SECODARY LIPID OXIDATIO PRODUCTS I FOOD AD COLO CACER Françoise Guéraud, Fabrice Pierre, Raphaëlle Santarelli, Sylviane Taché, Maryse Baradat, Nathalie Naud, Marc Audebert and Denis Corpet Laboratoire des Xénobiotiques, UMR 1089 INRA-ENVT, BP3, 180 ch. de Tournefeuille, 31027 Toulouse cedex 3, France.
Introduction
RESULTS •In vivo study: different diets (60% casein, chicken, beef or black pudding) on Fisher 344 female rats 20000 50
Control Chicken
45
16000
Beef
40
Beef
B. Pudding
35
B. Pudding
8-iso-PGF2 (ng/24h)
18000
14000
DHN-MA (ng/24h)
The recent dose-response meta-analysis of epidemiological studies by Norat et al. (1) suggests that red meat and processed meat intakes are associated with increased risks of colorectal cancer. The World Cancer Fund International recommends a limited intake of red meat (max: 300g /week) and to avoid processed meat (WCRF 2007). In a previous work, we have shown that dietary haem and red meat intake in a low-calcium diet with 5% safflower oil promote colon carcinogenesis in rats (2). Oxidative stress and lipid peroxidation are likely the causes of the promotion, as haem effect was inhibited by antioxidants or olive oil . 4-hydroxynonenal (HNE) is considered as the major end product of the oxidation of n-6 polyunsaturated fatty acids. HNE is a cytotoxic and genotoxic compound. It is also found in foodstuffs in concentrations reaching 500 ppm in food fried in thermally oxidized oils (3). The present study was designed to investigate the effects of diets containing haem and n-6 polyunsaturated fatty acids, on the urinary excretion of the major HNE metabolite (DHN-MA, 1,4dihydroxynonene mercapturic acid) and on preneoplastic lesions (MDF, mucin depleted foci). As HNE was shown to be present un colon lumen after feeding those heme rich diets, the cytotoxic and genotoxic effect of HNE on immortalized mouse colon cells lines was investigated too. Those cells were wild type cells (Apc +/+) or Apc mutated cells (Apc min/+). Apc mutation is an early event in human colon carcinogenesis.
12000 10000 8000 6000 4000
Control Chicken
30 25 20 15 10
2000
5
0
0
0
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4
6
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10
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14
0
16
2
4
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8
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14
16
Days
Days
8-isoPGF2α
DHN-MA
X 2 with black pudding diet
X 400 with black pudding diet X 30 with beef diet
Urinary excretion of DHN-MA (major HNE metabolite) and 8-isoPGF2α 25
Heme µmol/g of diet
20
Materials and Methods
*#
DHN-MA (µg/24h)
° 15
10
5
MDF DHN-MA
Long term study :100 days: cancer study
MDF crypts/colon
*#
•In vivo study: different diets (60% casein, chicken, beef or black pudding) on Fisher 344 female rats
° 0 Control
Chicken
Beef
Black pudding
Diets
azoxymethane
Relationship between heme content of the diet, urinary DHN-MA and colon cancer promotion in rats
Control Casein
30
•In vitro study : on Apc +/+ or Apc min/+ immortalized mouse colon cell lines 0
100 days
AIN 76 Control Casein
160 140
120
60 40
100
20
80 60 40 20
0
0
0 Promotion
Preneoplastic cells 140
80
•In vitro study : on Apc +/+ or Apc min/+ immortalized mouse colon cell lines Initiation
Normal cells Preneoplastic cells
100
% of viability
% of viability
Short term study :15 days: biomarker study
Normal cells
120
DHN-MA 8-isoPGF2α
50
100
150
200
250
300
CD
Chicken Beef Faecal water
B. pudding
(5xdilution)
µM HNE in feacal w ater of control group
Progression
(Pierre F, Taché S, Guéraud F, Rérole AL, Jourdan ML and Petit C. Apc mutation induces resistance of colonic cells to lipoperoxide-triggered apoptosis induced by faecal water from haem-fed rats, 2006 , Carcinogenesis, 28, 321-7.)
Cytotoxic effect of HNE (left) or of fecal extracts from rats fed the in vivo study diets (right) on colon cell lines using MTT assay
Aberrant Crypt Foci Normal Crypt
Apc +/+
Adenoma
Aberrant Crypt
Adenocarcinoma
Gamma-H2Ax is a marker of double-strand beaks in genomic DNA
Apc +/Forest V. et al, 2003, Nutrition Cancer, 45, 84-92.
Normal cells
Preneoplastic cells
Apc+/+
Apc Min/+
. Immortomouse F1 AgT Apc +/+
0 µM
Colon cells Apc +/+
C57Bl/6 Apc +/+
20 µM C57Bl6 Apc Min/+
.
Colon cells Apc Min/+
40 µM
F1 AgT Apc Min/+
Immortomouse
H2AX phosphorylation is dose-dependently increased with HNE and this effect is more pronounced in wild type cells/Apc mutated cells
Conclusion 14
• DHN-MA : a biomarker of food associated with increased colon cancer risk? Useful but reflects at the same time endogenous+food+ luminal lipid peroxidation HNE in fecal waters or HNE adducts in colon cells could be pertinent
12 10 8
Apc +/+ Apc Min/+
6 4 2
20
40
E HH
20
80 E
E HH
HH
0
40 DA
DA M
M
20 E
DA 8 M
HN
O
40
80 E
ET
E HN
HN
EM
0 DM
•Excretion of HNE urinary metabolite is associated with heme-rich diets. HNE was present in those diets (not shown). •Do secondary lipid oxidation products coming from food lipids play a role in colon cancer ? They have a promoting effect in vitro with selection of already mutated cells under luminal « peroxidizing » conditions. In vivo? Mechanism(s)? Apoptosis? DNA damage? Different biotransformations? What about 4-hydroxyhexenal (HHE) coming from the oxidation of n-3 fatty acids, malondialdehyde coming from fatty acids bearing more than two insaturations? Do they reach other tissues, what are the effects on other tissues? : Metabolism and biodisposition need to be studied.
4-hydroxyalkenals have a differential genotoxic effect on wild type cells compared to Apc mutated cells
Genotoxic effect of HNE (top) or HNE and other lipid peroxidation products (bottom) on colon cell lines using H2AX phosphorylation assay References: (1) Norat, T., Lukanova, A., Ferrari, P. & Riboli, E. (2002) Meat consumption and colorectal cancer risk: dose-response meta-analysis of epidemiological studies. International Journal of Cancer 98: 241-256. (2) Pierre, F., Tache, S., Petit, C. R., Van der Meer, R. & Corpet, D. E. (2003) Meat and cancer: hemoglobin and hemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt stage in rats. Carcinogenesis 24: 1683-1690. (3) Seppanen, C.M. and Csallany, A.S. (2004) Incorporation of the toxic aldehyde 4-hydroxy-2-trans-nonenal into food fried in thermally oxidized soybean oil. JAOCS 81,1137-1141.
