Borowicki, A.

Brenner-Weiss, G.

Glei, M.

Pool-Zobel, B.L.

Scharlau, D.

Scheu, K.

Stein, K.

English

Pool-Zobel, B. L.

KITopen

Results0 5 10 15 20050100150concentration [%]cellnumber[%]0 5 10 15 20050100150concentration [%]cellnumber[%]0 5 10 15 20050100150**concentration [%]cellnumber[%]§§§0 5 10 15 20050100150ox gall 4,16 g/l ox gall 0,06 g/lconcentration [%]cellnumber[%]0 5 10 15 20050100150***§§§§concentration [%]cellnumber[%]0 5 10 15 20050100150concentration [%]cellnumber[%]syntheticmixtureofDCAandCAoffsblanksyntheticmixtureofDCAandCAoffsinulin24 h 48 h 72 hBackgroundIn vitroin vitromodels simulating the digestive tract are commonly used to investigate degradation ofnutrients or development of gut flora. Aim of this work was to modify an established model(Aura, 1999, J Cer Sci) to allow further studying mechanisms of carcinogenesis andchemoprevention using human carcinoma cells . Therefore the modified model,characterised by changing ox gall concentration to a physiological level, should be compared tothe established model.MODIFICATION OF AN MODEL SIMULATING THE WHOLEDIGESTIVE PROCESS TO INVESTIGATE CELLULAR ENDPOINTS OFCARCINOGENESIS AND CHEMOPREVENTIONIN VITROKatrin Stein , Anke Borowicki , Michael Glei , Daniel Scharlau , Kerstin Scheu , Gerald Brenner-Weiss ,Beatrice L. Pool-Zobel1 1 1 1 2 2 11Department of Nutritional Toxicology, Institute for Nutrition, Friedrich-Schiller-University Jena, Dornburger Str. 24, 07743 Jena, GermanyResearch Centre Karlsruhe, Institute for Technical Chemistry, Group for Water- and Geotechnology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany2±ConclusionBy decreasing ox bile concentration secondary cytotoxic side effects of fs are reduced. Moreover fermentation products ofinulin suppress growth of HT29 cells. Fs obtained from the modified model (using 0.6 g/l ox gall in intestinal extract) canbe used for investigations on chemopreventive properties of complex food ingredients.in vitro24 h0 5 10 15 20050100150********§§§§concentration of fs [%]cellnumber[%]48 h0 5 10 15 20050100150************** ****§§§§§§ §§§blank (feces control) inulinconcentration of fs [%]cellnumber[%]72 h0 5 10 15 20050100150 §§§§§§ §§§******************concentration of fs [%]cellnumber[%]24 h0 5 10 15 20050100150 §§******concentration of fs [%]cellnumber[%]48 h0 5 10 15 20050100150 §§§**************blank (feces control) inulinconcentration of fs [%]cellnumber[%]72 h0 5 10 15 20050100150 §§§********* ********concentration of fs [%]cellnumber[%]Figure 2: Modulation of cell growth after incubation of HT29 cells with fs obtained by fermentation using 41.6 g/l ox gall in intestinalextract. Shown are means SD (n=3) and significant differences between fs blank and fs inulin (§ p<0.05, §§§ p<0.001) and differencesof fs concentrations to medium control (* p<0.5, ** p<0.01, *** p<0.001)±±Figure 3: Modulation of cell growth after incubation of HT29 cells with fs obtained by fermentation using 0.6 g/l ox gall in intestinalextract. Shown are means SD (n=3) and significant differences between fs blank and fs inulin (§ p<0.05, §§ p<0.01, §§§ p<0.001) anddifferences of fs concentrations to medium control (* p<0.5, ** p<0.01, *** p<0.001).±Figure 6: Modulation of cell growth after incubation of HT29 cells with synthetic mixtures of DCA and CA of corresponding fs yielded by fermentation using41.6 or 0.6 g/l ox gall. Shown are means SD (n=3) and significant differences between two synthetic mixtures of corresponding fs (§ p<0.05, §§§ p<0.001)and differences of synthetic mixtures to medium control (** p<0.01, *** p<0.001).±Figure 4: Concentrations of CA and DCA in fs afterfermenation of inulin using 41.6 ox gall in intestinal extract.Acknowledgements: For funding we would like to thank the Ministry of Education and Research, Germany (0313829A).Use of 0.6 g/l ox gall resulted in considerable loweramounts of CA and DCA in fs. Inulin decreased bile acidconcentrations (Fig. 5).Using 41.6 g/l ox gall both fs inhibited cell growthwhereas fs blank was more cytotoxic (Fig. 2).CA DCA050100150200250blank (feces control)inulinconcentration[µM]MethodsFermentation supernatant (fs) of inulin was obtained by fermentation (Fig. 1). Bile acids,cholic (CA) and deoxycholic acid (DCA), were determined in fs by HPLC-MS/MS. Cell growthof HT29 cells treated with different concentrations of fs and corresponding synthetic mixturesof CA and DCA were determined by quantifying DAPI-labelled DNA.in vitroCA DCA05001000150020002500blank (feces control)inulinconcentration[µM]Figure 1: Process of fermentation.in vitroUsing 0.6 g/l ox gall fs blank was less cytotoxic whereas fsinulin was more effective in reducing cell growth (Fig. 3).Use of 41.6 g/l ox gall resulted in unphysiological highamounts of CA and DCA in fs. Inulin decreased bile acidconcentrations (Fig. 4).Figure 5: Concentrations of CA and DCA in fs afterfermenation of inulin using 0.6 ox gall in intestinal extract.Cytotoxicity of fs blank is partly due to highamounts of CA and DCA (Fig. 6).10 g/l fermentable sample +fermentation buffermouth simulation ( -amylase, 5 min)stomach simulation (pepsin, pH 2, 2 h)duodenum simulation(intestinal extract, pH 6.5, 6 h)including dialysiscolon simulation (pH 6.5, 24 h)feces inoculumEstablished Model (high ox gall) Modified Model (low ox gall)

Modification of an in vitro model simulating the whole digestive process to investigate cellular endpoints of carcinogenesis and chemoprevention

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Modification of an in vitro model simulating the whole digestive process to investigate cellular endpoints of carcinogenesis and chemoprevention

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