targeting colon luminal lipid peroxidation limits …...2018/06/28 · 1 1 targeting colon luminal...
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1
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:
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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19
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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21
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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22
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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23
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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24
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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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
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709
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29
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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30
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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ReferenceMarinated Marinatedwith antioxydants
ReferenceMarinatedwith antioxydants
ReferenceReference Marinated Marinatedinform.with ax.with ax.
inform.
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Figure 4
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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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