Recombinant Saccharomyces cerevisiae Strain Expressing a Model ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2007, p. 3566–3574 0099-2240/07/$08.00⫹0 doi:10.1128/AEM.02091-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 73, No. 11

Recombinant Saccharomyces cerevisiae Strain Expressing a Model Cytochrome P450 in the Rat Digestive Environment: Viability and Bioconversion Activity䌤 G. Garrait, J. F. Jarrige, S. Blanquet, E. Beyssac, and M. Alric* Universite´ Clermont1, UFR Pharmacie, Centre de Recherche en Nutrition Humaine d’Auvergne, Institut Fe´de´ratif de Recherche Sante´-Auvergne, Equipe de Recherche Technologique Conception, Ingeńierie et De´veloppement de l’Aliment et du Me´dicament, F-63001 Clermont-Ferrand, France Received 5 September 2006/Accepted 2 April 2007

An innovative “biodrug” concept, based on the oral administration of living recombinant microorganisms, has recently emerged for the prevention or treatment of various diseases. An engineered Saccharomyces cerevisiae strain expressing plant P450 73A1 (cinnamate-4-hydroxylase [CA4H] activity) was used, and its survival and ability to convert trans-cinnamic acid (CIN) into p-coumaric acid (COU) were investigated in vivo. In rats, the recombinant yeast was resistant to gastric and small intestinal secretions but was more sensitive to the conditions found in the large intestine. After oral administration of yeast and CIN, the CA4H activity was shown in vivo, with COU being found throughout the rat’s digestive tract and in its urine. The bioconversion reaction occurred very fast, with most of the COU being produced within the first 5 min. The gastrointestinal sac technique demonstrated that the recombinant yeast was able to convert CIN into COU (conversion rate ranging from 2 to 5%) in all the organs of the rat’s 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 catalyzing a bioconversion reaction directly in the digestive tract. ganisms could thus be used to prevent multifactorial diseases that have been linked to anomalies in human detoxification processes. For example, a deficiency in glutathione S-transferase M1 has been associated with an increased susceptibility to different cancers, endometriosis, and chronic bronchitis (3). Another bioconversion application of recombinant cells is their use for controlling the activation of prodrug into drug directly in the digestive tract (4). This is of interest when the drug, but not the prodrug, is either toxic at high concentrations or damaged by digestive secretions. Both bacteria and yeasts have been suggested as potential hosts for this new biodrug concept. Each microorganism offers several advantages and disadvantages with regard to its metabolic activity in the digestive tract, the genetic construction, or how the biodrug is delivered. Recombinant bacteria, particularly lactic acid bacteria, have mostly been suggested as potential hosts (13). However, yeasts can be advantageous over bacteria (4), especially when a eukaryotic environment is required for the functional expression of heterologous genes. In addition, yeasts are not sensitive to antibacterial agents, allowing concomitant administration of the recombinant microorganisms and antibiotics. In our study, the yeast S. cerevisiae, already used in humans for probiotics (9), was chosen owing to its “generally recognized as safe” status, its eukaryotic status, its easy culture, and its high level of resistance to digestive secretions (5). In the present study, a recombinant S. cerevisiae strain expressing, as a model, a cytochrome P450 73A1 (cinnamate-4hydroxylase [CA4H] activity) of a plant (Helianthus tuberosus) and overexpressed yeast NADP-cytochrome P450 reductase was used (yeast strain WRP45073A1). The recombinant S. cerevisiae strain catalyzes the bioconversion of trans-cinnamic acid (CIN) into p-coumaric acid (COU). Using this strain, the

Recombinant DNA technology has been largely developed in microorganisms to produce substantial quantities of pharmacologically active compounds (41). An example is the production of several flavonoid compounds with therapeutic activities by reconstituting the phenylpropanoid pathway in microbes such as Escherichia coli (21, 49) and Saccharomyces cerevisiae (22, 52). Recently, an innovative “biodrug” concept has emerged based on the introduction of living recombinant microorganisms into the body by oral route for potential medical applications (2, 4, 13). The biodrug is produced inside the digestive tract by recombinant cells that can either produce compounds of interest or perform bioconversions. For instance, the recombinant microorganisms might produce active compounds, such as hormones (11), enzymes (15, 39, 40), interleukin (8, 45), and 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, and chemical additives [18]), by ingesting microorganisms expressing enzymes that play a major role in the human detoxication system (e.g., phase I xenobiotic metabolizing enzymes, such as cytochrome P450, or phase II enzymes, such as glutathione S-transferase [3]). Recombinant microor-

* Corresponding author. Mailing address: Equipe de Recherche Technologique Conception, Ingeńierie et De´veloppement de l’Aliment et du Me´dicament (ERT CIDAM), 5 ème Etage, CBRV, Faculte´ de Pharmacie, Universite´ d’Auvergne, 28 Place Henri-Dunant, 63001 Clermont-Ferrand, France. Phone: 33 473 17 79 52. Fax: 33 473 17 83 92. E-mail: [email protected]. 䌤 Published ahead of print on 6 April 2007. 3566

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VIABILITY AND CA4H ACTIVITY OF WRP45073A1 IN RATS

scientific feasibility of the biodrug concept has previously been shown in vitro (5) in the TNO gastrointestinal tract model (TIM), a multicompartmental, dynamic, computer-controlled system that closely mimics in vivo gastric and intestinal human conditions. The next step in development consists of the in vivo validation of the biodrug concept. The present study was undertaken to evaluate the viability and CA4H activity of the yeast strain WRP45073A1 in the rat. The experiments were conducted using different complementary approaches in the living rat, in the different digestive compartments of the sacrificed rat, and in ex vivo gastrointestinal sacs. MATERIALS AND METHODS Chemicals. Diethyl ether, glucose, galactose, hydrochloric acid, and all the chemicals (methanol, acetonitrile, and acetic acid) used for high-performance liquid chromatography (HPLC) analysis were gradient grade and were purchased from Acros Organics (Morris Plain, NJ). Krebs-Henseleit modified buffer, NaCl, trifluoroacetic acid, and tryptophan were supplied by Sigma Chemical Co. (St. Louis, MO). Disodium hydrogen citrate sesquihydrate buffer, CIN, and COU were provided by Fluka Chemical Corp. (Ronkonkoma, NY). All the products for yeast culture media were obtained 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 (MAT␣ ade2-1 his3-11,15 leu2-3,112 ura3-1; Canr; cyr⫹). The strain was genetically engineered to overexpress yeast NADP-cytochrome P450 reductase and Helianthus tuberosus CA4H when grown in the presence of galactose (47). The PCR-amplified CA4H open reading frame was inserted into the yeast expression vector pYeDP60. This plasmid was based on the origin of replication of the yeast 2-␮m minicircle, URA3 and ADE2 selection markers, and an expression cassette composed of the GAL10-CYC1 promoter and phosphoglycerate kinase terminator sequences. The resulting S. cerevisiae strain, called WRP45073A1, catalyzes the second step in the plant phenylpropanoid pathway (50), metabolizing CIN into COU. Another yeast strain with no CA4H gene in its plasmid, called WRpV60, was used in control experiments. Yeast culture conditions. The S. cerevisiae strain was precultured to stationary growth phase at 28°C in SGI broth (7 g/liter of yeast nitrogen base without amino acids, 1 g/liter of Bacto Casamino Acids, 20 mg/liter of tryptophan, and 20 g/liter of glucose). Preculture was carried out in YPGE (10 g/liter of yeast extract, 10 g/liter of Bacto peptone, 5 g/liter of glucose, and 3% [vol/vol] ethanol), and cells were grown in a shaking incubator (28°C, 220 rpm, 36 h). Induction was started by adding a 10% (vol/vol) aqueous solution of 200 g/liter of galactose and continued for 12 h (28°C, 220 rpm) until the cell density reached 108 cells/ml. The cells (10 ml) were then harvested (4°C, 3 min, 5,000 ⫻ g) and resuspended in 2 ml of disodium hydrogen citrate sesquihydrate buffer (0.1 mol/liter, pH 5.5) to obtain a final cell density of 109 cells/ml. The cell suspension was used in vitro, orally administered to rats, or introduced on the mucosal side of the gastrointestinal sacs. In vitro toxicity experiments. To check the potential toxicity of CIN towards the recombinant yeast strain, cells (yeast strain WRP45073A1) were added to 7, 70, or 700 ␮mol of CIN (n ⫽ 3 for each tested concentration) in 2 ml of disodium hydrogen citrate sesquihydrate buffer (0.1 mol/liter, pH 5.5). Throughout the experiment, the cell suspensions were incubated at 37°C in a 5-ml hemolysis tube. Samples were collected 5, 30, and 60 min after the beginning of the experiment. The survival rate of the recombinant S. cerevisiae strain was evaluated immediately by plating the samples onto SGI solid medium (yeast counts). Animals. Adult male Wistar rats (Elevage De´pre´, St. Doulchard, France) weighing 300 ⫾ 20 g at the beginning of the experiment were used. They were housed for an acclimatization period of 6 days with free access to food (A04, lot 50803; UAR, Epinay-sur-Orge, France) and tap water. Animals were maintained at constant room temperature (22°C) and exposed to natural light. All care and handling of animals were approved by the Institutional Authority for Laboratory Animal Care. Before the experiments, rats were housed alone in metabolic cages for 3 days, and food was withheld on the fourth day. Living rat experiments. After a 24-h fasting period, rats received a single oral dose of 109 recombinant S. cerevisiae cells with various amounts of CIN (0.23, 2.33, or 23.3 mmol/kg of body weight). The upper extreme of this dose range was the maximum dose tolerated without apparent discomfort to the animals (35). To

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ensure animal welfare, the volume administered by gavage did not exceed 10 ml/kg according to Diehl et al. (14). Hence, in our experiments, the rats, which weighed approximately 300 g, received 2 ml of disodium hydrogen citrate sesquihydrate buffer (0.1 mol/liter, pH 5.5) containing 7, 70, or 700 ␮mol of CIN plus the recombinant yeast strain (control strain WRpV60 or WRP45073A1). Thirty rats were used: five 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 2 h for 8 h and every 4 h for 16 h after gavage. Urine samples were stored at ⫺20°C until HPLC analysis. Sacrificed rat experiments. Animals (n ⫽ 45) were fasted for 24 h and received, by gavage, a single dose of cell (WRP45073A1 or WRpV60) suspension supplemented with 70 ␮mol of CIN diluted in 2 ml of disodium hydrogen citrate sesquihydrate buffer (0.1 mol/liter, pH 5.5). For each yeast strain, three rats were decapitated 5, 10, and 30 min and 2, 4, 8, and 24 h after oral administration. Also, three rats were sacrificed immediately after oral administration of 0.9% NaCl (2 ml) to determine the number of endogenous living yeasts (control experiments). After sacrifice, the blood samples were collected on heparin and immediately harvested (4°C, 10 min, 2,500 ⫻ g), and the plasma was collected. The different parts of the digestive tract, the stomach, duodenum (between the pylorus and the ligament of Treitz), jejunum (divided into three equal-length parts: proximal, median, and distal), ileum, cecum, and colon, were quickly removed. The mucosal fluid of each organ was collected on ice by gently scraping the luminal surface with a glass slide and diluted in 5 ml/g of 0.9% NaCl. Aliquots of 100 ␮l were then plated immediately onto SGI solid medium (see “Yeast counts” below) to evaluate the yeast survival rate (0 and 30 min and 2, 4, 8, and 24 h after gavage). All the samples (mucosal fluids and plasma samples) were stored at ⫺20°C until HPLC analysis to evaluate the amounts of CIN and COU. 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; AstraZeneca 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 stomach, duodenum, mid-jejunum (10 cm), ileum, cecum, and colon (10 cm beyond the cecum) were removed. The mucosal and serosal sides of each organ were washed, dried, and weighed. One end was ligated. The recombinant yeast strain (WRP45073A1 or WRpV60; n ⫽ 5 for each yeast strain) and 7 ␮mol of CIN suspended in 2 ml of disodium hydrogen citrate sesquihydrate buffer (0.1 mol/liter, pH 5.5) were simultaneously introduced on the mucosal side through the other end. The second end was then ligated. Gastrointestinal sacs were immediately transferred to a tissue chamber containing 10 ml of warmed (37°C), oxygenated (95% O2-5% CO2) KrebsHenseleit modified buffer (composition in mmol/liter: 118.1 NaCl, 4.7 KCl, 2.2 CaCl2 䡠 2H2O, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11.1 glucose, pH 7.5). The chamber was closed with a rubber stopper to prevent evaporation. Gastrointestinal preparations were incubated for 180 min, and 300-␮l samples were collected on the serosal side at 0, 15, 30, 60, 90, 120, and 180 min. The serosal fluid collections were stored at ⫺20°C until HPLC analysis. At the end of the experiment, the gastrointestinal sacs were immersed in 5 ml of 0.9% NaCl for 1 min to remove the CIN and COU adsorbed on the serosal surfaces. They were then dried and weighed. The mucosal fluid was collected on ice and diluted in 5 ml/g of 0.9% NaCl. The survival rate of the recombinant S. cerevisiae strain was evaluated immediately by plating the samples onto SGI solid medium (yeast counts). The amounts of CIN and COU in the mucosal fluids and in the organ walls were measured after an extraction step. The samples were homogenized in 3 ml/g of 0.9% NaCl (Ultraturax; 24,000 rpm/minute for 5 min on ice). Aliquots of 0.5 ml were acidified to pH 1 to 2 with 100 ␮l of 1 mol/liter HCl and extracted twice with 8 ml of diethyl ether. The mixture was agitated (Vortex; 2,750 rpm, 5 min), centrifuged (5,000 ⫻ g, 10 min, 4°C), and finally frozen (20 min, ⫺20°C). The ether fraction was removed and dried in a Speed Vac (depression, 15 hPa; ramp 3; 35 min; 45°C). The dried extracts were resuspended in 1 ml ethanoldiethyl ether (95:5, vol/vol) before HPLC analysis. Yeast counts. The mucosal fluid samples (sacrificed rats and ex vivo experiments) and cell suspensions used in in vitro toxicity experiments were diluted in 0.9% NaCl and plated onto SGI solid medium supplemented with ampicillin (100 ␮g/ml). The results were expressed as the number of cells or the percentage of the initial number of cells. Preparation and HPLC diode array detection analysis. CIN and COU were measured by HPLC as previously described by Blanquet et al. (5). The enzymatic reaction was stopped immediately after sampling by adding a solution of trifluoroacetic acid (2.5%, wt/vol). Before HPLC analysis, all the samples were filtered (hydrophilic polypropylene membrane, 0.45-␮m pore size). Aliquots of 10 ␮l of filtrate were analyzed on a Lichrospher 100 RP-18 (5 ␮m) column (125-mm by 4-mm inside diameter; Merck, Darmstadt, Germany). Elution was performed at

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APPL. ENVIRON. MICROBIOL.

FIG. 1. CA4H activity of WRP45073A1 yeast strain following simultaneous oral administration with 70 ␮mol of CIN in living rats. Values represent the mean amounts ⫾ SEM (n ⫽ 5) in nanomoles of CIN and COU recovered in urine samples.

a flow rate of 1 ml/minute and with a gradient of two solvents, A and B, composed of water-methanol-acetic acid (94.9:5:0.1, vol/vol/vol) and acetonitrilemethanol-acetic acid (94.9:5:0.1, vol/vol/vol), respectively. The HPLC analysis was started with 90% of solvent A and 10% of solvent B. After 16 min, solvent B was added, reaching 20% within 2 min. These conditions were maintained for 14 min, and the initial conditions were then restored within 2 min. CIN and COU were detected by UV absorbance at 280 and 314 nm, respectively, and quantified using standard curves. Statistical analysis. Values are presented as means ⫾ standard errors of the 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 a P value of ⬍0.05.

RESULTS Living rat experiments. No trace of CIN or COU was recovered in the urine samples of rats collected before gavage. After oral administration of the different amounts of CIN (7, 70, and 700 ␮mol) and the recombinant yeast strain WRpV60 or WRP45073A1 (Fig. 1), CIN was rapidly excreted and extensively detected in urine samples collected during the first 4 h and the excreted quantities over 24 h represented about 0.1% of the administered CIN (data not shown). Regardless of the dose levels used, no trace of COU was detected in urine collected 0 to 24 h after the administration of CIN and WRpV60. Likewise, no urinary trace of COU was discovered following gavage with WRP45073A1 and 7 or 700 ␮mol of CIN (data not shown). Conversely, when recombinant yeast strain WRP45073A1 and 70 ␮mol of CIN were simultaneously orally administered to fasted rats, COU was found in urine samples collected after 0 to 2 h (4.5 nmol) and 2 to 4 h (1.2 nmol) (Fig. 1). The amount of COU recovered in urine samples collected after 0 to 24 h represented 0.007% of the CIN orally administered (data not shown). In vitro toxicity experiments. To account for the absence of COU in urine after the administration of large 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 min of incubation with 7 ␮mol of CIN. In the presence of 70 ␮mol of CIN,

FIG. 2. In vitro toxicity of CIN towards WRP45073A1 yeast strain. Values are expressed as mean percentages ⫾ SEM (n ⫽ 3) relative to the initial amount of yeasts. Asterisks denote values significantly different from the yeast count at the beginning of the experiment (P ⬍ 0.05).

the survival rate decreased significantly (P ⬍ 0.05) with time, approximating to 30% after 1 h of incubation. Yeast viability fell dramatically when cells were incubated with 700 ␮mol of CIN: 15% of the initial yeast cells were recovered alive 5 min after the beginning of the experiment and 100% mortality was observed after 30 min of incubation. Sacrificed rat experiments. No yeast was detected after gavage with 0.9% NaCl (Fig. 3). Thirty minutes after their administration, WRP45073A1 yeast cells were recovered (Fig. 3A) in the stomach (2 ⫻ 108 CFU), duodenum (3 ⫻ 106 CFU), proximal jejunum (1 ⫻ 108 CFU), median jejunum (2 ⫻ 108 CFU), distal jejunum (9 ⫻ 108 CFU), and ileum (3 ⫻ 105 CFU). At 30 min, 77% ⫾ 10% of the total ingested yeasts were recovered alive (Fig. 3B). Two hours after administration (Fig. 3A), the yeasts colonized the large intestine (cecum and colon) and their survival rate decreased to 33% (Fig. 3B). The percentages of cells recovered 4 and 8 h after gavage were not significantly different and represented approximately 15% of the total ingested yeasts. At the end of the experiment (24 h), the recombinant cells were recovered only in the feces (7 ⫻ 106 CFU), accounting for less than 1% of the yeast initially administered. Similar survival rates were observed when cells were administered with 70 ␮mol of CIN (Fig. 4): the percentages of living cells ranged from 50% (time [t] ⫽ 30 min) to 0.2% (t ⫽ 24 h). Within the first 30 min of the experiment, CIN was found mainly in the upper part of the digestive tract and in plasma (Table 1). The rate of CIN recovered ranged from 33.5% (t ⫽ 5 min) to 1% (t ⫽ 2 h) of the amount initially administered to rats. CIN had 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 ␮mol), a CA4H activity was detected, since

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FIG. 3. In situ survival rate of WRP45073A1 yeast strain orally administered alone. Values are expressed as mean numbers ⫾ SEM (n ⫽ 3 for each time of sacrifice) of viable cells with a logarithmic scale (A) or mean percentages ⫾ SEM (n ⫽ 3) relative to the initial amount of yeasts (B). Control rats received 0.9% NaCl.

FIG. 4. In situ survival rate of WRP45073A1 yeast strain orally administered in the presence of 70 ␮mol of CIN. Values are expressed as mean percentages ⫾ SEM (n ⫽ 3) of viable yeasts relative to the initial amount of yeasts administered to rats. Control rats received 0.9% NaCl.

COU production was observed (Table 2) 5 min after gavage in the stomach (209 nmol), duodenum (94 nmol), proximal jejunum (144 nmol), and median jejunum (82 nmol) and weakly detected in the stomach and duodenum 10 and 30 min following ingestion. COU was also recovered in the plasma samples collected at 5, 10, and 30 min. COU was detected neither in the distal jejunum, ileum, cecum, or colon nor 2, 4, 8, and 24 h after gavage. Five minutes after gavage, the total amount of COU detected represented 0.76% of the CIN initially administered (Table 2). Ex vivo experiments. After a 180-min incubation period and in all the organs studied (stomach, duodenum, jejunum, ileum, cecum, and colon), about 30% of the initial yeast cells were recovered alive (data not shown). This survival rate of WRP45073A1 in the gastrointestinal sacs was similar to that observed in vivo 2 and 4 h after gavage (the survival rate was around 20%) (Fig. 4). CIN was absorbed by all the digestive organs, since this acid

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TABLE 1. In situ distribution of 70 ␮mol of CIN after simultaneous oral administration with the WRP45073A1 yeast strain in sacrificed ratsa Amt (␮mol) of CIN recovered in: Time after gavage

Stomach

Duodenum

Proximal jejunum

Median jejunum

Distal jejunum

Plasma

% of CIN recovered (% of intake)

5 min 10 min 30 min 2h 4h 8h 24 h

11.3 ⫾ 4.8 3.5 ⫾ 0.8 4.8 ⫾ 2.6 0.7 ⫾ 0.2 ND ND ND

4.3 ⫾ 0.4 3.1 ⫾ 1.2 0.2 ⫾ 0.1 ND ND ND ND

3.9 ⫾ 2.5 1.0 ⫾ 0.9 ND ND ND ND ND

2.1 ⫾ 1.9 0.2 ⫾ 0.1 ND ND ND ND ND

0.1 ⫾ 0.04 0.1 ⫾ 0.04 ND ND ND ND ND

1.8 ⫾ 0.7 1.3 ⫾ 0.3 0.4 ⫾ 0.1 ND ND ND ND

33.5 ⫾ 9.7 10.6 ⫾ 2.4 7.6 ⫾ 3.7 1.0 ⫾ 0.3 ND ND ND

a Values represent the means ⫾ SEM (n ⫽ 3 for each time of sacrifice) in ␮mol of CIN recovered in the different organs of the digestive tract and in plasma. ND, not detected.

was extensively recovered on the serosal side (data not shown). CIN was more efficiently absorbed in vivo than ex vivo. Only 8% of CIN was recovered on the mucosal side 30 min after its introduction (Table 1) in sacrificed rats, whereas the residual amount of CIN on the mucosal side reached 50% 30 min after its introduction ex vivo (data not shown). In control experiments (rats receiving CIN and the WRpV60 yeast strain), no trace of COU was detected in the various digestive contents (data not shown). In contrast, when WRP45073A1 and 7 ␮mol of CIN were simultaneously introduced in the gastrointestinal sacs, COU was rapidly found in all the digestive organs (Fig. 5). COU was detected on the serosal side of each organ after 15 min of incubation (except for the stomach, where COU appeared only at 180 min) 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 mucosal side: the amounts ranged from 56 nmol (duodenum) to 236 nmol (stomach). In comparison, the amounts of COU detected on the serosal side ranged from 4 nmol (stomach) to 83 nmol (ileum) and in the organ wall from 9 nmol (ileum) to 89 nmol (stomach). The total amount of COU detected in the three compartments of each organ represented 2% (duodenum) to 5% (stomach) of the CIN initially administered (data not shown). DISCUSSION The development of new drug delivery systems based on the oral administration of recombinant living microorganisms ac-

tive in the human digestive environment has recently been considered (2, 4). To validate the biodrug concept for a bioconversion application, a genetically engineered yeast catalyzing the 4-hydroxylation of CIN into COU was used. The survival rate and bioconversion activity of the recombinant yeast have previously been studied in vitro (5) in gastrointestinal tract models. A first system, named TIM1, reproduces the stomach and small intestine (31), while the large intestine model is called TIM2 (32). In these artificial digestive systems, the main parameters of digestion (temperature, peristaltic mixing, transit time, gastrointestinal secretions, pH, and microflora activity) are reproduced as faithfully as possible, and the digestive systems have been validated by many studies (29, 30, 44, 48). The next step in the development of the biodrug concept consists of 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. Bioconversion activity of recombinant yeast in living rats. To follow the yeast’s bioconversion activity in the living animals, recombinant yeasts (control strain WRpV60 and WRP45073A1) and various doses (7, 70, and 700 ␮mol) of CIN were orally administered to rats. It was recently shown (19) that orally administered CIN and COU rapidly travel from the gastrointestinal tract of the rats to the bloodstream and are then partially eliminated by the kidney and recovered in urine. Therefore, after gavage with CIN and recombinant yeasts, the CA4H activity inside the digestive tract should be revealed by the apparition of COU in the urine.

TABLE 2. In situ CA4H activity of the WRP45073A1 yeast strain after simultaneous oral administration with 70 ␮mol of CIN in sacrificed ratsa Amt (nmol) of COU recovered in: Time after gavage

Stomach

Duodenum

Proximal jejunum

Median jejunum

Plasma

% of COU recovered (% of ingested CIN)

5 min 10 min 30 min 2h 4h 8h 24 h

209 ⫾ 101 37 ⫾ 11 7⫾3 ND ND ND ND

94 ⫾ 20 2⫾1 0.3 ⫾ 0.3 ND ND ND ND

144 ⫾ 76 ND ND ND ND ND ND

82 ⫾ 64 ND ND ND ND ND ND

3⫾2 4⫾1 3⫾1 ND ND ND ND

0.76 ⫾ 0.15 0.06 ⫾ 0.02 0.010 ⫾ 0.004 ND ND ND ND

a Values represent the means ⫾ SEM (n ⫽ 3 for each time of sacrifice) in nmol of COU produced by the yeasts and recovered in the different organs of the digestive tract and in plasma. ND, not detected.

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FIG. 5. Ex vivo CA4H activity of WRP45073A1 yeast strain in gastrointestinal sacs. The values represent the mean amounts ⫾ SEM (n ⫽ 5) in nanomoles of COU produced by the yeasts and recovered on the serosal side of each organ.

COU was detected in the urine of rats (Fig. 1) 0 to 24 h following gavage with only 70 ␮mol of CIN and the yeast strain WRP45073A1. The absence of COU in the urine of rats given WRpV60 and CIN demonstrated the specificity of the enzyme reaction. When the lowest dose of CIN (7 ␮mol) was ingested, the CA4H activity could not be clearly demonstrated since the COU excreted in urine was too low to be detected. The absence of activity in the presence of 700 ␮mol of CIN could be due to the potential antimicrobial activity of CIN, which has been shown to possess antibacterial (36, 42) and antifungal (46) activities. To check for the potential toxicity of CIN towards WRP45073A1, cells were incubated with the different amounts of acid tested in living rats. CIN was nontoxic at 7 ␮mol, moderately toxic at 70 ␮mol, and lethal at 700 ␮mol (Fig. 2). These data led to the conclusion that the lack of bioconversion activity observed in rats receiving 700 ␮mol of CIN was due to extensive yeast death in the digestive tract. Therefore, all subsequent in vivo experiments (sacrificed rats) were performed using 70 ␮mol 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 previously observed in the artificial digestive system TIM1, in which about 41% of the initial CIN was converted into COU after 4 h of digestion (5). This large difference between in vivo and in vitro results may be explained by higher yeast mortality in the rat digestive environment than in the gastrointestinal system. To test this hypothesis, the survival rate of orally administered recombinant yeast in the rat digestive tract was studied. Viability of recombinant yeasts. The recombinant yeasts showed a high survival rate in the upper part of the digestive tract. As previously demonstrated for Saccharomyces boulardii (6), WRP45073A1 is resistant to gastric environmental conditions. Also, the yeasts leaving the stomach alive seem to resist pancreatic and hepatic secretions, as their survival rate was not modified from the duodenum to the ileum. When yeasts reached the large intestine, their survival rate decreased strongly (less than 1% 24 h after ingestion) (Fig. 3B). Our in vivo results are consistent with those obtained in vitro (5) in gastrointestinal tract models (TIM1 and TIM2). Few studies evaluating the viability of Saccharomyces spp.

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FIG. 6. Ex vivo repartition of COU in the digestive organs after a 180-min incubation period. The values represent the mean amounts ⫾ SEM (n ⫽ 5) in nanomoles of COU produced by the yeasts and recovered in the three compartments of each organ. Asterisks denote values significantly different from the amounts recovered in the organ wall and serosal side of the same organ (P ⬍ 0.05).

throughout the length of the gastrointestinal tract of rats or humans are available. A distribution similar to ours has previously been shown in rats 30 min after oral administration of 109 S. boulardii cells (1). Yeast viability is evaluated mainly in feces. In the present study, the survival rate observed in feces is consistent with that obtained for rats (7) and healthy humans (23, 37). Several biochemical processes can explain the high mortality of yeasts in the large intestine. Some studies (17, 28, 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 ␤-1,3-glucanases by Bacteroides, the most prevalent genus of intestinal bacteria in humans (51). Also, a “barrier” role for the endogenous colonic microflora towards orally administered S. boulardii has been demonstrated by Ducluzeau and Bensaada (16). This effect induces an extensive elimination of yeasts from the digestive tract. The high viability of WRP45073A1 in the rat upper digestive tract encouraged us to further investigate the potential efficiency of the recombinant yeasts to perform, in vivo, the model reaction of bioconversion. Since the search for the appearance of COU in urine is limited by large losses between the production and excretion of COU in living rats (absorption, body distribution, metabolism, and renal elimination), two other techniques were devised to determine whether recombinant yeasts were able to convert CIN into COU in the rat digestive tract: (i) in situ detection of COU (sacrificed rats) after oral administration of yeasts and CIN and (ii) ex vivo experiments. The former technique allows for the direct detection of COU potentially produced in situ. The latter is a useful screening tool for studying the digestive absorption and metabolic behavior of substances introduced in the various parts of the gastrointestinal tract (10). In addition, this last technique allows the CA4H activity of recombinant yeasts to be tested using the lowest nontoxic dose of CIN (7 ␮mol). Whatever the time after gavage, the in situ survival rates of

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WRP45073A1 strains were similar after their oral administration alone or with 70 ␮mol of CIN. The moderate antifungal activity of the 70 ␮mol of CIN observed in vitro was not found in sacrificed rats, probably owing to the rapid and extensive absorption of CIN by the rat digestive tract. The yeast viability in the gastrointestinal sacs did not differ from that observed in sacrificed rats. 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 rats than that found in gastrointestinal sacs. Several hypotheses can explain this difference of absorption between in situ and ex vivo experiments. First, after its intestinal absorption in vivo, CIN passes into the bloodstream and is converted mainly into hippuric acid before its elimination in urine (35). Thus, the disappearance of CIN from the systemic circulation favors its absorption across the digestive epithelium. Second, the area of the rat digestive wall is larger in vivo than in gastrointestinal sacs. For instance, the length of the jejunum is up to 1 m in rats (20) whereas it measures only 10 cm ex vivo. Bioconversion activity in sacrificed rats. Following its ingestion, the gastrointestinal absorption of CIN (70 ␮mol) induced a rapid disappearance of CIN in the immediate vicinity of the recombinant yeasts. Consecutively to the rapid absorption of their substrate (CIN), recombinant yeasts were unable to exert a very high CA4H activity throughout the rat digestive tract. Nevertheless, the bioconversion reaction occurred very fast, with most of the COU being produced within the first 5 min. COU was detected only in the upper digestive part, and the amounts of COU recovered in sacrificed rats were 100 times higher than those excreted in the urine of living rats. This result may account for the small losses of in situ-detected COU compared with those observed in living rats. Bioconversion activity in ex vivo experiments. A CA4H activity was detected in the gastrointestinal sacs. COU was produced inside the lumen and then absorbed and recovered on the serosal side, 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 seven 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. As a consequence, CIN remains in the immediate vicinity of the yeasts for a longer time on the mucosal side of gastrointestinal sacs than in sacrificed rats, explaining the higher CA4H activity detected ex vivo. A closely similar effect was observed in the artificial gastrointestinal tract model TIM1, in which CIN was not absorbed via a monocarboxylic acid transporter but exclusively absorbed from the jejunum and ileum via passive diffusion (5). The yeasts were therefore in close contact with CIN for a longer time, enhancing their CA4H activity. In our ex vivo study, a higher activity of bioconversion was observed in the cecum and colon (CIN conversion of around 2%) than that


reported for the artificial large intestinal model TIM2, which was too weak to be quantified (5). The reason is that in TIM2, the recombinant yeasts were rapidly deprived of CIN due to its metabolization by the colonic microflora (5) whereas most of the rat endogenous microflora was probably removed from the mucosal side of the gastrointestinal sacs by rinsing during their preparation. In conclusion, these new results indicate that after oral administration in rats, the recombinant yeast strain WRP45073A1 was resistant to gastric, hepatic, pancreatic, and small intestinal secretions but was more sensitive to the conditions of the large intestine. For the first time, it is shown that the recombinant yeasts are able to exert a CA4H activity directly in the rat digestive tract. In sacrificed rats, the reaction of bioconversion was very fast, with most of the COU produced within the first 5 min, and was detected only in the upper digestive tract. Using the gastrointestinal sac technique, a CA4H activity was found in all the organs of the digestive tract: stomach, duodenum, jejunum, ileum, cecum, and colon. Compared with results previously obtained with 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 versus 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 yeasts were quickly deprived of their substrate, probably explaining their weak CA4H activity in vivo. The recombinant yeast used to validate the biodrug concept for bioconversion is less well tailored for in vivo experiments than for in vitro experiments, owing to the extensive and rapid absorption of CIN by the rat gastrointestinal tract. However, the results obtained in this study do not challenge the biodrug concept, since a high yeast survival rate and a CA4H activity were still observed in the rat digestive environment. These in vivo results support the possibility of using genetically modified S. cerevisiae as a potential host for the development of biodrugs, in particular to perform biodetoxication by metabolizing xenobiotics that are poorly or slowly absorbed by the human digestive tract. Soon, once the therapeutic target has been identified, model genes will be replaced by candidate genes. Of course, heterologous gene expression strategies will have to be adapted for safe use in humans, as the presence of mobilizable vectors, antibiotic selection markers, and bacterial sequences liable to promote gene transfer to host microflora is prohibited. In addition, environmental confinement of recombinant cells (24, 34) will have to be achieved by introducing a suicide process (e.g., activation of a toxic protein or repression of an essential gene) that triggers the destruction of the yeast as soon as it leaves the human digestive tract. ACKNOWLEDGMENTS This work was supported by the De´le´gation Geńe´rale pour l’Armement. We gratefully acknowledge the skillful technical assistance of Astrid Vega, Marianne Collange, Aı¨ssa Sangare, Sabrina Maquaire, and Ange´lique Gardes. REFERENCES 1. Albert, O., J. Massot, and M. C. Cortois. 1977. Etude cine´tique quantitative de la re´partition d’une levure vivante du genre Saccharomyces à diffe´rents niveaux du tractus digestif. La Vie Me´dicale 18:1604–1606.

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