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Lack of aberrant crypt promotion andof mutagenicity in extracts of cookedcasein, a colon cancer‐promoting foodDenis E. Corpet a b & Pierrette Cassand ca Laboratoire de Sécurité des Aliments , Ecole NationaleVétérinaire , Toulouse, 31076, Franceb Laboratoire des Xénobiotiques , Institut National de laRecherche Agronomique , BP.3, Toulouse, F‐31931, Francec Laboratoire de Toxicologie Alimentaire , Université BordeauxI , Talence, 33405, FrancePublished online: 04 Aug 2009.

To cite this article: Denis E. Corpet & Pierrette Cassand (1995) Lack of aberrant crypt promotionand of mutagenicity in extracts of cooked casein, a colon cancer‐promoting food, Nutrition andCancer, 24:3, 249-256, DOI: 10.1080/01635589509514414

To link to this article: http://dx.doi.org/10.1080/01635589509514414

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Lack of Aberrant Crypt Promotion and ofMutagenicity in Extracts of Cooked Casein, a

Colon Cancer-Promoting Food

Denis E. Corpet and Pierrette Cassand

Abstract

Dietary casein cooked at 180°C promotes the growth of aberrant crypt foci and colon cancerin rats initiated with azoxymethane. We speculated that promotion was due to a product thatcould be extracted by a solvent, such as 5-hydroxymethyl-2-furaldehyde (HMF), with tumor-promoting activity or the carcinogenic heterocyclic aromatic amines (HAA). This hypothesis wastested by extracting cooked casein with solvents and water. The extracts were then 1) assayedby high-performance liquid chromatography for HMF and HAA, 2) measured for mutagenicityon a frame-shift-sensitive strain of Salmonella typhimurium, and 3) fed for 100 days toazoxymethane-initiated rats to test the promoting effect on aberrant crypt foci. Data show that1) no HMF or HAA was detected in cooked casein, 2) no mutagenicity was detected on strainTA98, with or without metabolic activation, and 3) promotion was not associated with the extractsbut with the cooked casein residue. Therefore the promotion by cooked casein would not appearto be associated with a product that can be extracted by solvents.

(Nutr Cancer 24, 249-256, 1995)

Introduction

Colon cancer might be prevented by modifying the diet in Western countries, but the neededchanges are not precisely known. The current hypothesis is that a high-fat low-fiber dietincreases the risk of colon cancer (1). However, recent intervention trials assessing therecurrence of polyps in volunteers do not support this hypothesis (2,3). An alternate hypothesisis that the risk of colon cancer might increase with the intake of meats cooked at hightemperature because of their mutagen or promoter content (4,5). This hypothesis is supportedby case-control studies (6,7) and a cohort study (8). Indeed, a diet containing casein cookedfor two hours at 180°C in a kitchen oven increases colon carcinogenesis in mice and ratsinitiated with azoxymethane (9,10).

We speculated that the cooking of casein gives rise to a promoting substance that could beextracted from the protein. Pyrolysis of proteins can produce heterocyclic aromatic amines(HAA) that can be activated to frame-shift mutagens (11,12). Most HAA are colon carcino-gens, and at low doses they enhance large intestinal carcinogenesis in the promotion stage(13). At a high temperature (300-450°Q, the pyrolysis of casein does produce the mutagenic

D. E. Corpet is affiliated with the Laboratoire de Sécurité des Aliments, Ecole NationaleVétérinaire, 31076 Toulouse, France, and the Laboratoire des Xénobiotiques, Institut Nationalde la Recherche Agronomique, 31931 Toulouse, France. P. Cassand is affiliated with theLaboratoire de Toxicologie Alimentaire, Université Bordeaux I, 33405 Talence, France.

Copyright © 1995, Lawrence Erlbaum Associates, Inc.

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HAA Glu-P-2 (14). Besides, the pyrolysis of sucrose yields 5-hydroxymethyl-2-furaldehyde(HMF), which promotes aberrant crypt foci (ACF) (15). We therefore looked for HMF andHAA, bacterial mutagens, and tumor promoters in cooked casein extracts.

Promotion of cancer was assessed with the ACF assay (16). ACF are putative precursorsof colon cancer (17), specifically induced by colon carcinogens (18), promoted by promotingdiets (19), and inhibited by inhibitors of carcinogenesis (20,21). In rodents and humans, ACFdisplay mutations and histological changes in colonic tumors (22-24), and the growth of ACFcorrelates with the adenocarcinoma yield (10,25,26). The number of ACF per animal is anassay for initiators of colon cancer, although the number of crypts per focus (multiplicity) isa measure of promotion effect (10,26,27).

Materials and Methods

Extraction of Cooked Casein

From a 10-kg batch of casein (vitamin free; ICN, Buckingamshire, UK), 5 kg were cookedfor two hours at 180°C as a thin layer on a Pyrex glass pan in an electric oven (10). One-half(2.5 kg) of the batch was directly included in a diet (20% wt/wt) called "cooked casein"; theother half was extracted sequentially by dichloromethane, methanol, and water. Portions (400g) of cooked casein were mixed for 10 minutes in 750 ml of dichloromethane [high-performanceliquid chromatography (HPLC) grade; Prolabo, Paris, France] with an Ultra Turrax grinder(model T25IKA, Stauffen, Germany). It was then filtered through a fritted-glass funnel, andthe extraction was repeated twice. The casein residue was then extracted three times withmethanol (Prolabo). Finally, the casein residue was extracted three times with ultrafiltereddeionized water (pH of water plus casein was 3.3). Solvents (14 liters of each) were evaporatedto dryness under vacuum, while the water extract was freeze-dried. Dry extracts obtained withdichloromethane, methanol, and water weighed 0.079%, 0.22%, and 0.38% of the casein,respectively. The combined extracts were added to raw casein. This casein with extracts andthe casein dried residue were added to the diets (20% wt/wt) called "extracts" and "residue,"respectively. Raw casein was included in a diet called "control."

Assay of HMF and HAA

Extract of cooked casein was analyzed for HMF by reverse-phase liquid chromatography(28). Standard HMF was obtained from Sigma Chemical (St. Quentin, France). Some cookedcasein was extracted by acetonitrile (Prolabo) by grinding 1 g in 5 ml for five minutes, thenevaporating the solvent to dryness after centrifugation of the solid phase (7,000 g for 4 mins).The dried extract was dissolved in 200 ul of water-acetonitrile (19:1 vol/vol). Twenty microliterswere injected onto an RP18 (Merck, 5 urn) column for isocratic HPLC, with water-acetonitrile(19:1) as mobile phase (1 ml/min), and ultraviolet (UV) detection at 280 nm. Cooked caseinwas also analyzed for HAA at the Lawrence Livermore National Laboratory (Livermore,CA) with use of solid-phase extraction and HPLC. The recovery of specific HAA was testedin a cooked casein sample spiked with IQ (2-amino-3-methylimidazo-[4,5-fJquinoline), MelQ(2-amino-3,4-dimethylimidazo-[4,5-fJquinoline), MelQx (2-amino-3,8-dimethylimidazo-[4,5-fjquinoxaline), DiMelQx (2-amino-3,4,8-trimethylimidazo-t4,5-fJquinoxaline), and PhIP (2-amino-l-methyl-6-phenylimidazo-[4,5-b]pyridine) (29,30).

Mutagenicity Assay

Heterocyclic amines are known to induce guanosine-cytosine DNA frame-shift changesafter metabolic activation, and the strains TA98 and TA1538 are the most sensitive testersfor these mutations (12). Therefore the mutagenicity of dichloromethane, methanol, and water

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extracts of cooked casein, separated and pooled, was determined with the Salmonella ty-phimurium TA98 tester strain, with and without S9 activation (31). The TA98 strain wasobtained from Dr. B. N. Ames (University of California, Berkeley, CA). Liver S9 mix wasprepared from Sprague-Dawley rats induced with Aroclor 1254. To 2.5 ml of molten top agarwere added sequentially 0.1 ml of extract in dimethyl sulfoxide (except the dichloromethaneextract dissolved in ethyleneglycol methyl ether), 0.1 ml of bacterial suspension, and 0.5 mlof 20% S9 mix (3.3 mg protein/plate). It was poured on minimal glucose agar plates andincubated at 37°C for 48 hours. Then the number of histidine-independent (His+) revertantcolonies of TA98 was scored. The experiment was carried out in triplicate with seven concen-trations of each extract and of the pool of extracts (1 ng-1 mg/plate). Positive controls were2-nitrofluorene without S9 mix and benzo[a]pyrene with S9 mix.

Animals

Thirty-six five-week-old female F344 rats were obtained from Iffa-Credo (Lyon, France).They were acclimatized to the colony for one week, housed four rats per plastic cage on woodchips at 22°C with 12:12-hour light-dark cycle, and fed a laboratory chow (6% fat; UAR,Villemoisson, France) and water ad libitum. The rats were initiated with one injection ofazoxymethane (Sigma Chemical) at 20 mg/kg ip in NaCl (9 g/1). They were maintained on thesame diet for a further seven days and were then randomly allocated to the diets: 12 rats inthe control group and 8 rats in the three experimental groups. Food intake was measured percage per week. The animals were fed the experimental diets for 100 days, then sacrificed bychloroform anesthesia and asphyxiation, weighed, and scored for colonic ACF.

Diets

The experimental diets for ACF promotion were based on the standard AIN-76 diet (32),which was modified to contain 20% beef tallow and 3.5% corn oil. Fat was added to the dietat the expense of sucrose on a caloric basis, and the diets differed only in their proteincomponent, as previously described (9). The four diets contained 3.5% unheated casein plus20% of one of the following: raw casein (control diet), cooked casein, extracts of cooked caseinadded to raw casein, or the residue of this cooked casein after extraction. Casein or caseinproducts were thoroughly mixed with the powdered food.

Assay of ACF

ACF were scored after 100 days on the diets with use of the procedure described by Bird(17). Immediately after the animals were sacrificed, colons were removed and flushed withKrebs-Ringer solution, then opened longitudinally and fixed flat between filter paper in 10%buffered formalin (Sigma Chemical). The colons were stained with methylene blue (0.1%) for10-15 minutes, then the mucosal side was observed at x32 magnification. ACF were distin-guished by their slitlike opening, increased staining, size, and pericryptal zone. The multiplicity(number of crypts per ACF) was recorded for each ACF in each colon. Those were scoredblindly by a single observer.

Statistics

The statistical analysis of ACF assay for colon cancer promotion was based on ACFmultiplicity, as defined by the mean number of aberrant crypts per focus (26). Data werefirst examined by one-way analysis of variance, then by Student's f-test. Another approachthat used the quadratic nature of the variance of ACF multiplicity (27) yielded similarresults.

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Results

HMFandHAA

The HPLC profile of authentic HMF showed a single UV peak at 9.6-9.8 minutes. TheHPLC profile of the cooked casein extract showed >10 peaks, two of them with retentiontimes of 9.3-9.4 and 10.1-10.2 minutes. Those two peaks were collected. Their UV spectrashowed maxima below 250 run, whereas HMF exhibited a maximum at 280 nm. The HPLCprofile of an extract of cooked casein, spiked with 1 ug of HMF/g before extraction, showedthat the procedure extracted about 75% of HMF. It also showed that the HMF peak arosein the valley between the two peaks cited above (9.3 and 10.1 mins retention times). Thereforeno HMF or <0.5 ug was extracted from 1 g of unspiked cooked casein.

The recoveries of IQ, MelQ, MelQx, DiMelQx, and PhIP in a spiked cooked casein sampleranged from 21% to 64% after solid-phase extraction and HPLC analysis. No known HAAwas detected in unspiked cooked casein (M. G. Knize, personal communication).

Mutagenicity

The number of TA98 revertants was not increased by the extracts of cooked casein at anydose, either single or pooled, with or without activation by S9 mix. All plates spiked withextracts yielded <75 revertants, and no dose relationship was seen. For illustration, 60.5, 62.5,53.5, 67, 61, 66.5, and 55 revertants were counted on plates containing 1, 10, and 100 ng, 1,10, and 100 ug, and 1 mg, respectively, of the pooled extracts with S9 mix (data are meansof 3 plates). Spontaneous revertants were 44/plate (range 39-47), and positive controls, 1 ugof 2-nitrofluorene without S9 and 2.5 ug of benzo[a]pyrene with S9, yielded 286 and 298revertants, respectively. A basic extract of cooked casein was also tested for mutagenicity inanother laboratory with use of strain TA98 with metabolic activation, and no detectableactivity was found (M. G. Knize, personal communication). Therefore the extracts of cookedcasein were not likely to contain a frame-shift mutagen similar to HAA.

Promotion ofACF

Groups of rats were given azoxymethane for initiation of colon carcinogenesis. Subsequentlyfor 100 days they were fed diets containing raw casein as a control, cooked casein, the extractsof the cooked casein, or the residue after preparation of the extracts. Rats fed cooked caseinor residue gained less weight than control or extract-fed rats (p < 0.001), although their foodintake was higher (Table 1). A total of 4,141 ACF were detected in the 36 colons. The numberof ACF per colon, which is not associated with tumor promotion, was smaller in rats fed thecooked casein than in controls (p = 0.01; Table 1).

By contrast, the crypt multiplicity (or number of crypts/focus), a predictor of tumorincidence, was higher for the cooked casein and the residue of extraction than for the control(p < 0.001; Figure 1). The crypt multiplicity was not different in the extracts and the controlgroups (p = 0.74). Another way of analyzing the data, according to Magnuson and co-workers(26), gave similar conclusions: when all ACF from each diet group were pooled, higher cryptmultiplicity was shown (F= 24, p < 0.001) in groups given cooked or residue diets (both 3.7crypts/ACF) than in groups given extracts or control diets [both 3.2 ± 0.05 (SE) crypts/ACF].Moreover, although tumors were not a designed end point in this study (and no histology wasdone), we observed macroscopically more tumors in rats fed the cooked casein and the residuethan in control or extract-fed rats (p = 0.03; Table 1).

The whole extraction and promotion experiment was repeated in another laboratory (datanot shown) with use of CF1 mice rather than rats and with a different extraction protocolbut with similar results: extracts did not promote ACF growth in mice, cooked casein residue

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Table 1. Body Weight, Food Intake, ACF, and Macroscopic Tumors in the Colonof Rats"-6

Tumors

GroupRat Wt, g

(mean ± SD)Food Intake, g/day

(mean)ACF /colon(mean ± SD) Rats with tumor

Raw casein controlCooked caseinResidue from cooked

caseinExtracts from cooked

caseinp (ANOVA)

1288

176 ±11147 ±8154 ± 13

182 ±4

0.001

7.59.29.9

7.5

128 ± 4181 ±30

124 ± 36

121 ±44

0.06

033

0

0.03c

a: Rats were initiated with azoxymethane (20 mg/kg) and fed diets containing 20% casein, raw or cooked, orextracts or residue of cooked casein for 100 days.

b: Abbreviations are as follows: ACF, aberrant crypt focus; n, no. of rats; ANOVA, analysis of variance.c: Kruskal-Wallice x2 = 8.7.

Figure 1. Aberrant crypt focus (ACF)multiplicity in rats given azoxymethane (20mg/kg), then fed diets containing 20% ofcasein, raw or cooked, or extracts or resi-due of cooked casein for 100 days. ACFmultiplicity was calculated for each rat(mean 45-203 ACF). Values (means ± SE)represent ACF multiplicity among rats (12rats in control group and 8 in others). Pvalues show differences from control groupgiven raw casein.

Control Cooked Residue Extractsraw casein casein (from cooked casein)

did (W. R. Bruce, personal communication). Thus data confirm that cooked casein promotesthe ACF growth and show that extracts of cooked casein do not.

Discussion

The growth of ACF and colon cancers is increased in animals consuming cooked casein.We speculated that this promoting activity was due to a promoter that could be extractedfrom cooked casein with solvents. According to previous studies, we moreover supposed thatthis promoter might be HMF, as in cooked sucrose, or a mutagen, i.e., HAA in cooked meat.The results show that cooked casein extracts contain no HMF, no HAA, no mutagen onTA98, and no promoter on ACF. First, it is unlikely that HMF could be responsible for thepromoting effect of cooked casein, because no HMF could be extracted from the promotingcooked casein. In contrast, cooked sucrose contains about 1% HMF and promotes the growth

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of ACF (9). A diet supplemented with 1% pure HMF promotes ACF growth, and HMF canalso initiate ACF in rats (15).

Second, no known HAA was detected in cooked casein, and both solvent and water extractsof cooked casein failed to show a mutagenic effect on 5. typhimurium TA98 strain. Becauseheterocyclic amines produced by heating meat are extracted by solvents and by acidic waterand are very mutagenic to strain TA98 (33), it is unlikely that known HAA or similar mutagenscould be responsible for the promotion by dietary cooked casein.

Third, we compared the ACF promotion by cooked casein with that of an organic andaqueous extract of the heated protein. We observed a very significant promotion of ACF bycooked casein (p < 0.001). The crypt multiplicity increased by a factor of 1.2, exactly as inZhang and co-workers (10) and Magnuson and colleagues (26). According to their data, thediets that increase crypt multiplicity by a factor of 1.2 at 100 days also increase adenocarcinomaincidence by a factor of 3.3 at 240 days (10) and by a factor of 2.7 at 126 days (26). Unlikemultiplicity, numbers of ACF per colon are reduced in cholic acid-fed rats (26), as in thecooked casein-fed rats, suggesting that the number of ACF is not correlated with tumorpromotion. Here, the promoting activity was not associated with the extracts of cooked casein,but with the residue. These results show that the promoter cannot be extracted easily fromcooked casein to be analyzed and identified. We cannot exclude, however, that the extractionprocedure was not efficient enough (e.g., a hydrolysis step might have been useful) or that thedetection systems were not sensitive enough.

Cooked casein promotes colon tumor, but no promoter could be extracted from it bysolvents or water. We therefore may speculate that the promoter in cooked casein is retainedin the protein backbone itself. Thermolysis of proteins can result in the dehydration of aminoacids such as serine, resulting in the formation of dehydroalanine in the protein chain.Dehydroalanine alkylates lysine to yield the cross-linking amino acid lysinoalanine. Thesemodified amino acids can be toxic (34), and we are currently studying whether they couldpromote colon carcinogenesis. Another possibility is that cooking affects the structure of caseinso that it becomes less digestible. Indeed, in rats, most cooked casein is not digested (35).Thus, in rats fed cooked casein, much protein reaches the cecum, and bacterial metabolitesof amino acids increase in the gut, namely, ammonia and branched-chain fatty acids (35),phenols, indole, and indican, which might have promoting properties (36). However, recentresults suggest that protein fermentation products do not play an important role in coloncancer promotion (37). Most research on cooking and cancer focuses on HAA, but the presentresults show that other compound(s) might be involved as well.

Acknowledgments and Notes

The authors thank M. G. Knize for analysis of HAA at the Lawrence Livermore National Laboratory, W. R.Bruce for stimulating discussions, R. Gazel for care of the rats, and J. P. Cravedi for advice on the HPLC analysis.This study was supported in part by the Institut National de la Recherche Agronomique and by a grant from theAssociation de la Recherche contre le Cancer. Address reprint requests to Prof. D. Corpet, INRA Xenobiotiques,BP.3, F-31931 Toulouse, France.

Submitted 13 March 1995; accepted in final form 22 June 1995.

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] at

00:

00 1

2 N

ovem

ber

2014