AEM Accepts, published online ahead of print on 6 April 2007 Appl. Environ. Microbiol. doi:10.1128/AEM.02091-06 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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Recombinant Saccharomyces cerevisiae expressing a model P450
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in the rat digestive environment: viability and bioconversion activity
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G. Garrait, J. F. Jarrige, S. Blanquet, E. Beyssac and M. Alric*.
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Université Clermont1, UFR Pharmacie, Centre de Recherche en Nutrition Humaine (CRNH)
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d'Auvergne, Institut Fédératif de Recherche (IFR) Santé-Auvergne, Equipe de Recherche
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Technologique "Conception, Ingénierie et Développement de l'Aliment et du Médicament"
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(ERT CIDAM), Clermont-Ferrand, F-63001, France
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* Corresponding author
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Prof. Monique ALRIC
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Mailing address: Equipe de Recherche Technologique "Conception, Ingénierie et
Développement de l'Aliment et du Médicament" (ERT CIDAM) 5ème étage, CBRV, Faculté de Pharmacie, Université d'Auvergne, 28, place Henri-Dunant
63001 CLERMONT-FERRAND FRANCE
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Email:
[email protected]
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Phone number: +33 / (0)4 73 17 79 52
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Fax number: +33 / (0)4 73 17 83 92
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ABSTRACT
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An innovative “biodrug” concept, based on the oral administration of living
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recombinant microorganisms, has recently emerged for the prevention or treatment of various
31
diseases. A engineered Saccharomyces cerevisiae strain expressing plant P450 73A1
32
(cinnamate-4-hyroxylase-CA4H activity) was used, and its survival and ability to convert
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trans-cinnamic acid (CIN) into p-coumaric acid (COU) were investigated in vivo. In rats, the
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recombinant yeast was resistant to gastric and small intestinal secretions but was more
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sensitive to the conditions found in the large intestine. After oral administration of yeast and
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CIN, the CA4H activity was shown in vivo, COU being found throughout the rat digestive
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tract and in its urine. The bioconversion reaction occurred very fast, most of the COU being
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produced within the first 5 minutes. The gastrointestinal sac technique demonstrated that the
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recombinant yeast was able to convert CIN into COU (conversion rate ranging from 2 to 5%) in all the organs of the rat digestive tract: stomach, duodenum, jejunum, ileum, cecum and colon. These results promise new opportunities for the development of drug delivery systems based on engineered yeasts catalysing a bioconversion reaction directly in the digestive tract.
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INTRODUCTION
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Recombinant DNA technology has been largely developed in microorganisms to
55
produce substantial quantities of pharmacologically active compounds (41). An example is
56
the production of several flavonoid compounds with therapeutic activities by reconstituting
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the phenylpropanoid pathway in microbes such as Escherichia coli (21, 49) and
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Saccharomyces cerevisiae - S. cerevisiae (22, 52).
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Recently, an innovative "biodrug" concept has emerged, based on the introduction by
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oral route of living recombinant microorganisms into the body for potential medical
61
applications (1, 4, 13). The "biodrug" is produced inside the digestive tract by recombinant
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cells that can either produce compounds of interest or perform bioconversions. For instance,
63
the recombinant microorganisms might produce active compounds such as hormones (11),
64 65 66 67 68
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enzymes (15, 39, 40), interleukin (8, 45) or antigens for the development of oral vaccines (12, 38, 53). For bioconversion, recombinant cells might be administered to carry out "biodetoxication" in the gut. The objective would be to increase the body's protection against environmental xenobiotics, particularly those borne by food (e.g. pesticides, procarcinogens or chemical additives [18]), by ingesting microorganisms expressing enzymes that play a
69
major role in the human detoxication system (e.g. phase I xenobiotic metabolizing enzymes
70
such as cytochrome P450 or phase II such as glutathione-S-transferase [3]). Recombinant
71
microorganisms could thus be used to prevent multifactorial diseases that have been linked to
72
anomalies in human detoxification processes. For example, a deficiency in glutathione-S-
73
transferase M1 has been associated with an increased susceptibility to different cancers,
74
endometriosis and chronic bronchitis (3). Another bioconversion application of recombinant
75
cells is their use to control the activation of pro-drug into drug, directly in the digestive tract
-3-
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(4). This is of interest when the drug, but not the pro-drug, is either toxic at high
77
concentrations or damaged by digestive secretions.
78
Both bacteria and yeasts have been suggested as potential hosts for this new "biodrug"
79
concept. Each microorganism offers several advantages and disadvantages with regard to its
80
metabolic activity in the digestive tract, the genetic construction or how the "biodrug" is
81
delivered. Recombinant bacteria, particularly lactic acid bacteria, have been mostly suggested
82
as potential hosts (13). However, yeasts can be advantageous over bacteria (4) especially
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when a eukaryotic environment is required for the functional expression of heterologous
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genes. In addition, yeasts are not sensitive to antibacterial agents, allowing concomitant
85
administration of the recombinant microorganisms and antibiotics. In our study, the yeast S.
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cerevisiae, already used in humans for probiotics (9), was chosen owing to its "generally
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recognized as safe" (GRAS) status, its eukaryotic status, its easy culture and its high level of
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resistance to digestive secretions (5).
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In the present study, a recombinant S. cerevisiae expressing, as a model, a cytochrome
P450 73A1 (cinnamate-4-hydroxylase activity or CA4H activity) of a plant (Helianthus tuberosus) and overexpressed yeast NADP-cytochrome P450 reductase (CPR) was used
(yeast strain WRP45073A1). The recombinant S. cerevisiae catalyses the bioconversion of trans-cinnamic acid (CIN) into p-coumaric acid (COU). Using this strain, the scientific
94
feasibility of the "biodrug" concept had been previously shown in vitro (5) in the TNO
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gastrointestinal tract model (TIM), a multicompartmental, dynamic, computer-controlled
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system that closely mimics in vivo gastric and intestinal human conditions. The next step in
97
development consists in the in vivo validation of the "biodrug" concept.
98
The present study was undertaken to evaluate the viability and CA4H activity of the
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yeast strain WRP45073A1 in the rat. The experiments were conducted using different
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complementary approaches: in the living rat, in the different digestive compartments of the
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sacrificed rat and in ex vivo gastrointestinal sacs.
102 103 104
Materials and Methods
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Chemicals. Diethyl ether, glucose, galactose, hydrochloric acid and all the chemicals
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(methanol, acetonitrile and acetic acid) used for high-performance liquid chromatography
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(HPLC) analysis were gradient grade and were purchased from Acros Organics (Morris Plain,
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NJ). Krebs Henseleit modified buffer, NaCl, trifluoroacetic acid and tryptophan were supplied
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by Sigma Chemical Co. (St Louis, MO). Disodium hydrogen citrate sesquihydrate buffer,
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trans-cinnamic acid (CIN) and p-coumaric acid (COU) were provided by Fluka Chemical
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Corp. (Ronkonkoma, NY). All the products for yeast culture media were from Difco (Le Pont de Claix, France).
Yeast strains. The S. cerevisiae strain (kindly provided by Denis Pompon, CNRS, Gif-
sur-Yvette, France) was derived from the haploid strain W303-1B (MATα; ade2-1; his3-11,-
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15; leu2-3,-112; ura3-1; Canr; cyr+). The strain was genetically engineered to overexpress
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yeast NADP-CPR and Helianthus tuberosus CA4H when grown in the presence of galactose
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(47). The PCR-amplified CA4H open reading frame was inserted into the yeast expression
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vector pYeDP60. This plasmid was based on the origin of replication of the yeast 2µ
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minicircle, URA3 and ADE2 selection markers, and an expression cassette composed of
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GAL10-CYC1 promoter and phosphoglycerate kinase terminator sequences. The resulting S.
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cerevisiae strain, called WRP45073A1, catalyzes the second step in the plant
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phenylpropanoid pathway (50), metabolizing CIN into COU.
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Another yeast strain with no CA4H gene in its plasmid, called WRpV60, was used in control experiments.
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Yeast culture conditions. The S. cerevisiae strain was precultured to stationary growth
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phase at 28°C in SGI broth (7 g/L of yeast nitrogen base without amino acids, 1 g/L of bacto
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casamino acids, 20 mg/L of tryptophan, and 20 g/L of glucose). Preculture was carried out in
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YPGE (10 g/L of yeast extract, 10 g/L of Bacto peptone, 5 g/L of glucose, and 3% [vol/vol]
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ethanol), and cells were grown in a shaking incubator (28°C, 220 rpm, 36 h). Induction was
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started by adding a 10% (vol/vol) aqueous solution of 200 g/L of galactose and continued for
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12 h (28°C, 220 rpm) until the cell density reached 108 cells/mL. The cells (10 mL) were then
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harvested (4°C, 3 minutes, 5000g) and resuspended in 2 mL of disodium hydrogen citrate
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sesquihydrate buffer (0.1 mol/L, pH 5.5) to obtain a final cell density of 109 cells/mL. The
cell suspension was used in vitro or orally administered to rats or introduced on the mucosal side of the gastrointestinal sacs.
In vitro toxicity experiments. To check potential toxicity of CIN towards the
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recombinant yeast, cells (yeast strain WRP45073A1) were added to 7, 70 or 700 µmol of CIN
143
(n = 3 for each tested concentration) in 2 mL of disodium hydrogen citrate sesquihydrate
144
buffer (0.1 mol/L, pH 5.5). Throughout the experiment, the cell suspensions were incubated at
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37°C in a 5 mL haemolysis tube. Samples were collected 5, 30 and 60 minutes after the
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beginning of the experiment. The survival rate of the recombinant S. cerevisiae was evaluated
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immediately by plating the samples onto SGI solid medium (yeast counts).
148
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Animals. Adult male Wistar rats (Elevage Dépré, St-Doulchard, France) weighing
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300 ± 20 g at the beginning of the experiment were used. They were housed for an
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acclimatization period of 6 days with free access to food (A04, lot 50803, UAR, Epinay-sur-
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Orge, France) and tap water. Animals were maintained at constant room temperature (22°C)
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and exposed to natural light. All care and handling of animals was approved by the
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Institutional Authority for Laboratory Animal Care. Before experiments rats were housed
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alone in metabolic cages for 3 days, and food was withheld on the fourth day.
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Living rats experiments. After a 24 h fasting period, rats received a single oral dose of
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109 recombinant S. cerevisiae with various amounts of CIN (0.23, 2.33 or 23.3 mmol/kg of
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body weight). The upper extreme of this dose range was the maximum dose tolerated without
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apparent discomfort to the animals (35). To ensure animal welfare, the volume administered
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by gavage did not exceed 10 mL/kg according to Diehl et al. (14). Hence in our experiments
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the rats, which weighed approximately 300 g, received 2 mL of disodium hydrogen citrate sesquihydrate buffer (0.1 mol/L, pH 5.5) containing 7, 70 or 700 µmol of CIN plus the recombinant yeasts (control strain WRpV60 or WRP45073A1). Thirty rats were used: 5 rats for each yeast strain and each dose of CIN. Urine samples were collected every 4 h for 24 h before oral administration and every
167
2 h for 8 h and every 4 h for 16 h after gavage. Urine samples were stored at -20°C until
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HPLC analysis.
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Sacrificed rat experiments. Animals (n = 45) were fasted for 24 h and received, by
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gavage, a single dose of cell (WRP45073A1 or WRpV60) suspension supplemented with
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70 µmol of CIN diluted in 2 mL of disodium hydrogen citrate sesquihydrate buffer
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(0.1 mol/L, pH 5.5). For each yeast strain, three rats were decapitated 5, 10, 30 minutes, and
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2, 4, 8 and 24 h after the oral administration. Also, three rats were sacrificed immediately
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after oral administration of 0.9% NaCl (2 mL) to determine the number of endogenous living
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yeasts (control experiments). After sacrifice, the blood samples were collected on heparin and
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immediately harvested (4°C, 10 minutes, 2500g), and the plasma was collected. The different
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parts of the digestive tract were quickly removed: the stomach, duodenum (between the
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pylorus and the ligament of Treitz), jejunum (divided in three equal-length parts: proximal,
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median and distal), ileum, cecum and colon. The mucosal fluid of each organ was collected
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on ice by gently scraping the luminal surface with a glass slide and diluted in 5 mL/g of 0.9%
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NaCl. Aliquots of 100 µL were then plated immediately onto SGI solid medium (see below
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yeast count) to evaluate the yeast survival rate (0, 30 minutes, 2, 4, 8 and 24 h after gavage).
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All the samples (mucosal fluids and plasma samples) were stored at -20°C until HPLC
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analysis to evaluate the amounts of CIN and COU.
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Ex vivo experiments. After a 24 h fasting period, animals (n = 10) were anesthetized
with an intraperitoneal injection of ketamine (Imalgene 1000, Merial, Lyon, France) at 0.15 g/kg body weight and with a subcutaneous injection of lidocaine (Xylocaine, AstraZaneca laboratory, Rueil-Malmaison, France) at 0.04 g/kg body weight. Different parts of the digestive tract were quickly removed as previously described (19). Briefly, the
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stomach, duodenum, mid-jejunum (10 cm), ileum, cecum and colon (10 cm beyond the
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cecum) were removed. The mucosal and serosal sides of each organ were washed, dried and
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weighed. One end was ligated. The recombinant yeasts (WRP45073A1 or WRpV60, n = 5 for
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each yeast strain) and 7 µmol of CIN suspended in 2 mL of disodium hydrogen citrate
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sesquihydrate buffer (0.1 mol/L, pH 5.5) were simultaneously introduced on the mucosal side
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through the other end. The second end was then ligated. Gastrointestinal sacs were
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immediately transferred to a tissue chamber containing 10 mL of warmed (37 °C), oxygenated
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(95% O2 / 5% CO2) Krebs Henseleit modified buffer (KHMB, composition in mmol/L: 118.1
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NaCl, 4.7 KCl, 2.2 CaCl2 2H2O, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11.1 glucose, pH 7.5).
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The chamber was closed with a rubber stopper to prevent evaporation. Gastrointestinal
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preparations were incubated for 180 minutes, and 300 µL samples were collected on the
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serosal side at 0, 15, 30, 60, 90, 120 and 180 minutes. The serosal fluid collections were
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stored at -20°C until HPLC analysis. At the end of the experiment, the gastrointestinal sacs
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were immersed in 5 mL of 0.9% NaCl for 1 minute to remove CIN and COU adsorbed on the
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serosal surfaces. They were then dried and weighed. The mucosal fluid was collected on ice
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and diluted in 5 mL/g of 0.9% NaCl. The survival rate of the recombinant S. cerevisiae was
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evaluated immediately by plating the samples onto SGI solid medium (yeast counts). The
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amounts of CIN and COU in the mucosal fluids and in the organ walls were measured after an
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extraction step. The samples were homogenized in 3 mL/g of 0.9% NaCl (Ultratturax,
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24000 rpm/minute, 5 minutes, on ice). Aliquots of 0.5 mL were acidified to pH 1-2 with
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100 µL of 1 mol/L HCl and extracted twice with 8 mL of diethyl ether. The mixture was agitated (Vortex, 2750 rpm, 5 minutes), centrifuged (5000g, 10 minutes, 4 °C) and finally frozen (20 minutes, -20 °C). The ether fraction was removed and dried in a speed-vac (depression 15 hPa, ramp 3, 35 minutes, 45 °C). The dried extracts were resuspended in 1 mL ethanol/diethyl ether (95/5, vol/vol) before HPLC analysis.
217 218
Yeast counts. The mucosal fluid samples (sacrificed rats and ex vivo experiments) and
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cell suspensions used in in vitro toxicity experiments were diluted in 0.9% NaCl and plated
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onto SGI solid medium supplemented with ampicillin (100 µg/mL). The results were
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expressed as number of cells or percentage of initial number of cells.
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Preparation and HPLC-diode array detection (DAD) analysis. CIN and COU were
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measured by HPLC as previously described by Blanquet et al. (5). The enzymatic reaction
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was stopped immediately after sampling by adding a solution of trifluoroacetic acid (2.5%
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wt/vol.). Before HPLC analysis all the samples were filtered (GHP membrane, 0.45 µm pore
227
size). Aliquots of 10 µL of filtrate were analyzed on a Lichrospher 100 RP-18 (5 µm) column
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(125 mm by 4 mm inside diameter; Merck, Darmstadt, Germany). Elution was performed
229
with a flow rate of 1 mL/minute and a gradient of two solvents, A and B, composed of water-
230
methanol-acetic
231
(94.9/5/0.1, vol/vol/vol), respectively. The HPLC analysis was started with 90% of solvent A
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and 10% of solvent B. After 16 minutes, solvent B was added, reaching 20% within
233
2 minutes. These conditions were maintained for 14 minutes, and initial conditions were then
234
restored within 2 minutes. CIN and COU were detected by UV absorbance at 280 and
235
314 nm, respectively, and quantified using standard curves.
236 237 238 239 240
acid
(94.9/5/0.1,
vol/vol/vol)
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and
acetonitrile-methanol-acetic
acid
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Statistical analysis. Values are presented as means ± SEM. Comparisons between
groups were performed using Student's t test. All statistical evaluations were performed on a computer using the SAS system program (Software Version 8.1. SAS Institute Inc., Cary, NC). The level of statistical significance was set at P < 0.05.
241 242 243
RESULTS
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Living rat experiments. No trace of CIN or COU was recovered in urine of rats
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collected before gavage. After the oral administration of the different amounts of CIN (7, 70
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or 700 µmol) and the recombinant yeast strains WRpV60 or WRP45073A1 (illustrated with
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WRP45073A1 and 70 µmol; Fig. 1), CIN was rapidly excreted and extensively detected in
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urine samples collected during the first 4 h and the excreted quantities over 24 h represented
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about 0.1% of the administered CIN (data not shown). Regardless of the dose levels used, no
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trace of COU was detected in 0-24 h urine after administration of CIN and WRpV60.
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Likewise, no urinary trace of COU was discovered following gavage with WRP45073A1 and
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7 or 700 µmol of CIN (data not shown). Conversely, when recombinant yeast strain
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WRP45073A1 and 70 µmol of CIN were simultaneously orally administered to fasted rats,
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COU was found in 0-2 h (4.5 nmol) and 2-4 h (1.2 nmol) urine collections (Fig. 1). The
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amount of COU recovered in 0-24 h urine represented 0,007% of the CIN orally administered
258
(data not shown).
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In vitro toxicity experiments. To account for the absence of COU in urine after the
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administration of high amounts of CIN with WRP45073A1, the toxicity of CIN towards the yeast was investigated. The cell viability was evaluated in vitro following incubation with various concentrations of CIN (Fig. 2). No significant mortality was observed after 5 to 60 minutes incubation with 7 µmol of CIN. In the presence of 70 µmol of CIN, the survival rate decreased significantly (P < 0.05) with time, approximating to 30% after 1 h incubation.
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The yeast viability fell dramatically when cells were incubated with 700 µmol of CIN: 15% of
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the initial yeast cells were recovered alive 5 minutes after the beginning of the experiment and
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100% mortality was observed after 30 minutes incubation.
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Sacrificed rat experiments. No yeast was detected after gavage with 0.9% NaCl
271
(Fig. 3). Thirty minutes after their administration, WRP45073A1 yeast cells were recovered
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(Fig. 3A) in the stomach (2×108 CFU), duodenum (3×106 CFU), proximal jejunum (1×108
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CFU), median jejunum (2×108 CFU), distal jejunum (9×108 CFU) and ileum (3×105 CFU). At
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30 minutes, 77 ± 10% of the total ingested yeasts were recovered alive (Fig 3B). Two hours
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after administration (Fig. 3A), the yeasts colonised the large intestine (cecum and colon) and
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their survival rate decreased to 33% (Fig. 3B). The percentages of cells recovered 4 and 8 h
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after gavage were not significantly different and represented approximately 15% of the total
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ingested yeasts. At the end of the experiment (24 h), the recombinant cells were only
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recovered in the feces (7×106 CFU) accounting for less than 1% of the yeast initially
280
administered. Similar survival rates were observed when cells were administered with
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70 µmol of CIN (Fig. 4): the percentages of living cells ranged from 50% (t = 30 minutes) to
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0.2% (t = 24 h).
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Within the first 30 minutes of the experiment, CIN was mainly found in the upper part
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of the digestive tract and in plasma (Table 1). The rate of CIN recovered ranged from 33.5%
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(t = 5 minutes) to 1% (t = 2 h) of the amount initially administered to rats. CIN had
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completely disappeared from the mucosal fluids 4 h after the beginning of the experiment. No trace of COU was recovered in various samples collected after gavage with CIN
alone or added to yeast strain WRpV60 (data not shown). When WRP45073A1 was orally administered with CIN (70 µmol), a CA4H activity was detected, since COU production was observed (Table 2) 5 minutes after gavage in the stomach (209 nmol), duodenum (94 nmol),
291
proximal jejunum (144 nmol), median jejunum (82 nmol) and weakly detected in the stomach
292
and duodenum 10 and 30 minutes following ingestion. COU was also recovered in the plasma
293
samples collected at 5, 10 and 30 minutes. COU was not detected in the distal jejunum, ileum,
294
cecum or colon, or 2, 4, 8 and 24 h after gavage. Five minutes after gavage, the total amount
295
of COU detected represented 0,76% of the CIN initially administered (Table 2).
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Ex vivo experiments. After a 180 minute incubation period and in all the organs
299
studied (stomach, duodenum, jejunum, ileum, cecum and colon), about 30% of the initial
300
yeast cells were recovered alive (data not shown). This survival rate of WRP45073A1 in the
301
gastrointestinal sacs was similar to that observed in vivo 2 and 4 h after gavage (survival rate
302
around 20%; Fig. 4).
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CIN is absorbed by all the digestive organs, since this acid was extensively recovered
304
on the serosal side (data not shown). CIN was more efficiently absorbed in vivo than ex vivo.
305
Only 8% of CIN was recovered on the mucosal side 30 minutes after its introduction (Table
306
1) in sacrificed rats, whereas the residual amount of CIN on the mucosal side reached 50% 30
307
minutes after its introduction ex vivo (data not shown).
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In control experiments (rats receiving CIN and WRpV60 yeast strain), no trace of
309
COU was detected in the various digestive contents (data not shown). In contrast, when
310
WRP45073A1 and 7 µmol of CIN were simultaneously introduced in the gastrointestinal
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sacs, COU was rapidly found in all the digestive organs (Fig. 5). COU was detected on the serosal side of each organ from 15 minutes of incubation (except for the stomach, where COU only appeared at 180 minutes) and increased slowly and regularly over time. The distribution of COU among the three compartments (mucosal side, organ wall and serosal side) is illustrated in Fig. 6. At the end of the experiment, most of the COU was recovered on the
316
mucosal side: the amounts ranged from 56 nmol (duodenum) to 236 nmol (stomach). In
317
comparison, the amounts of COU detected on the serosal side ranged from 4 nmol (stomach)
318
to 83 nmol (ileum) and in organ wall from 9 nmol (ileum) to 89 nmol (stomach). The total
319
amount of COU detected in the three compartments of each organ represented from 2%
320
(duodenum) to 5% (stomach) of the CIN initially administered (data not shown).
321 322
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323
DISCUSSION
324 325 326
The development of new drug delivery systems based on the oral administration of
327
recombinant living microorganisms active in the human digestive environment has recently
328
been considered (1, 4). To validate the "biodrug" concept for a bioconversion application, a
329
genetically engineered yeast catalyzing the 4-hydroxylation of CIN into COU was used. The
330
survival rate and bioconversion activity of the recombinant yeast had been previously studied
331
in vitro (5) in gastrointestinal tract models. A first system named TIM1 reproduces the
332
stomach and small intestine (31), while the large intestine model is called TIM2 (32). In these
333
artificial digestive systems the main parameters of digestion (temperature, peristaltic mixing,
334
transit time, gastrointestinal secretions, pH and microflora activity) are reproduced as
335
faithfully as possible and the digestive systems have been validated by many studies (29, 30,
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The next step in the development of the "biodrug" concept consists in its validation in
vivo in the rodent. The aim of this study was to evaluate the viability and heterologous CA4H
activity of the recombinant yeast in the rat digestive environment.
341
Bioconversion activity of recombinant yeast in living rats. To follow the yeast's
342
bioconversion activity in the living animals, recombinant yeasts (control strain WRpV60 or
343
WRP45073A1) and various doses (7, 70 or 700 µmol) of CIN were orally administered to
344
rats. It was recently shown (19) that orally administered CIN and COU rapidly travel from the
345
gastrointestinal tract of the rats to the bloodstream and are then partially eliminated by the
346
kidney and recovered in urine. Therefore, after gavage with CIN and recombinant yeasts the
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347
CA4H activity inside the digestive tract should be revealed by the apparition of COU in the
348
urine.
349
COU was detected in the 0-24 h urine of rats (Fig. 1) following gavage, only with 70
350
µmol of CIN and the yeast strain WRP45073A1. The absence of COU in the urine of rats
351
given WRpV60 and CIN demonstrated the specificity of the enzyme reaction. When the
352
lowest dose of CIN (7 µmol) was ingested, the CA4H activity could not be clearly
353
demonstrated since the COU excreted in urine was too low to be detected. The absence of
354
activity in the presence of 700 µmol of CIN could be due to the potential antimicrobial
355
activity of CIN, which has been shown to possess antibacterial (36, 42) and antifungal (46)
356
activities. To check for potential toxicity of CIN towards WRP45073A1, cells were incubated
357
with the different amounts of acid tested in living rats. CIN was non-toxic at 7 µmol,
358
moderately toxic at 70 µmol and lethal at 700 µmol (Fig. 2). These data led to the conclusion
359
that the lack of bioconversion activity observed in rats receiving 700 µmol of CIN was due to
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extensive yeast death in the digestive tract. Therefore all subsequent in vivo experiments (sacrificed rats) were performed using 70 µmol of CIN. For the first time, the ability of recombinant yeast to exert CA4H activity has been
shown in the rat digestive environment, since COU was detected in urine, representing 0.007% of the ingested CIN. This in vivo CA4H activity was very weak compared with that
365
previously observed in the artificial digestive system TIM-1 in which about 41% of the initial
366
CIN was converted into COU after 4 h digestion (5). This large difference between in vivo
367
and in vitro results may be explained by higher yeast mortality in the rat digestive
368
environment than in the gastrointestinal system. To test this hypothesis, the survival rate of
369
orally administered recombinant yeast in the rat digestive tract was studied.
370
- 15 -
371
Viability of recombinant yeasts. The recombinant yeasts showed a high survival rate
372
in the upper part of the digestive tract. As previously demonstrated for S. boulardii (6),
373
WRP45073A1 are resistant to gastric environmental conditions. Also, the yeasts leaving the
374
stomach alive seem to resist pancreatic and hepatic secretions, as their survival rate was not
375
modified from duodenum to ileum. When yeasts reached the large intestine, their survival rate
376
decreased strongly (less than 1% 24 h after ingestion; Fig. 3B). Our in vivo results are
377
consistent with those obtained in vitro (5) in gastrointestinal tract models (TIM1 and TIM2).
D E
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378
Few studies evaluating the viability of Saccharomyces spp. throughout the length of
379
the gastrointestinal tract of rat or humans are available. A similar distribution to ours has been
380
previously shown in rats 30 minutes after the oral administration of 109 S. boulardii (2). Yeast
381
viability is mainly evaluated in feces. In the present study, the survival rate observed in feces
382
is consistent with that obtained in rat (7) and healthy humans (23, 37). Several biochemical
383
processes can explain the high mortality of yeasts in the large intestine. Some studies (17, 28,
384 385 386 387 388
E C
C A
33) have established the prevalence of polysaccharides (glucans and mannan) among the components of the cell wall of yeasts. These polysaccharides can be hydrolyzed by degrading enzymes present in the large intestine. For instance, Salyers et al. (43) have demonstrated the production of β 1–3 glucanases by Bacteroides, the most prevalent genus of intestinal bacteria in humans (51). Also, a "barrier" role of the endogenous colonic microflora towards orally
389
administered S. boulardi, has been demonstrated by Ducluzeau et al. (16). This effect induces
390
an extensive elimination of yeasts from the digestive tract.
391
The high viability of WRP45073A1 in the rat upper digestive tract encouraged us to
392
further investigate the potential efficiency of the recombinant yeasts to perform, in vivo, the
393
model reaction of bioconversion. Since the search for appearance of COU in urine is limited
394
by high losses between production and excretion of COU in living rats (absorption, body
395
distribution, metabolism, renal elimination), two other techniques were devised to determine
- 16 -
396
whether recombinant yeasts were able to convert CIN into COU in the rat digestive tract: (i)
397
in situ detection of COU (sacrificed rats) after oral administration of yeasts and CIN and (ii)
398
ex vivo experiments. The former technique allows direct detection of COU potentially
399
produced in situ. The latter is a useful screening tool for studying the digestive absorption and
400
metabolic behaviour of substances introduced in the various parts of the gastrointestinal tract
401
(10). In addition, this last technique allows the CA4H activity of recombinant yeasts to be
402
tested using the lowest non-toxic dose of CIN (7 µmol).
D E
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403
Whatever the time after gavage, the in situ survival rates of WRP45073A1 were
404
similar after their oral administration alone or with 70 µmol of CIN. The moderate antifungal
405
activity of the 70 µmol of CIN observed in vitro was not found in sacrificed rats, probably
406
owing to the rapid and extensive absorption of CIN by the rat digestive tract. The yeast
407
viability in the gastrointestinal sacs did not differ from that observed in sacrificed rats.
408 409 410 411 412 413
E C
C A
CIN absorption. In sacrificed rats, CIN was rapidly absorbed by the digestive tract
and, as previously reported (19), the gastrointestinal sac technique shows that all the digestive organs absorb CIN. It is known that CIN is partially transported across either the various digestive organs (19) or Caco-2 cell monolayers (25) by a carrier-mediated transport process. The present study demonstrates a faster and more extensive absorption of CIN in sacrificed
414
rats than that found in gastrointestinal sacs. Several hypotheses can explain this difference of
415
absorption between in situ and ex vivo experiments. First, after its intestinal absorption in
416
vivo, CIN passes into the bloodstream and is mainly converted into hippuric acid before its
417
elimination in urine (35). Thus the disappearance of CIN from the systemic circulation favors
418
its absorption across the digestive epithelium. Second, the area of the rat digestive wall is
419
higher in vivo than in gastrointestinal sacs. For instance, the length of the jejunum is up to 1 m
420
in rats (20) whereas it measures only 10 cm ex vivo.
- 17 -
421 422
Bioconversion activity. (i) In sacrificed rats. Following its ingestion, the
423
gastrointestinal absorption of CIN (70 µmol) induced a rapid disappearance of CIN in the
424
immediate vicinity of the recombinant yeasts. Consecutively to the rapid absorption of their
425
substrate (CIN), recombinant yeasts were unable to exert a very high CA4H activity
426
throughout the rat digestive tract. Nevertheless, the bioconversion reaction occurred very fast,
427
most of the COU being produced within the first 5 minutes. COU was detected only in the
428
upper digestive part and the amounts of COU recovered in sacrificed rats were 100 times
429
higher than those excreted in the urine of living rats. This result may account for the low
430
losses of in situ detected COU compared with those observed in living rats.
D E
T P
E C
431 432
(ii) In ex vivo experiments. A CA4H activity was detected in the gastrointestinal sacs.
433
COU was produced inside the lumen, and then absorbed and recovered on the serosal side
434 435 436 437 438
C A
where its amount regularly increased with time (Fig. 5). It is known that COU is absorbed according to passive and active transport, across either Caco-2 cell monolayers (25, 26) or the intestinal digestive epithelium of rats (19, 27). The CA4H activity observed ex vivo was some 7 times higher than that detected in sacrificed rats. This difference is certainly a consequence of a faster and more extensive absorption of CIN in sacrificed rats than that observed ex vivo.
439
As a consequence, CIN remains in the immediate vicinity of the yeasts for a longer time on
440
the mucosal side of gastrointestinal sacs than in sacrificed rats, explaining the higher CA4H
441
activity detected ex vivo. A closely similar effect was observed in the artificial gastrointestinal
442
tract model TIM1, in which CIN was not absorbed via a monocarboxylic acid transporter but
443
exclusively absorbed from the jejunum via a passive diffusion (5). The yeasts were therefore
444
in close contact with CIN for a long time, enhancing their CA4H activity. In our ex vivo
445
study, a higher activity of bioconversion was observed in the cecum and colon (CIN
- 18 -
446
conversion of around 2%), than that reported in the artificial large intestinal model TIM2
447
which was too weak to be quantified (5). The reason is that in TIM2, the recombinant yeasts
448
were rapidly deprived of CIN due to its metabolization by the colonic microflora (5) whereas
449
most of the rat endogenous microflora was probably removed from the mucosal side of the
450
gastrointestinal sacs by rinsing during their preparation.
D E
451 452
In conclusion, these new results indicate that after oral administration to rats, the
453
recombinant yeasts WRP45073A1 were resistant to gastric, hepatic, pancreatic and small
454
intestinal secretions, but were more sensitive to the conditions of the large intestine. For the
455
first time it is shown that the recombinant yeasts are able to exert a CA4H activity directly in
456
the rat digestive tract. In sacrificed rats, the reaction of bioconversion was very fast, with most
457
of the COU produced within the first 5 minutes, and was only detected in the upper digestive
458
tract. Using the gastrointestinal sac technique, a CA4H activity was found in all the organs of
459 460 461 462 463
T P
E C
C A
the digestive tract: stomach, duodenum, jejunum, ileum, cecum and colon. Compared with results previously obtained in the gastrointestinal tract model TIM1, the bioconversion activity of WRP45073A1 observed in sacrificed rats and in ex vivo experiments was very low
(CIN conversion rate of 41% in vitro vs 0.7% in sacrificed rats and 5% ex vivo). An extensive absorption of CIN by the digestive tract was shown in the rat. Consequently, the recombinant
464
yeasts were quickly deprived of their substrate, probably explaining their weak CA4H activity
465
in vivo. The recombinant yeast used to validate the "biodrug" concept for bioconversion is
466
less well tailored for in vivo than for in vitro experiments, owing to the extensive and rapid
467
absorption of CIN by the rat gastrointestinal tract. However, the results obtained in this study
468
do not challenge the "biodrug" concept, since a high yeast survival rate and a CA4H activity
469
were still observed in the rat digestive environment. These in vivo results support the
470
possibility of using genetically modified S. cerevisiae as a potential host for the development
- 19 -
471
of "biodrugs", in particular to perform biodetoxication by metabolizing xenobiotics that are
472
poorly or slowly absorbed by the human digestive tract. Soon, once the therapeutic target has
473
been identified, model genes will be replaced by candidate genes. Of course, heterologous
474
gene expression strategies will have to be adapted for safe use in humans, as the presence of
475
mobilisable vectors, antibiotic selection markers and bacterial sequences liable to promote
476
gene transfer to host microflora are prohibited. In addition, environmental confinement of
477
recombinant cells (24, 34) will have to be achieved by introducing a suicide process (e.g.
478
activation of a toxic protein or repression of an essential gene) that triggers the destruction of
479
the yeast as soon as it leaves the human digestive tract.
480
483 484 485 486 487 488
T P
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481 482
D E
ABBREVIATIONS USED
C A
CIN, trans-cinnamic acid; COU, p-coumaric acid; MCT, Monocarboxylic acid
transporter; CA4H, cinnamate-4-hydroxylase; CPR, cytochrome P450 reductase; CFU, colony-forming unit.
489 490
ACKNOWLEDGMENTS
491 492 493
This work was supported by Délégation Générale pour l'Armement (D.G.A.).
494
The authors gratefully acknowledge the skilful technical assistance of Astrid Vega,
495
Marianne Collange, Aïssa Sangare, Sabrina Maquaire and Angélique Gardes.
- 20 -
496 497 498
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655 656 657 658 659
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TABLE AND FIGURE LEGENDS
660 661 662
Table 1 In situ distribution of CIN (70 µmol) after simultaneous oral administration
663
with WRP45073A1 yeast strain in sacrificed rats. Values represent the mean amounts ±
664
SEM (n = 3 for each time of sacrifice) in micromoles of CIN recovered in the different organs
665
of the digestive tract and in plasma. n.d.: non-detected.
666
- 27 -
667
Table 2 In situ CA4H activity of WRP45073A1 yeast strain after simultaneous oral
668
administration with 70 µmol of CIN in sacrificed rats. Values represent the mean amounts
669
± SEM (n = 3 for each time of sacrifice) in nanomoles of COU produced by the yeasts and
670
recovered in the different organs of the digestive tract and in plasma. n.d.: non-detected.
671
D E
672
Figure 1 CA4H activity of WRP45073A1 yeast strain following simultaneous oral
673
administration with 70 µmol of CIN in living rats. Values represent the mean amounts ±
674
SEM (n = 5) in nanomoles of CIN and COU recovered in urine samples.
T P
675
In vitro toxicity of CIN towards WRP45073A1 yeast strain. Values are
676
Figure 2
677
expressed as mean percentages ± SEM (n = 3) of initial amount of yeasts. Asterisks denote
678
values significantly different from the yeast count at the beginning of the experiment (P
