residue saponified in NaOH (1 M, 20 ml), extracted using SPTE, and designated PK + SPTE. In each case residues (8 g beef equivalent) remaining after the evaporation of solvent were re-suspended in DMSO prior to incorporation into assays at a maximum concentration of 1% (v/v). Single-cell gel electrophoresis (‘Comet’) assay. MCL-5 cells (obtained under licence from the Gentest, Woburn, MA) suspended in PBS (1 105 cells=75 ll) were incubated in the presence of the DNArepair inhibitors, hydroxyurea (HU, 10 mM) and cytosine arabinoside (ara-C, 1.8 mM), at 37 °C for 30 min [15,16] with or without the addition of beef extracts. Cell lysis and alkaline nuclear electrophoresis were performed exactly as described [15,16]. Briefly, frosted microscope slides (Curtin Matheson Scientific, Houston, TX), on which cells were embedded in an agarose sandwich, were submerged in cold alkaline lysis solution (2.5 M NaCl, 100 mM EDTA disodium salt,
Fig. 1. Scheme showing sequence of steps carried out to yield extracts of homogenates of cooked or uncooked beef that were tested for genotoxic activity as described in the text (SPTE, solid-phase tandem extraction; DCM, dichloromethane).
10 mM Tris, 1% N-lauroyl sarcosine, adjusted to pH 10 with NaOH; then made up to 1% Triton X-100 and 10% DMSO before use), protected from light, and stored at 4 °C for at least 1 h. Under red light, slides were transferred to a horizontal electrophoresis tank, covered in alkaline electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH > 13:0), and stored in a chilled incubator at 10 °C for 40 min to allow unwinding of the DNA before electrophoresis at 0.8 V/cm and 300 mA for 36 min. Slides were neutralised (Tris, 0.5 M, pH 7.5), stained with an aqueous solution of ethidium bromide (20 ng/ml) and nuclear material was visualised by epifluorescence using a Leitz Laborlux S microscope. Images were digitized, and DNA damage was expressed as comet tail length (CTL, lm) [15,16]. A total of 50 nuclei/ data point from two slides was scored. Increases in CTL were assessed for significance using the Mann–Whitney test. Bacterial mutagenicity. Extracts were assessed for mutagenicity towards Salmonella typhimurium TA1538 and YG1019 by the plateincorporation assay. Assays, in triplicate, were performed in the presence of Aroclor 1254-induced rat-liver S9 prepared from male Wistar rats with the final S9 mix (30% v/v) containing 33 mM KCl, 8 mM MgCl2 , 5 mM glucose-6-phosphate, 3.3 mM NADPþ , 0.1 M phosphate buffer, pH 7.4 [14]. Revertants/plate were recorded following 72 h incubation at 37 °C, using a calibrated Biotran II Automatic Colony Counter. Cell viability. Trypan blue exclusion was used. Individual cell suspensions were gently mixed 1:1 with trypan blue (0.4% solution in 0.85% saline, Flow Laboratories, Scotland), allowed to stand for 5–10 min and placed in a haemocytometer. The percentage of cells that excluded trypan blue was used as an indicator of cell viability and was estimated both before and after treatment of cells.
Results Comet formation in MCL-5 cells Fig. 2 shows that comet-formation induced in MCL-5 cells by cooked beef extracts (SPTE-1) was reduced if a series of solvent extractions with DCM are performed prior to the preparation of a second set of SPTE extracts (SPTE-2). In the presence of HU/ara-C, SPTE-1 induced increases in CTL (median ¼ 49:5 lm, P < 0:0001) as opposed to control CTL ðmedian ¼ 14:0 lmÞ. However, following solvent extraction SPTE-2 was not comet-forming ðmedian ¼ 10:0 lmÞ. This suggests that ‘‘free’’ genotoxins were extracted by the solvent from cooked beef samples. However, proteinase K digestion of these previously extracted cooked beef samples generated DCM extracts (PK) that were significantly cometforming (median ¼ 81:5 lm, P < 0:0001). The level of comet-forming activity released by enzymic digestion was similar to, if not greater than, the levels of activity in the original extracts (SPTE-1). A significant proportion of this activity was removed from PK extracts by SPTE (extracts designated PK + SPTE; median ¼ 35:0 lm). These results were reproducible in subsequent experiments using other freshly cooked beef patties (data not shown). Parallel extracts (SPTE-1, SPTE-2, PK, and PK + SPTE) of homogenates of uncooked beef samples were all inactive (Fig. 3), suggesting that it is the cooking process that leads to the production of both ‘‘free’’ and ‘‘bound’’ genotoxic principles. Neither extracts of
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Fig. 2. Comet-forming activities of extracts of cooked beef (8 g equivalent), measured in MCL-5 cells. Cell suspensions in PBS (1 105 cells=75 ll) were incubated in the presence of the DNArepair inhibitors, hydroxyurea, and cytosine arabinoside (HU/ara-C) (10 mM/1.8 mM final concentration), at 37 °C for 30 min. Comet tail length ðlmÞ was used as a measure of DNA damage. MCL-5 cells were treated with vehicle control (DMSO): A, in the absence of HU/ara-C and B, in the presence of HU/ara-C. Cells were also treated, in the presence of HU/ara-C, with extracts of cooked beef, added as solutions in DMSO as follows: C, SPTE-1; D, SPTE-2; E, PK 3; F, PK+SPTE. Incubations and the Comet assay were carried out as described in Materials and methods. P , as compared with HU/ara-C control; P , PK + SPTE compared with PK.
cooked nor uncooked beef samples were observed to induce cytotoxicity, as measured by trypan blue exclusion (data not shown). Bacterial mutagenicity An exogenous metabolic activation system (Aroclor 1254-induced rat-liver S9) was found to be a requirement for the detection of bacterial mutagenicity in cooked beef extracts, since no mutagenic activity was observed in its absence (data not shown). Results for each extraction procedure were normalised (revertants/ plate/g-beef equivalent) by subtracting the mean number of background revertant colonies and then dividing the mean number of induced-revertant colonies by the g-meat equivalent for that extract. Levels of spontaneous revertants were 35 6 and 52 6 (n ¼ 12) for the strains TA1538 and YG1019, respectively. A positive control (PhIP, 0:5 lg=plate) was employed and levels of induced revertants were 844 125 and 1231 217
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Fig. 3. Comet-forming activities of extracts (8 g equivalent) of uncooked beef, measured in MCL-5 cells. Cell suspensions in PBS (1 105 cells=75 ll) were incubated in the presence of the DNArepair inhibitors, hydroxyurea, and cytosine arabinoside (HU/ara-C) (10 mM/1.8 mM final concentration), at 37 °C for 30 min. Comet tail length ðlmÞ was used as a measure of DNA damage. MCL-5 cells were treated with vehicle control (DMSO): A, in the absence of HU/ara-C and B, in the presence of HU/ara-C. Cells were also treated in the presence of HU/ara-C with extracts of uncooked beef, added as solutions in DMSO as follows: C, SPTE-1; D, SPTE-2; E, PK 3; F, PK + SPTE. Incubations and the Comet assay were carried out as described in Materials and methods.
(n ¼ 12) for the strains TA1538 and YG1019, respectively. Fig. 4 shows that levels of bacterial mutagenicity induced by a cooked beef extract (SPTE-1) are reduced, but not eliminated, if a series of solvent extractions with DCM are performed prior to the preparation of the second set of extracts (SPTE-2), suggesting that free mutagens are extractible from the aqueous homogenates of cooked beef samples. However, following a proteinase K digestion of these previously solvent-extracted beef samples, the subsequent DCM extracts (PK) induce levels of bacterial mutagenicity in strains TA1538 and YG1019 similar to, if not greater than, the levels induced by the original extracts (SPTE-1). Not all of this activity is extractable by SPTE as demonstrated by the levels of bacterial mutagenicity remaining in the PK + SPTE extracts. These results were reproducible in subsequent experiments using other freshly cooked beef patties: no significant increases above background mutagenicity were seen with any of the extracts prepared from uncooked beef (data not shown).
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Fig. 4. Normalised levels of bacterial mutagenicity induced by extracts (8 g equivalent) of cooked beef in S. typhimurium TA1538 or YG1019. Bacterial mutagenicity assays were performed in the presence of S9 as described in Materials and methods. Each point represents normalised mean revertants per plate (SD < 20%) from three plates.
Discussion Dietary and/or environmental factors are important determinants in the aetiology of both non-hereditary and hereditary cancers [17]. The best evidence for this observation comes from studies in which incidence rates or mortality have been determined for migrants moving from lower-risk to higher-risk countries [18]. For example, data from 1962–1971 showed that, although breast cancer mortality in recently arrived Italian migrants to Australia was low, it rose over time to match that of the host population [19]. Similarly, breast cancer risk in females of Chinese, Japanese, or Filipino origin migrating to the United States increased by 80% after 10 years of residence [20]. Cancers which occur with higher frequency in Western societies have been associated with the consumption of higher levels of cooked meats, especially cooked red meat [2,7,10,17]. Well-done meat contains protein-derived mutagens [21], many of which have been identified as HAAs. These compounds are formed at parts-per-billion levels as the products of protein pyrolysis or Maillard reactions [2]. They have also been produced in modelling reactions in which amino acids such as glycine or alanine are heated, either dry or in solution, in the presence of creatinine or creatine [3,4]. HAAs have induced tumours
in rodent bioassays [9] and are strongly suspected of contributing to human cancer incidence [7,10]. Their characteristic structure includes one or two heterocyclic rings fused to an aminoimidazo ring. These imidazole mutagens include the aminoimidazo-quinoxaline derivatives, the aminoimidazo-quinoline derivatives, and the pyrido-indole derivatives. The quinolines and quinoxalines are strongly mutagenic to bacteria whereas the pyridine derivatives are much less potent [5,6]. However, the pyridines seem to possess greater genotoxicity in test systems involving mammalian cells [5,6]. Although some 24 HAAs have either been identified or partially identified [10], more than 70% of the mutagenic activity extractable from grilled, fried, or broiled meat samples remains unaccounted for [22]. The present study has investigated the possibility that there are dietary genotoxins that have so far avoided detection and quantitation. When proteinaceous foods are heated, ‘‘free’’ HAAs are formed that are extractable by well-documented methods [11–13]. Higher yields can be produced if the protein is hydrolysed to its amino acid constituents prior to heating [23,24], in agreement with the results obtained in modelling reactions [25]. We have now examined the notion that other genotoxins are formed in heated proteins that remain integral with, and covalently bound to, the protein until hydrolysis occurs in the gastro-intestinal tract. Reactions between, for example, C- or N-terminal aromatic amino acid residues and creatine or creatinine might be expected to yield HAA-like compounds. The products would not be extractable prior to proteolysis and would therefore have remained undetected to date: they would not necessarily be identical to known HAAs since either the carboxylic acid or the amino group will have been part of a peptide bond when, or if, condensation reactions occurred (with ‘internal’ amino acid residues both the –COOH and the –NH2 groups would be unavailable for reactions). Results from the preliminary experiments described here have lent support to this idea. When aqueous homogenates of well-done beef were proteinase K-digested, materials that were genotoxic in the Comet assay employing MCL-5 cells became extractable that were not apparently solvent soluble before proteolysis. Quantitatively, the results obtained using the Comet assay (Fig. 2) showed that less than half the total genotoxic activity present in a cooked beef homogenate was extractible prior to proteolysis and similar but less marked results were obtained using the mutagenicity assays (Fig. 4). Some of the solvent-soluble genotoxic activities released by proteolysis were also extractible using SPTE (Figs. 2 and 4, PK + SPTE) suggesting that it may include heterocyclic or aromatic amino compounds. In addition, although no more comet-forming activity could be extracted using SPTE after five sequential DCM extractions (Fig. 2C and D), some mutagenic
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activity was present in the SPTE-2 extract (Fig. 4). The reasons for the differences between the results obtained with the two biological test systems are not known. It may be worth noting that, for some HAAs, genotoxicity in the Comet assay appears to be more closely correlated with their ability to induce morphological transformation of cells in culture than with their mutagenicity in S. typhimurium [5]. Further work is clearly required to confirm the findings reported here, to identify the genotoxins released by the enzymic digestion of heattreated proteins and to assess the contribution that such putative ‘‘closet carcinogens’’ may make to the levels of genotoxins that are ingested in the diet. Acknowledgments This study was supported by research grants from the Association for International Cancer Research, the Cancer Research Campaign, and the Department of Health. We wish to thank Professor Nitya Kundu and Drs. Wolfgang Pfau and Terry Jones for helpful discussions.
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