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1 Targeting colon luminal lipid peroxidation limits colon carcinogenesis 1 associated with red meat consumption 2 Océane Martin 1,9 , Nathalie Naud 1 , Sylviane Taché 1 , Laurent Debrauwer 1 , Sylvie Chevolleau 1 , Jacques Dupuy 1 , Céline Chantelauze 2 , Denis Durand 2 , Estelle Pujos-Guillot 3 Florence Blas-Y-Estrada 1 , Christine Urbano 4 , Gunter GC Kuhnle 5 , Véronique Santé- Lhoutellier 6 , Thierry Sayd 6 , Didier Viala 6 , Adeline Blot 7 , Nathalie Meunier 7 , Pascal Schlich 8 , Didier Attaix 3,7 , Françoise Guéraud 1 , Valérie Scislowski 9 , Denis E. Corpet 1 and Fabrice HF Pierre 1,* Authors’ Affiliations: 3 1 INRA UMR1331, TOXALIM (Research Centre in Food Toxicology), Université de Toulouse, ENVT, INP-Purpan, UPS, Toulouse, France 2 INRA, UMR1213 Herbivores, Saint-Genès-Champanelle, France 3 Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, Clermont Ferrand, France. 4 Sensostat, Dijon, France 5 Department of Food & Nutritional Sciences, University of Reading, Reading, United Kingdom. 6 INRA UR0370, QuaPA, QuaPA, Saint-Genès-Champanelle, France 7 CHU Clermont Ferrand, CRNH Auvergne, France 8 Centre des Sciences du Goût et de l'Alimentation, CNRS, INRA, Univ. Bourgogne Franche-Comté, Dijon, France. 9 ADIV, 10 rue Jacqueline Auriol, 63039 Clermont-Ferrand, France Running Title: Fresh red meat, lipoperoxidation and colon cancer. 4 Keywords: colorectal cancer, red meat, heme iron, lipoperoxidation, marinade 5 Financial support: This work was supported by the French National Agency of Research (ANR-10-ALIA-14, SecuriViande project), the French National Institute for Agricultural Research (INRA), and by the French Technical Center of Meat (ADIV). Cancer Research. on April 15, 2020. © 2018 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 28, 2018; DOI: 10.1158/1940-6207.CAPR-17-0361

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Page 1: Targeting colon luminal lipid peroxidation limits …...2018/06/28  · 1 1 Targeting colon luminal lipid peroxidation limits colon carcinogenesis 2 associated with red meat consumption

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Targeting colon luminal lipid peroxidation limits colon carcinogenesis 1

associated with red meat consumption 2

Océane Martin1,9, Nathalie Naud1, Sylviane Taché1, Laurent Debrauwer1, Sylvie

Chevolleau1, Jacques Dupuy1, Céline Chantelauze2, Denis Durand2, Estelle Pujos-Guillot3

Florence Blas-Y-Estrada1, Christine Urbano4, Gunter GC Kuhnle5, Véronique Santé-

Lhoutellier6, Thierry Sayd6, Didier Viala6, Adeline Blot7, Nathalie Meunier7, Pascal

Schlich8, Didier Attaix3,7, Françoise Guéraud1, Valérie Scislowski9, Denis E. Corpet1 and

Fabrice HF Pierre1,*

Authors’ Affiliations: 3

1 INRA UMR1331, TOXALIM (Research Centre in Food Toxicology), Université de

Toulouse, ENVT, INP-Purpan, UPS, Toulouse, France

2 INRA, UMR1213 Herbivores, Saint-Genès-Champanelle, France

3 Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, Clermont

Ferrand, France.

4 Sensostat, Dijon, France

5 Department of Food & Nutritional Sciences, University of Reading, Reading, United

Kingdom.

6 INRA UR0370, QuaPA, QuaPA, Saint-Genès-Champanelle, France

7 CHU Clermont Ferrand, CRNH Auvergne, France

8 Centre des Sciences du Goût et de l'Alimentation, CNRS, INRA, Univ. Bourgogne

Franche-Comté, Dijon, France.

9 ADIV, 10 rue Jacqueline Auriol, 63039 Clermont-Ferrand, France

Running Title: Fresh red meat, lipoperoxidation and colon cancer. 4

Keywords: colorectal cancer, red meat, heme iron, lipoperoxidation, marinade 5

Financial support: This work was supported by the French National Agency of

Research (ANR-10-ALIA-14, SecuriViande project), the French National Institute for

Agricultural Research (INRA), and by the French Technical Center of Meat (ADIV).

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Correspondence: Fabrice H.F. Pierre; INRA UMR1331, 180 chemin de Tournefeuille, F-

31027 Toulouse, France; Phone: +33 582 066 370; Fax : +33 561 285 244; E-mail:

[email protected]

Conflict of Interest: Océane Martin and Valérie Scislowski were employed by the French

Technical Center of Meat (ADIV).

N Naud, S Taché, L Debrauwer, S Chevolleau, J Dupuy, C Chantelauze, D Durand, E Pujos-

Guillot, F Blas-Y-Estrada, Ch Urbano, GGC Kuhnle, V Santé-Lhoutellier, Th Sayd, D Viala, A

Blot, N Meunier, F Guéraud, P Schlich, D Attaix, DE Corpet and FHF Pierre declare no

conflicts of interest.

Authors’ Contributions

Conception and design: O Martin, L Debrauwer, D Durand, V Santé-Lhoutellier, P

Schlich, D Attaix, F Guéraud, V Scislowski, DE Corpet, FHF Pierre.

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): O Martin, N Naud, S Taché, S Chevolleau, J Dupuy, C Chantelauze, E

Pujos-Guillot, F Blas-Y-Estrada, Ch Urbano, GGC Kuhnle, Th Sayd, D Viala, A Blot, N

Meunier, F Guéraud

Analysis and interpretation of data: O Martin, DE Corpet, P Schlich, V Santé-

Lhoutellier, FHF Pierre.

Writing, review, and/or revision of the manuscript: O Martin, DE Corpet, FHF Pierre.

Study Supervision: FHF Pierre, DE Corpet

Information pertinent to Clinical trial registry: The human volunteer cross-over

study was registered at Clinicaltrials.gov under the number NCT02473302.

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ABSTRACT

Red meat is probably carcinogenic to humans (WHO/IARC class 2A), in part through 6

heme iron-induced lipoperoxidation. Here, we investigated whether red meat promotes 7

carcinogenesis in rodents and modulates associated biomarkers in volunteers, 8

speculating that an antioxidant marinade could suppress these effects via limitation of 9

the heme induced lipid peroxidation. We gave marinated or non-marinated beef with 10

various degrees of cooking to azoxymethane-initiated rats, Min mice, and human 11

volunteers (crossover study). Mucin-depleted foci were scored in rats, adenoma in Min 12

mice. Biomarkers of lipoperoxidation were measured in the feces and urine of rats, mice, 13

and volunteers. The organoleptic properties of marinated meat were tested. Fresh beef 14

increased colon carcinogenesis and lipoperoxidation in rats and mice and 15

lipoperoxidation in humans. Without an adverse organoleptic effect on meat, marinade 16

normalized peroxidation biomarkers in rat and mouse feces, reduced peroxidation in 17

human feces and reduced the number of Mucin-depleted foci in rats and adenoma in 18

female Min mice. This could lead to protective strategies to decrease the colorectal 19

cancer burden associated with red meat consumption. 20

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INTRODUCTION 21

Colorectal cancer is the third most common type of cancer and fourth most common 22

cause of death from cancer worldwide (1). In 2007 and confirmed in 2011 and 2017, the 23

World Cancer Research Fund (WCRF) and American Institute for Cancer Research 24

(AICR) panel stated that there is strong evidence that consuming red meat increases the 25

risk of colorectal cancer (2,3). In 2015, the International Agency for Research on Cancer 26

(IARC), an agency of the World Health Organization (WHO), classified red meat 27

consumption as “probably carcinogenic to humans” (4). 28

Epidemiological studies incited the WCRF to recommend limiting red meat consumption 29

to less than 500g/w. (1,2). These recommendations may reduce the colorectal cancer 30

burden, but sociological studies have demonstrated that people with a lower 31

socioeconomic status are less receptive to nutritional messages and are less likely to 32

change risky behaviors than affluent people (5-9). Simply conveying information on 33

risks and benefits has almost no effect on food choices among less-educated people and 34

tends to enlarge the health inequalities to which colorectal cancer contributes (10,11). 35

Furthermore, adherence to the WCRF recommendation of limiting red meat intake is 36

low in cancer survivors (8%) (12). Based on these observations, we wanted to propose 37

an alternative to the current recommendation of limiting consumption by adding 38

protective additives directly in the commercial product. 39

Different hypotheses have been explored to explain the link between red meat intake 40

and colorectal cancer, including heterocyclic amine formation during high-temperature 41

cooking (13), N-nitroso compound formation by endogenous nitrosation, and high heme 42

iron content, principally through the catalysis of dietary lipid oxidation and formation of 43

N-nitroso compounds involving gut microbiota (14,15). We recently showed that, at 44

nutritional doses, heme iron is the major actor in meat-induced promotion of colon 45

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cancer without an additive or synergistic effect of heterocyclic amines and endogenous 46

N-nitroso compounds (16). The promoting effect of heme iron has been validated in 47

rodent models using purified molecules (hemoglobin or hemin) and lyophilized red 48

meat (16-20). Furthermore, this promoting effect of heme was confirmed at the 49

epidemiological level in the French E3N prospective cohort of women (21). In humans 50

and in rodent models, meat and heme intake have been associated with an increase in 51

two lipoperoxidation biomarkers: thiobarbituric acid reactive substances (TBARS) and 52

1,4-dihydroxynonane mercapturic acid (DHN-MA) (18-20,22-24). Heme iron can 53

catalyze dietary lipoperoxidation, a key downstream feature of ferroptosis an iron-54

dependent form of non apoptotic regulated cell death (25-27) and a reaction leading to 55

the production of genotoxic and/or cytotoxic aldehydes that could be involved in the 56

promotion of carcinogenesis (16,28-31). Moreover, we have shown that the addition of 57

antioxidants to diet is efficient to decrease meat-induced lipoperoxidation and 58

carcinogenesis in animal models (19,20,32). The protective effect of the dietary 59

antioxidant capacity was confirmed in humans in the French E3N prospective cohort of 60

women (21). 61

We designed a project which aimed to limit the heme-induced colonic luminal 62

lipoperoxidation in order to prevent the risk of colon cancer associated with red meat 63

consumption. The project was divided in six sequential studies including an industrial 64

task of meat production and analysis, short-term and carcinogenesis animal studies and 65

clinical and organoleptic acceptability studies in human volunteers (Supplementary Fig. 66

S1). 67

68

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MATERIALS AND METHODS 69

Ethics 70

All animal studies were performed in an accredited animal facility by approved staff and 71

animal care conducted in accordance with the ARRIVE and European Council on Animals 72

used in Experimental Studies guidelines. The human volunteer crossover study was 73

allowed by a written decision of the ethical committee (French CPP Sud Est VI) and 74

authorized by the French Ministry of Health (No. IDRCB 2013-A01692-43). The study 75

was registered at Clinicaltrials.gov under the number NCT02473302. 76

77

Study design 78

First, we selected the most effective marinade among the 12 tested (see Supplementary 79

Materials for details) on the basis of a DPPH assay. The selected grape and olive 80

marinade was then incorporated into cooked or uncooked meats to test the modulation of 81

peroxidation and iron content in meats. A 14-days nutritional experiment (see 82

Supplementary Materials for details) was then performed in Fischer 344 male rats to 83

test the effect of different marinated and non-marinated and cooked or uncooked meats 84

on the modulation of peroxidation and iron content in fecal water, two biomarkers 85

associated with the promotion of colon carcinogenesis by red meat. These six meats 86

were then tested in colorectal carcinogenesis studies in chemically-induced rats, and 87

four were selected for study in Apc Min mice. From these results, the biological effect of 88

the most effective production process was tested in healthy human volunteers, as well 89

as its organoleptic acceptability. A flowchart of the experiments is presented in 90

Supplementary Fig. S1. 91

92

93

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Carcinogenesis study in azoxymethane-induced rats 94

Male Fischer 344 rats (N=92) purchased at 5 weeks of age from Charles River (France) 95

were housed individually in stainless steel wire-bottom cages. The rats were kept at 96

22°C with a 12 h-12 h light-dark cycle and allowed free access to standard AIN76 semi-97

purified diet and tap water. After acclimatization, the rats received a single i.p. injection 98

of azoxymethane (Sigma, 20 mg/kg) in NaCl (9 g/L of water). Seven days later, the rats 99

were randomly allocated to seven groups. Groups of twelve rats were given meats 100

following the same 3 x 2 factorial protocol as for study 2: meats were raw, rare, or well-101

done and marinated or not-marinated for each level of cooking. As in the short-term 102

experiment, pieces of meat were completed with low calcium modified AIN76. After 103

evaluating the consumption of the pieces of meat and powder, we calculated that diets 104

contained 47.3 g of meat (dry weight, per 100g total diet) and 52.7 g of low calcium 105

modified AIN76 (See Supplementary Materials for details). One group (N=20) received 106

control diet without meat, composed only of low calcium modified AIN76. Each diet was 107

stored at –20°C under vacuum and dispensed daily at 5:00 p.m. Rats were fed the 108

experimental diets daily for 98 days before CO2 euthanasia. Body weight was monitored 109

every week during the first 4 weeks, then every 2 weeks. Food and water intake was 110

measured on days 25 and 60. Feces were collected on days 88-91 and kept at -20°C. 111

Each rat was placed in a metabolic cage and urine collected on days 67-70 and kept at -112

20°C. 113

114

Carcinogenesis study in Apc Min mice 115

Five to 8-week old male and female Apc Min mice (N=97; Jackson Laboratory, USA) were 116

housed 2 to 5 mice per cage under standard laboratory conditions with free access to 117

food and water. After 3 days of acclimatization, the mice were randomly allocated to five 118

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experimental groups balanced for age and sex. Groups of 18 mice were given meats 119

following a 2 x 2 factorial protocol: meats were raw or rare and marinated or not 120

marinated for each level of cooking. As we could not have the same number of 121

experimental groups as in rats, we decided to eliminate the well-done meat group 122

because it is not as representative of human consumption (33). Unlike for rats, for the 123

mice we had to grind and mix the meat with the modified AIN76 to ensure it was eaten 124

entirely. The meat diets contained 60% (dry weight) meat and 40% low calcium 125

modified AIN76-base powder (see Supplementary Materials for more details on diets). 126

One group (N= 25) received a control diet without meat, composed only of modified 127

AIN76. Each diet was stored at –20°C under vacuum and dispensed daily at 5:00 p.m. 128

Mice were fed the experimental diets for 45 days before euthanasia by cervical 129

dislocation. Body weight was monitored every week during the first 3 weeks, then every 130

2 weeks. Feces were collected on days 35-39 and urine on day 39 and kept at -20°C. 131

132

The consumption of raw beef is too unusual to be representative of human 133

consumption; therefore, for studies in humans, the meat was cooked to be consistent 134

with the most common consumption methods (rare and well-done). 135

136

Cross over study with human volunteers 137

The human study was performed in the Nutritional Investigation Unit of the Human 138

Nutrition Center of Auvergne (Clermont-Ferrand, France). A single-blind randomized, 139

controlled cross-over trial was performed in 24 human volunteers after obtaining 140

written informed consent (inclusion criteria in Supplementary Materials). During the 1-141

week run-in period for adaptation volunteers, were asked to eat a diet without beef or 142

pork and low in antioxidant products. Volunteers were then randomly submitted to 143

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three alternating 4-day intervention periods with 110g/d of rare beef, marinated rare 144

beef or marinated well-done beef in a random order. During the intervention periods, 145

the volunteers must not eat meat outside the provided beef, fish, or eggs. To facilitate 146

the comparison between human and rodents studies of this project, meats for the 147

human study were obtained from the same production batch as those given to rats and 148

mice. Intervention periods were separated by a wash-out period of at least 3 days with 149

the same diet as the run-in period. Urine and stool were collected during the last 3 days 150

of each intervention period and at the end of each washout period. Each subject came to 151

the Nutrition Investigation Unit four times in addition to the screening visit. Before the 152

first intervention period (visit 1), volunteers were given cooking instructions. At each 153

visit, meats were distributed for the following intervention period. During visits 2, 3, and 154

4, each volunteer brought back the meat packaging and frozen urine and stool samples. 155

Compliance to diet was assessed after the collection of feces and urine samples at the 156

end of the intervention periods. 157

158

Organoleptic acceptability of marinated meats by a consumer panel 159

Two consumer studies were organized in INRA-Dijon, France (see Supplementary 160

Materials for details). The first study aimed at assessing a reference beef meat (beef) 161

and the two marinated products (one marinade without grape-olive extracts and one 162

marinade with these extracts) for overall liking by consumers during blind tasting. The 163

second study aimed to replicate the first study (reference versus grape-olive marinade) 164

but also aimed to assess whether providing the consumers with information about the 165

protective effect of the marinated product could improve their overall liking of the 166

product. Consumers from both groups were regular eaters of beef. The consumers from 167

the treatment group were first (at recruitment and at the beginning of the session) 168

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instructed about the risk of colon cancer promotion by excessive red meat consumption. 169

Both consumer studies were designed according to the same rules. Data were collected 170

by the Fizz system in a sensory room equipped with 16 booths and maintained 171

according to ISO Standard (NF EN ISO 8589). Sessions occurred at lunch time. The order 172

of serving the samples was balanced using Williams Latin squares, ensuring that each 173

product was evaluated equally often in the different position orders and preceded each 174

other product equally often. Each consumer had the products served either rare or well 175

done according to his/her usual way of consuming beef. The liking scale was the 9-point 176

hedonic scale with the first tick-box on the left labeled “I don’t like it at all” and the ninth 177

on the right labeled “I like it very much”. 178

In the second study, the information about colon cancer risk and the protective effect of 179

new products was provided to the consumer of the informed group (see Supplementary 180

Table S9). This text was sent to them by mail before the experiment and included in a 181

preliminary screen of the sensory session. For each product and subject, the difference 182

of liking scores given in the informed condition (protective effect) versus no information 183

was computed. The 3 differences of each subject were averaged providing individual 184

scores of the effect of information on liking. A number of individual variables 185

(demographics, usages, attitudes, …) were screened for differences in information 186

scores among their levels. 187

188

Carcinogenesis endpoints 189

MDF scoring 190

At euthanasia, the rats’ colons were removed, washed with cold Ringer, opened, coded, 191

and fixed flat between two sheets of filter paper in 10% buffered formalin (Sigma). The 192

number of MDF per colon and the number of crypts per MDF, as a measure of their size, 193

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were scored by two blinded investigators after high-iron diamine alcian blue staining 194

according to Caderni et al. and Femia et al. (34,35). 195

Tumor scoring 196

At euthanasia, the small intestine (from duodenum to ileum) and the colon of mice were 197

removed. Sections of the duodenum, jejunum, ileum and colon were opened along the 198

longitudinal axis and washed in PBS. After fixation, the different sections were stained 199

for 48 hours in a 300 ppm solution of methylene blue in formalin. Two blinded 200

investigators scored tumors and determined their diameters using a binocular 201

microscope at x25 magnification. All tumors in each section of the intestines were 202

counted; the smallest tumors that could be counted were approximately 0.5 mm in 203

diameter. 204

Fecal biomarkers 205

Fecal water preparation 206

To prepare fecal water of induced rats and mice, 1 mL of distilled water and 50 µL of 207

butylated hydroxytoluene (BHT, Sigma) 0.45 M were added to 0.4 g of fresh feces. The 208

feces were ground using Fast-Prep® (MP Biomedicals, Illkirch, France) for 30 s three 209

times and then centrifuged at 5500 g for 20 min. The fecal water was collected and kept 210

at –20˚C until use. For humans, 1 mL of sterilized water and 25 µL of BHT (0.90M) were 211

added to 0.25 g of fresh feces and the same protocol followed. 212

Heme and TBARS assays 213

Heme was measured by fluorescence in fecal water according to Sesink et al. as 214

described previously (20,36). TBARS were measured in fecal water according to Ohkawa 215

et al. as previously described (37). 216

HNE assay 217

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Samples were prepared for free HNE determination as described previously by Lesgards 218

et al. (38) and adapted for the analysis of rat fecal water samples (see Supplementary 219

Materials for details). 220

Urinary biomarker 221

Urine preparation 222

Urine samples were diluted at 1:200 for animal studies and at 1:20 for human study in 223

an EIA/BSA solution containing phosphate buffer (0.1M at pH 7.4) with NaCl (0.15 M), 224

0.1% BSA and 0.01% sodium azide. 225

DHN-MA assay 226

DHN-MA assay was performed by competitive enzyme immunoassay as described 227

previously using DHN-MA-linked acetylcholinesterase enzyme as tracer (24). Urine 228

samples from non-induced rats study were pooled and analysed in duplicate, whereas 229

other urine samples were assayed individually. 230

Statistical analysis 231

Data were analyzed using Systat 12 software for Windows and reported as mean ± SEM 232

or mean ± SEM as noted in legends. 233

In study with induced rats, the effect of meat intake was analyzed by one-way ANOVA 234

comparing the control animals to the meat-fed animals. Next, the control animals were 235

removed and the effect of marinade and cooking analysed in two-way ANOVA. The 236

interaction between the two factors (marinade and cooking) was always checked. In 237

study with Min mice, the effect of meat intake was analysed by two-way ANOVA 238

including the factor sex. Next, the effect of the marinade, cooking, and gender was 239

analyzed in three-way ANOVA. The interaction between the two or three factors was 240

always checked. MDF and tumor scoring was performed in duplicate by two 241

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independent readers; therefore, these variables were tested first by 2-factor ANOVA 242

(groups and readers). The group x reader interaction was never significant, and when 243

total ANOVA was significant (P<0.05), pairwise differences between groups were 244

analyzed. 245

Human volunteer data were analyzed using the Wilcoxon’s signed rank test with each 246

volunteer being his own control. No Bonferronni correction was made for the multiple 247

comparison analysis, as only three pairwise comparisons were made that had been 248

decided beforehand (i.e., rare-cooked meat vs. no-meat control period, rare-cooked meat 249

vs. rare-cooked meat marinated, and rare-cooked meat marinated vs. well-done meat 250

marinated). 251

Liking data from the first panel and control group of the second panel were analyzed 252

separately using two-way ANOVA model including the product and the consumer 253

effects. The information (classical/protective) and its interaction with the product effect 254

were added to the model for the informed group of the second panel. Finally, the 255

difference between liking scores with and without the protective information averaged 256

over the two products was used to screen the items on the consumer questionnaires by 257

one-way ANOVAs for the effect of this information. 258

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RESULTS 259

Associated to fecal and urinary lipoperoxidation biomarkers, consumption of red 260

meat increases the size of colonic preneoplastic lesions in azoxymethane-initiated 261

rats, but marinating meat protects against these modulations. 262

Rats fed meats had significantly larger MDF (P=0.028) and more fecal heme (P<0.0001), 263

TBARS (P<0.0001), free and bound 4-hydroxynonenal (HNE; P=0.002 and P<0.0001, 264

respectively), and urinary DHN-MA (P<0.0001) than rats given the control diet with no 265

meat (Table 1). Proteomic analysis showed that raw no-marinated meat fed rats had 266

significantly more ferritin and annexin in the colon mucosa than control no-meat diet 267

fed rats (Table S6). All proteomics results as well as the metabolic pathway affected are 268

presented in Supplementary data (Table S6 and Fig. S3). Rats given marinated meats 269

had significantly fewer MDF (P=0.018) and less fecal heme (P=0.013), TBARS (P=0.015), 270

free and bound HNE (P=0.003 and P=0.031, respectively), and urinary DHN-MA 271

(P=0.001) than rats given non-marinated meats (Table 1). Finally, rats given cooked 272

meats had significantly fewer MDF (P=0.042) and less fecal heme (P<0.0001) and 273

TBARS (P=0.034) than rats given uncooked meats, without a significant difference in 274

free and bound HNE (P=0.8 and P=0.2, respectively) and urinary DHN-MA (P=0.2; Table 275

1). We identified significant interactions between marinade and cooking factors for fecal 276

heme (P=0.014) and fecal bound HNE (P=0.042). 277

278

Red meat intake induces a simultaneous increase in the number of intestinal 279

tumors, the tumor load and the fecal biomarker of peroxidation in Apc Min mice. 280

Marinating red meat protects against these increases in female mice. 281

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The tumoral character of the enumerated lesions in the intestine after methylene blue 282

staining (Fig. 1A) was evaluated in histological sections (Fig. 1B). Mice given meats had 283

significantly more intestinal tumors and higher tumor load (P=0.048 and P=0.001, 284

respectively; Fig. 1C, D) and more fecal heme (P=0.02, Fig. 2A) and TBARS (P<0.0001, 285

Fig. 2B) than mice given the control diet with no meat. Taking into account the size of 286

the tumors, the significant effect of meat in increasing the number of tumours was 287

confirmed for medium and large tumors (Supplementary Table S7). Furthermore, 288

female mice given meats had significantly more and larger intestinal tumors (P=0.047, 289

Fig. 1C and P=0.009, respectively) than male mice given meats. 290

We found a significant interaction between marinade and sex factors for tumor number 291

(P=0.015). Female mice given marinated meats had significantly fewer intestinal tumors 292

than female mice given non-marinated meats (P=0.019, Fig. 1E), but no protecting effect 293

of marinade was observed for male mice (P=0.47, Fig. 1F). 294

Mice given marinated meats had significantly less fecal TBARS (P=0.043) than rats given 295

non-marinated meats without a significant effect on fecal heme (P=0.4), and urinary 296

DHN-MA (P=0.2, Fig. 2). Proteomic analysis showed that rare marinated meat fed mice 297

had significantly less vimentin in the colon mucosa than control no-meat diet fed mice 298

(Table S8). All proteomics results as well as the metabolic pathway affected are 299

presented in Supplementary data (Table S8 and Fig. S5). Mice given cooked meats had 300

significantly more intestinal tumors and higher tumor load (P=0.001 and P=0.001, 301

respectively; Fig. 1C, D) than mice given uncooked meats, without a significant 302

difference for fecal heme (P=0.2), TBARS (P=0.1), and urinary DHN-MA (P=0.1, Fig. 2). 303

We found no significant interaction between marinade and cooking factors. 304

305

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Rare cooked meat consumption increases luminal peroxidation in healthy 306

volunteers and a grape-olive marinade reduces this effect 307

The nutritional intervention was scheduled with 24 healthy volunteers (see 308

Supplementary Materials for details). Biomarker measurements revealed a significant 309

increase in the heme content of fecal water for the two rare meat periods (Fig. 3A) and a 310

tendency to increase with the well-done meat. We also observed a significant increase in 311

the concentrations of TBARS in fecal water from human volunteers fed 110 g of non-312

marinated meat for 4 days compared to control periods (P=0.038, Fig. 3B). Marinade 313

significantly decreased fecal TBARS for rare cooking (P=0.046) but not well-done 314

cooking (P=1). We found no significant difference between volunteers in regards to HNE 315

content in fecal water, urinary DHN-MA, or fecal ATNC and fecal and genotoxicity 316

(Supplementary Fig. S6). 317

318

The olive-grape marinade does not alter the organoleptic acceptability of red 319

meat but conversely increases the preference of the meat 320

To quantify the contribution of antioxidants in the effect of grape-olive marinade on 321

organoleptic acceptability, an antioxidant-free marinade was compared to a marinade 322

with antioxidants. A two-way (product and consumer) ANOVA model of liking scores 323

from the first study demonstrated that the marinated meats were both significantly 324

preferred to the reference meat (P=0.004, Fig. 4A). A very slight similar trend was 325

observed in the second study (Fig. 4B), although it did not reach statistical significance 326

(P=0.283). The marinated meat with antioxidants was slightly less appreciated than the 327

marinated meat without antioxidants, though this difference was not significant (Fig. 328

4A). 329

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The effect of information about colon cancer risk and the protective effect of new 330

products was not significant and had almost (P=0.182) no interaction with the product 331

effect (Fig. 4C). However, looking precisely at the mean liking scores of the four products 332

tested in the test group, a potential positive effect of the information on the liking of the 333

marinated meat was observed (P=0.086). 334

None of the variables on the usage, attitude, and knowledge questionnaire influenced 335

the effect of the information on product-liking. However, the level of education and age 336

of the consumers seemed to have impacted that effect. Indeed, the difference between 337

liking scores with and without information is significantly (p=0.022) higher in primary 338

and secondary versus higher school levels (Fig. 4D). Also, Fig. 4E shows that the effect of 339

information would be negative in consumers aged less than 35, about null in consumers 340

between 36 et 55 and positive in consumer older than 56; these differences among the 341

three age categories were almost significant (p=0.053). 342

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DISCUSSION 343

The present study is the first to show that consumption of fresh red meat for few days 344

increased fecal lipid oxidation biomarkers associated with heme-induced promotion of 345

colon carcinogenesis in human volunteers and in two complementary animal models. 346

These findings agree with the epidemiological data and the conclusions of the WCRF and 347

WHO (3,4). This study also shows that this increase and the promotion of carcinogenesis 348

in rats and female mice were decreased when meat was treated with an antioxidant 349

marinade. 350

As people of a lower socioeconomic status are not receptive to nutritional messages and 351

less likely to change risky behaviors than affluent people, we wanted to propose an 352

alternative to the current recommendation of limiting consumption by working directly 353

on the commercial product. To achieve this objective, we designed a project (Flow chart 354

in Supplementary Fig. S1) allowing to highlight a beef antioxidant marinade limiting the 355

heme-iron induced lipoperoxidation, a key step in the promotion of colon cancer 356

associated with red meat consumption (11). 357

For this project, we associated two animal models of carcinogenesis: the AOM-induced 358

rat and the Apc Min mice. Indeed, for 30 years investigators have searched for dietary 359

agents that could impact colon carcinogenesis tumors. For that, the azoxymethane 360

(AOM) model was widely used and more recently associated to Apc Mice models. If 361

carcinogenesis in the rat model results of a large bolus of AOM, it has many 362

morphological as well as molecular similarities to human sporadic colorectal cancer 363

(39). Similarly, if the main location of tumors in the Apc Min mice is the small intestine, 364

this mice is considered to be a model for human familial adenomatous polyposis with 365

applications in order to better understand the molecular mechanisms of multistage 366

carcinogenesis in the large bowel of humans (40). Furthermore, review of experimental 367

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data between diet and colon cancer demonstrated that there is a close agreement 368

between many results obtained in the colons of AOM-initiated rats and in the small 369

intestine of Min mice (41) and that there is a reasonable agreement between the results 370

of these animal studies and the more limited clinical studies (42). 371

First, we defined an antioxidant marinade able to decrease lipoperoxidation in meats 372

and rat fecal water after meat consumption. Several studies have shown that natural 373

antioxidants can be used to reduce lipoperoxidation in meat, and that the antioxidant 374

activity of a marinade can be determined by a DPPH assay (43-46). Twelve marinades 375

were prepared to test the antioxidant activity of eight molecules, alone or in mixture, 376

and grape-olive marinade had the highest (Supplementary Table S2). The addition of 377

this marinade to beef sirloin led to a significant decrease in the concentrations of TBARS 378

in meat (study 1, Supplementary Table S3). Although the grape-olive mixture has never 379

been tested, these results are consistent with the literature regarding the properties of 380

grapes and olives (47-49). In a short-term study in rats, we confirmed that the marinade 381

can also decrease meat-induced colonic luminal lipoperoxidation (study 2, 382

Supplementary Table S4), which is consistent with our previous studies (19,20). 383

Thus, we wanted to check the effect of marinated and non-marinated meat on two 384

stages of colorectal carcinogenesis using two complementary animal models: 385

azoxymethane-initiated rats for the preneoplastic stage and Apc Min mice for the 386

tumoral stage. Finally, we explored the impact of these meats on fecal and urinary 387

biomarkers in human volunteers given 110 g of meat per day for 4 days. 388

Our results showed that fresh red meat intake significantly increased the size of pre-389

cancerous lesion MDF in initiated rats (Table 1) and the number and size of intestinal 390

tumors and tumor load in Apc Min mice (Fig. 1). More precisely, fresh red meat intake 391

significantly increased the number of medium and large tumors (Supplementary Table 392

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S7). In the two carcinogenesis animal models, meat-induced promotion was associated 393

with a significant increase in fecal heme and lipoperoxidation biomarkers (TBARS, HNE 394

in fecal water, and urinary DHN-MA in rats [Table 1]; TBARS in Apc Min mice [Fig. 2]). In 395

mucosa, promotion in initiated rats was positively associated with the level of ferritin 396

and annexin (Supplementary Table S6), two proteins for which expression was 397

positively associated with the degree of dysplasia (50,51). In our study, the level of 398

ferritin protein in the colon mucosa correlated with the size of MDF (r=0.497, P=0.002), 399

with the TBARS level in fecal water (r=0.511, P=0.001) and with the DHN-NA content in 400

urine (r=0.409, P=0.013). The level of annexin protein in mucosa positively correlated 401

with the size of MDF (r=0.452, P=0.006), with the heme content in fecal water (r=0.394, 402

P=0.017) and with the TBARS level in fecal water (r=0.402, P=0.015) . These results on 403

fecal and urinary biomarker modulations are consistent with previous studies showing a 404

promoting effect of hemoglobin or lyophilized red meat on pre-cancerous lesions or on 405

tumors in association with increased fecal lipoperoxidation (16,18-20). In human 406

volunteers, the present crossover study showed that eating fresh red meat for 4 days 407

was sufficient to increase lipoperoxidation biomarker concentrations in fecal water, 408

compared to the control period without meat (Fig. 4). We did not observe a significant 409

increase in urinary DHN-MA, which is consistent with our previous results with red 410

meats (22). In humans, unlike in rodents, meat intake correlates with endogenous 411

formation of fecal ATNC (52,53). In this study, we did not observe a significant increase 412

of fecal ATNC (Supplementary Fig. S6) and the level of ATNC was low overall, but this 413

result is consistent with other human studies in which an increase in fecal ATNC was 414

found only in subjects consuming at least 240g of red meat per day and for a longer 415

consumption period (52,54,55). Thus, the intake of fresh red meat can modulate 416

lipoperoxidation biomarkers associated with the promotion of colon carcinogenesis in 417

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initiated rats and Apc Min mice, which gives experimental support to the epidemiology-418

based conclusion that red meat may be a cause of colorectal cancer (1,2,4). 419

Grape-olive marinade was efficient in initiated rats to decrease the number of pre-420

cancerous lesion MDF and was associated with a decrease in heme and lipoperoxidation 421

biomarkers (Table 1). In Apc Min mice study, we observed a significant protective effect 422

in female mice (Fig. 1E). To the best of our knowledge, this study is the first in which an 423

antioxidant marinade was used to reduce the promotion of colorectal cancer by fresh 424

red meat. Our results support epidemiological studies showing that adhesion to a 425

Mediterranean diet, rich in olive oil and antioxidants, may reduce colorectal cancer risk, 426

mainly in women (56,57). The fact that we only observed a protective effect of the 427

marinade only in female may be consistent with epidemiologic evidences showing a 428

gender-specific associations between antioxidants and rectal cancer risk, pointing a 429

protective effect in women while no effect in men, probably due to the estrogen status 430

(58). In our study, the protection due to the marinade in female Apc Min mice was 431

associated with decreased expression of vimentin in the colon mucosa (Supplementary 432

Table S8). Vimentin activates the Wnt signaling pathway, which is detectable as 433

increased β-catenin accumulation in the nucleus with concomitant activation of β-434

catenin-dependent transcription of Wnt signaling downstream targets (59); thus, a 435

decrease in the activation of this pathway largely involved in the colon carcinogenesis 436

could participate in the observed protection. In human volunteers, as in the two 437

carcinogenesis animal models, the marinade reduced significantly the increase of fecal 438

TBARS when meat was cooked rare (Fig. 3). However, the protective effect was lost 439

when the meat was well-done, likely reflecting an alteration in the antioxidant capacity 440

during cooking at high temperature. In accordance with our initial aim to limit the risk 441

in big meat eaters, in whom the risk of colon cancer increases the most, this absence of 442

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effect in well-done meat will not greatly limit the efficiency of the marinade because a 443

majority of big eaters in France (60), in Europe (33) and in USA (61) consume rare or 444

medium red meat. Moreover, in parallel to the addition of antioxidant via a marinade, it 445

may be interesting to test other solutions to provide the antioxidants with the meat: sell 446

antioxidant extracts with the meat for example. These extracts could be used to dress 447

the meat after cooking. However, if it is not certain that a sufficient amount of 448

antioxidant is attained, this solution could be useful in order to limit the loss of 449

antioxidant activity during “well-done” cooking. 450

The biological effectiveness of modifying a product is not useful if consumers do not like 451

the product. For the marinated meat, it is important to note that the product had higher 452

liking scores than the unmodified product (Fig. 4). Thus, modifying the product to limit 453

the risk of cancer does not affect the acceptability of the product. Furthermore, we 454

observed a potential positive effect of the information about colon cancer risk and the 455

protective effect of new products on the liking score for the marinated meat. This 456

suggests that, if the consequences of the modification of the meat product are 457

appreciated, then the consumers can positively value the information. Though the 458

current communication of only limiting consumption does not seem to be well accepted 459

(12), the strategy of associating the modification of the product with the communication 460

on the potential benefit could be very effective in limiting the risk. 461

We have also evaluated the effect of cooking on meat-induced colorectal cancer. We 462

studied rare and well-done meat in initiated rats and meat cooked rare in Apc Min mice 463

because it is the cooking most used by consumers (62). In a previous study, in which we 464

demonstrated the promotive effect of heme iron (16), we did not observe a positive 465

association between the number of MDF and PhiP and Me-IQx in an AIN-76-based diet. 466

In the present study, cooking had a protective effect on the number of pre-cancerous 467

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lesions in initiated-rats. In contrast, in Apc Min mice, cooking had a promoting effect on 468

the number and size of intestinal tumors. In our study, this promoting effect did not 469

come from potentially carcinogenic heterocyclic amine formation because 470

concentrations found in meats were very low. Among the thirteen tested, 8 heterocyclic 471

amines were not detected or under the quantification limit, only PhIP, IQx, MeIQx, 472

DiMeIQx and DMIP were quantifiable in rare and well-cooked meats, but at low doses and 473

well below the concentrations usually used to promote colorectal carcinogenesis in Apc 474

Min mice (Supplementary Table S5) (63,64). In initiated rats, the protective effect of 475

cooking was associated with a significant decrease in fecal heme, and in Apc Min mice 476

the promoting effect of cooking was associated with a tendency of increase in fecal 477

heme. Thus, as observed in our previous studies (16,19), the promotion of 478

carcinogenesis appears to be associated with the luminal heme content. Differences 479

observed between rats and Apc Min mice suggest a difference in heme bioavailability in 480

these two different rodent models. These opposing results make it difficult to come to a 481

conclusion on the effect of cooking on heme bioavailability and the link with 482

lipoperoxidation and the promotion of carcinogenesis. However, in human volunteers, if 483

meat consumption increased the level of heme in fecal water, the cooking method did 484

not significantly affect this level. Thus, the impact of marinated meat on lipoperoxidation 485

in human volunteers could be assigned to the antioxidant capacity of the marinade and 486

not the decrease of heme bioavailability, the catalyzer of lipid peroxidation. 487

In conclusion, consumption of fresh red meat for few days increased biomarkers 488

associated with heme-induced promotion of colon carcinogenesis. Grape-olive marinade 489

counteracted this promoting effect in carcinogen-injected rats and female Apc Min mice. 490

This protection was associated with normalization of fecal biomarkers in the two animal 491

models and a significant limitation in human volunteers. As in one of our previous 492

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studies with processed meat (32), these results suggest that it is possible to modify fresh 493

red meat products by adding antioxidants to reduce the cancer-promoting properties of 494

red meat, which could also improve, or at least not deteriorate, its sensory acceptability. 495

The protective effect of meat antioxidant marinades need now to be validated by 496

epidemiological studies and mechanisms need to be explored more precisely with for 497

example the monitoring of the levels of DNA damage and DNA adducts to 498

lipoperoxidation endproducts. Their effectiveness could lead to protective strategies to 499

decrease the colorectal cancer burden in all populations, especially those who need it 500

most. 501

502

ACKNOWLEDGMENTS 503

We thank all of the volunteers who generously gave their time for this research. We 504

thank the ADIV workshop staff for the meat preparation. This work was conducted in 505

the framework of the French network for Nutrition And Cancer Research (NACRe 506

network), www.inra.fr/nacre. 507

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Meat intake, cooking methods and doneness and risk of colorectal tumours in the Spanish 615 multicase-control study (MCC-Spain). Eur J Nutr 2016 doi 10.1007/s00394-016-1350-6. 616

34. Caderni G, Femia AP, Giannini A, Favuzza A, Luceri C, Salvadori M, et al. Identification of 617 mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with 618 carcinogenesis. Cancer Res 2003;63(10):2388-92. 619

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40. Yamada Y, Mori H. Multistep carcinogenesis of the colon in Apc(Min/+) mouse. Cancer Sci 635 2007;98(1):6-10 doi 10.1111/j.1349-7006.2006.00348.x. 636

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42. Corpet DE, Pierre F. How good are rodent models of carcinogenesis in predicting efficacy in 640 humans? A systematic review and meta-analysis of colon chemoprevention in rats, mice and 641 men. Eur J Cancer 2005;41(13):1911-22 doi S0959-8049(05)00476-4 [pii] 642

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antioxidants. J Sci Food Agric 2011;91(5):963-8 doi 10.1002/jsfa.4274. 652 46. Viegas O, Amaro LF, Ferreira IM, Pinho O. Inhibitory effect of antioxidant-rich marinades on 653

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48. Jongberg S, Skov SH, Torngren MA, Skibsted LH, Lund MN. Effect of white grape extract and 659 modified atmosphere packaging on lipid and protein oxidation in chill stored beef patties. 660 Food Chem 2011;128(2):276-83 doi S0308-8146(11)00396-7 [pii] 661

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55. Hughes R, Cross AJ, Pollock JR, Bingham S. Dose-dependent effect of dietary meat on 680 endogenous colonic N-nitrosation. Carcinogenesis 2001;22(1):199-202. 681

56. Jones P, Cade JE, Evans CEL, Hancock N, Greenwood DC. The Mediterranean diet and risk of 682 colorectal cancer in the UK Women's Cohort Study. Int J Epidemiol 2017;46(6):1786-96 doi 683 10.1093/ije/dyx155. 684

57. Bamia C, Lagiou P, Buckland G, Grioni S, Agnoli C, Taylor AJ, et al. Mediterranean diet and 685 colorectal cancer risk: results from a European cohort. Eur J Epidemiol 2013;28(4):317-28 doi 686 10.1007/s10654-013-9795-x. 687

58. Murtaugh MA, Ma KN, Benson J, Curtin K, Caan B, Slattery ML. Antioxidants, carotenoids, 688 and risk of rectal cancer. Am J Epidemiol 2004;159(1):32-41. 689

59. Satelli A, Hu J, Xia X, Li S. Potential Function of Exogenous Vimentin on the Activation of Wnt 690 Signaling Pathway in Cancer Cells. J Cancer 2016;7(13):1824-32 doi 10.7150/jca.15622. 691

60. AFSSA. Etude Individuelle Nationale des Consommations Alimentaires 2 (INCA 2) 2006-07. 692 2009. 225 p. 693

61. Reicks AL, Brooks JC, Garmyn AJ, Thompson LD, Lyford CL, Miller MF. Demographics and beef 694 preferences affect consumer motivation for purchasing fresh beef steaks and roasts. Meat 695 Sci 2011;87(4):403-11 doi 10.1016/j.meatsci.2010.11.018. 696

62. INCA-2. National Individual Study of Dietary Consumption-2 697 2006-2007. 2009. 698 63. Andreassen A, Vikse R, Mikalsen A, Adamovic T, Steffensen IL, Hjertholm H, et al. 2-Amino-1-699

methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces genetic changes in murine intestinal 700 tumours and cells with ApcMin mutation. Mutat Res 2006;604(1-2):60-70 doi S1383-701 5718(06)00044-1 [pii] 702

10.1016/j.mrgentox.2006.01.004. 703 64. Svendsen C, Meinl W, Glatt H, Alexander J, Knutsen HK, Hjertholm H, et al. Intestinal 704

carcinogenesis of two food processing contaminants, 2-amino-1-methyl-6-705 phenylimidazo[4,5-b]pyridine and 5-hydroxymethylfurfural, in transgenic FVB min mice 706 expressing human sulfotransferases. Mol Carcinog 2012;51(12):984-92 doi 707 10.1002/mc.20869. 708

709

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LEGENDS OF FIGURES AND TABLE 710

Figure 1. Effect of experimental meats on small intestine tumors in Apc Min mice fed for 711

45 days. A, Adenoma after methylene blue staining. B, Histological section of adenoma 712

(H&E staining). C, Number of tumors per mouse. D, Tumor load per mouse. E, Number of 713

tumors per female mouse. F, Number of tumors per male mouse. Scale bars = 500μm. 714

Data are presented in the graphs as mean ± SEM and the circle are the individuals. The 715

effect of meat intake was evaluated by two-way ANOVA and the effect of marinated and 716

cooked meat intake by three-way ANOVA. # P<0.05; # # P<0.01 vs. the no-meat-fed mice, 717

* P<0.05 vs. the raw condition with the same marinade status, °° °° P<0.01 vs. the no-718

marinated condition. 719

720

Figure 2. Effect of experimental meats on fecal and urinary biomarkers associated with 721

meat-induced promotion in Apc Min mice fed for 45 days. A, Heme in fecal water. B, 722

TBARS in fecal water. C, DHN-MA in urine. Data are presented as mean ± SEM. The effect 723

of meat intake was evaluated by two-way ANOVA and the effect of marinated and 724

cooked meat intake evaluated by three-way ANOVA. * * P<0.05; ** P<0,0001 vs. no 725

meat-fed mice, ° P<0.05 vs. no-marinated condition. 726

727

Figure 3. Effect of experimental meats on fecal biomarkers associated with meat-induced 728

promotion in human volunteers after 4 days of 110g/day of meat. A, Heme. B, TBARS in 729

fecal water. Data are presented as mean ± Min/Max, N=21.* P<0.05 vs. the no-meat 730

control period (Wilcoxon’s test). §: P<0.05, vs. the no-marinated meat period 731

(Wilcoxon’s test). 732

733

Figure 4. Product liking means in the first sensory study (A), second sensory study (B), 734

test groups (C). Effect of the information according to education level (D) and age (E). 735

Data are presented as mean ± SEM. Statistical analysis was three-way ANOVA. * P<0.05, 736

vs. the reference. ° P<0.05, vs. the reference Higher school level; ax. : antioxidants; 737

inform. : after information. 738

739

Table 1 Effect of experimental meats on size and number of preneoplastic Mucin 740

depleted foci (MDF) lesions and on fecal and urinary biomarkers associated with meat-741

induced promotion in rats previously injected with azoxymethane and fed for 98 days. 742

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Data are presented as mean ± SD. a The effect of meat intake was evaluated by a one-743

way ANOVA and the effect of marinated and cooked meats intake by a two-way ANOVA. 744

FW: fecal water, WD: well-done. 745

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Table 1 Effect of experimental meats on size and number of preneoplastic Mucin depleted foci (MDF) lesions and on fecal and urinary biomarkers associated with meat-induced promotion in rats previously injected with azoxymethane and fed for 98 days. Data are presented as mean ± SD. a The effect of meat intake was evaluated by a one-way ANOVA and the effect of marinated and cooked meats intake by a two-way ANOVA. FW: fecal water, WD: well-done.

CONTROL

DIET

(no meat)

EXPERIMENTAL MEATS STATISTICS a

No marinade Marinade

Meat Marinade Cooking Raw Rare WD

Raw Rare WD

MDF size 2.3 ± 0.3

2.5 ± 0.6 2.7 ± 0.9 2.4 ± 0.6

2.6 ± 0.5 2.5 ± 0.5 2.7 ± 0.7

P=0.028 P=0.855 P=0.944

MDF number 19 ± 9 21 ± 11 17 ± 10 15 ± 6 16 ± 9 14 ± 7 13 ± 7 P=0.067 P=0.018 P=0.042

Heme

(µM in FW) 2 ± 2

193 ± 72 122 ± 43 90 ± 30

120 ± 58 101 ± 44 99 ± 26

P<0.0001 P=0.013 P<0.0001

TBARS

(µM MDA eq. in FW) 13 ± 12

84 ± 21 75 ± 15 91 ± 17

67 ± 19 68 ± 14 81 ± 27

P<0.0001 P=0.015 P=0.034

HNE free

(nmol/g FW) 1.1 ± 0.4

1.7 ± 0.4 1.8 ± 0.5 1.7 ± 0.6

1.5 ± 0.3 1.2 ± 0.4 1.3 ± 0.6

P=0.002 P=0.003 P=0.824

HNE bound

(nmol/g proteins) 44 ± 17

72 ± 23 97 ± 28 66 ± 32

63 ± 21 62 ± 22 69 ± 28

P<0.0001 P=0.031 P=0.202

DHN-MA

(µg/24 h in urine) 178 ± 62

406 ± 173 564 ± 237 485 ± 229

335 ± 86 372 ± 170 310±149

P<0.0001 P=0.001 P=0.172

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Figure 2

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Published OnlineFirst June 28, 2018.Cancer Prev Res   Océane C.B. Martin, Nathalie Naud, Sylviane Taché, et al.   carcinogenesis associated with red meat consumptionTargeting colon luminal lipid peroxidation limits colon

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 28, 2018; DOI: 10.1158/1940-6207.CAPR-17-0361