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Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads
Journal of Bioscience and Bioengineering VOL. 111 No. 4, 454 – 458, 2011 www.elsevier.com/locate/jbiosc
Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads Naoko Yamamoto,1,2 Kikuo Komori,1,⁎ Kevin Montagne,1,3 Hitoshi Matsui,2 Hidenari Nakayama,1 Shoji Takeuchi,1,2 and Yasuyuki Sakai1,2 Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan, 1 Life BEANS Center BEANS Project, Komaba, Meguro-ku, Tokyo 153-8505, Japan, 2 and LIMMS/CNRS-IIS, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan 3 Received 20 August 2010; accepted 1 December 2010 Available online 15 January 2011
We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cytotoxicity test; Rat liver homogenate microsome; Gel microbeads; Encapsulation; Reactive metabolite]
A rapid and simple way to evaluate the toxicity of xenobiotics, such as drugs, pesticides, and food additives, has been increasingly sought for the early stages of their development. During xenobiotic metabolism, a xenobiotic introduced into the human body is often converted into a reactive metabolite, mainly by detoxification enzymes produced in the liver, resulting in expression and/or enhancement of toxicity. Reactive metabolites are widely recognized as mutagens and carcinogens based on their covalent binding with DNA and proteins (1,2). For instance, it is generally known that aflatoxin B1, which is a natural carcinogen, is itself lowly toxic, while its reactive metabolites, aflatoxin B1-8,9-epoxide, exhibit severe toxicity to the liver (3,4). Therefore, toxicities of not only xenobiotics but also their metabolites should be determined. In drug metabolism and detoxification pathways, cytochrome P450 (CYP) enzymes are generally involved in the formation of reactive metabolites and can be isolated in microsomes of human and animal cells. The S9 microsomal fraction of rat liver homogenate is widely used in in vitro bioassays to evaluate the toxicity of reactive metabolites (5–7). Since S9 contains enzymes of the CYP superfamily, phase I metabolism in the liver is partially replicable in the presence of cofactors, such as NADP+, which is used as an electron donor. Therefore, comprehensive evaluation of CYP-metabolized xenobiotic toxicity may become possible. However, it is difficult to determine net cytotoxicity of the xenobiotic metabolites, because lipids that make up ⁎ Corresponding author. Tel./fax: + 81 3 5452 6349. E-mail address: [email protected] (K. Komori).
S9 microsomes are known to be metabolized by CYPs, resulting in the formation of toxic microsomal lipid peroxides (8,9). Therefore, due to the different sensitivity of target cells to S9 and its metabolites, the concentration of S9 is adjusted in cytotoxicity tests in order to minimize cell damage. To overcome this problem, encapsulation of S9 in hydrogel microbeads may be an effective way of preventing leakage of the microsomal lipid peroxides. It has been reported that hydrogel microbeads are extensively employed as supporting materials for biocatalysts (10), proteins (11,12), and cells (13,14). In particular, there has been considerable research on gel microbeads of alginic acid, which is a well known as polysaccharide, thanks to its possible handling in mild conditions and high biocompatibility (15,16). Moreover, coating the beads with poly-L-lysine (PLL) can control diffusion of chemicals due to the polyion complex-based polymer network (17,18). This means that high-molecular-weight chemicals larger than the polymer network pores can be retained in the gel microbeads, whereas low-molecular-weight chemicals can move in and out of the gel microbeads. Therefore, it is expected that metabolic activity of CYPs in S9 will be retained and leakage of microsomal lipid peroxides prevented by encapsulation of S9 in alginate gel microbeads coated with PLL. Such S9-encapsulated gel microbeads may lead to the formation of artificial liver cells. In this work, using a coaxial two-fluid nozzle, we fabricated non toxic S9-encapsulated alginate gel microbeads, the surfaces of which were coated with PLL. Moreover, we established a simple in vitro cytotoxicity test using an incubation system based on a membrane
1389-1723/$ - see front matter © 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.12.004
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culture insert, on which the S9-encapsulated gel microbeads were seeded. Here, we used a mouse fibroblast NIH3T3 cells as a cell model and the anti-cancer drug cyclophosphamide (CPA) as an example of a reactive metabolite precursor, as CPA is known as an indirect mutagen. Cytotoxicity was determined from the ratio between cell growth after exposure to the xenobiotic and that observed in a standard culture system. The present test might readily permit direct comparison of the cytotoxicity of various S9-generated xenobiotic metabolites under the same concentration of S9, regardless of the type of cell. MATERIALS AND METHODS Preparation of gel microbeads Using a coaxial two-fluid nozzle, 2.5 wt.% alginate (Wako Pure Chemical Industries, Ltd., Japan, Mw = 64 000) aqueous solution with 0 or 50% S9 of rat liver homogenate suspension (Kikkoman, Japan), containing 14 μM CYPs, was discharged from the inner nozzle (0.27 mm in internal diameter (ID)) into 5 wt.% CaCl2 aqueous solution stirred at 700 rpm under running N2 gas (2.0 L min− 1) from the outer nozzle (1.3 mm in ID). Microbeads thus obtained were suspended in 1.5 wt.% PLL (Sigma-Aldrich, USA, Mw = 24 000) aqueous solution, followed by suspension in D-MEM/F12 (Invitrogen, USA) containing 10% fetal bovine serum (FBS; Gemini Bio-Products, USA). Consequently, we obtained normal and S9encapsulated gel microbeads, the surfaces of which were electrostatically coated with PLL and proteins. Determination of metabolic activity The metabolic activity of CYP 1A1/2 in S9 was determined on the basis of fluorescence intensity of resorufin (λex = 530 nm and λem = 585 nm), which is enzymatically converted from ethoxyresorufin (ER, SigmaAldrich) (19–21). 5.0× 105 S9-encapsulated gel microbeads L− 1 and 20 μM dicumarol (Sigma-Aldrich) were added to D-MEM/F12-based culture medium containing 10% FBS, 25 mM hydroxyethylpiperadine-N′ 2-ethanesulfonic acid (Dojindo Laboratories, Japan), 100 units mL− 1 penicillin (Wako), 100 μg mL− 1 streptomycin (Wako), and 0.25 μg mL− 1 amphotericin B (Sigma-Aldrich). Then, the culture medium containing S9-encapsulated gel microbeads was gently stirred at 37°C, followed by the addition of culture medium containing 35 μM ER and cofactors, such as 0.83 mM D-glucose-6-phosphate disodium salt hydrate (Sigma-Aldrich), 0.67 mM β-NADP+ (Oriental Yeast Co., Ltd, Japan), and 0.83 mM MgCl2 (final concentrations). A spectrofluorophotometer RF-5300PC (Shimadzu, Japan) was used for spectrofluorometric measurements. Cytotoxicity tests To evaluate the cytotoxicity of S9-encapsulated gel microbeads, we used a culture insert-based incubation system as illustrated in Fig. 1. A polyester (PE) membrane culture insert (4.67 cm2 with 0.4 μm pores) filled with S9 or S9-encapsulated gel microbeads in culture mediums was set in the accompanying culture dish, on which NIH3T3 cells were incubated for 24 h after cell seeding at an initial density of 2.0 × 104 cells cm− 2. NIH3T3 cells were incubated in the culture medium (4.1 mL in total) containing cofactors for 4 h in a shaking incubator (5% CO2, 37°C), followed by further incubation in the culture medium without cofactors for 7 days in a stationary incubator (5% CO2, 37°C) after removal of the culture insert. As a control experiment, NIH3T3 cells were incubated in the culture medium containing S9 and cofactors for 4 h, followed by incubation in the culture medium. Based on 4′,6-diamino-2-phenylindole (DAPI) fluorometry (22), the number of cells on day 7 of cell culture was measured. The number of cells obtained in a standard culture conditions was also measured as a reference. To evaluate the cytotoxicity of CPA, the measurement system was essentially the same as the one described above. The PE membrane culture insert filled with 4.4 × 102 S9-encapsulated gel microbeads per cm2 and culture medium was set in the accompanying dish with NIH3T3 cells. NIH3T3 cells were incubated in the culture medium (4.1 mL in total) containing cofactors and CPA for 4 h in the shaking incubator, followed by further incubation in the culture medium without cofactors and CPA for
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7 days in the stationary incubator after removal of the culture insert. The number of cells on day 7 of cell culture was measured on the basis of DAPI fluorometry.
RESULTS AND DISCUSSION Shape of gel microbeads It is known that spherical alginate microbeads are formed when the microbeads are prepared at 1.2 wt.% or higher alginate concentrations (9). The normal and S9-encapsulated gel microbeads prepared for this study and observed under a microscope were spherical (Fig. 2), consistent with the fact that a 2.5 wt.% alginate aqueous solution was used. In addition, the size of normal and S9encapsulated gel microbeads was found to be 480 ± 20 μm and 720 ± 30 μm in diameter, respectively. This difference may be explained as follows: normal microbeads are densely packed with alginate polymers thanks to coordinate bonds between carboxyl groups via Ca2+. Regarding S9-encapsulated microbeads, since the surface of rat liver microsomes is known to be negatively charged at neutral pH (23), microbeads swelled likely due to electrostatic repulsion between parts of carboxyl groups in the gel microbeads and the surfaces of S9 microsomes. Note that reversible expansion/contraction of a coordination bond-based hydrogel is known to be controllable by electrostatic interaction based on dissociation/formation of the coordination bond (24). Biochemical characteristics of S9-encapsulated gel microbeads To determine the metabolic capacity of the S9-encapsulated gel microbeads, we measured the activity of CYP 1A1/2 contained in S9 as an example of the CYPs superfamily by measuring the fluorescence intensity of resorufin, the metabolite of ER by CYP1A1/2 activity (19–21). The plot a in Fig. 3 shows that resorufin production in the culture medium containing 5.0×105 beads L− 1 of S9-encapsulated gel microbeads, 35 μM ER, and cofactors increased for up to 2 h and then leveled off. As a control experiment, plot b in Fig. 3 shows resorufin production in the culture medium containing 5% S9, 35 μM ER, and cofactors. Note that the S9 concentration in the culture medium containing 5% S9 was roughly equal to that containing 5.0×105 beads L− 1 of S9-encapsulated gel microbeads. As can be seen in plot a and b of Fig. 3, the final amount of resorufin produced by 5.0×105 beads L− 1 of S9-encapsulated gel microbeads was actually equal to that by 5% S9. However, the initial rate of resorufin production in the former was slower than in the latter. Based on plot a and b in Fig. 3, each initial rate was determined to be 15 and 19 nM min− 1, respectively. This difference is likely due to the lower diffusion rates of ER and resorufin in the gel microbeads. In addition, we examined leakage of S9 from gel microbeads. After S9-encapsulated gel microbeads were removed from the culture medium containing 35 μM ER and cofactors after 30 min, resorufin production was further measured. If resorufin production increased after removal of the S9-encapsulated gel microbeads from the culture medium, S9 might have leaked out of the gel microbeads. However, resorufin production did not increase after 60 min (Fig. 3, plot c). This
FIG. 1. Schematic illustration of the PE membrane culture insert-based incubation system for the metabolic activation test. (A) S9 is present in both culture insert and culture dish. (B) S9 is present only in the culture insert. (C) S9-encapsulated gel microbeads are present only in the culture insert.
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J. BIOSCI. BIOENG., coating. Correspondingly, S9 (ca. 0.1 μm in diameter or larger) (8) was apparently larger than PLL (Mw = 2.5 × 104) employed in the present work, resulting in stably encapsulation in the gel microbeads. In contrast, the molecular weights of cofactors, ER, and resorufin are smaller than 750, so that they might move in or out of the microbeads regardless of surface modification by PLL. In the present investigation, we focused on the activity of only CYP 1A1/2 in S9. In contrast, it is well-known that S9 contains several CYP enzymes, such as CYP 2B6, CYP 2C19, CYP 2D6, CYP 2E1, and CYP 3A4/ 5 (25). Therefore, S9-encapsulated gel microbeads prepared here might be expected to exhibit the metabolic capacity based on not only CYP 1A1/2 but also other CYP enzymes.
FIG. 2. Microphotographs of (A) normal and (B) S9-encapsulated gel microbeads. Scale bar indicates 500 μm.
result suggests that S9 was retained in the gel microbeads thanks to the formation of fine polymer networks based on a polyion complex between the alginate gel microbead surface and PLL. It has been reported that leakage of hemoglobin (Mw = 6.8 × 104) does not occur from alginate gel microbeads coated with PLL (Mw = (1.7 or 2.5) × 104), whereas chymotrypsin (Mw = 2.5 × 104) can enter the alginate gel microbeads coated with PLL (Mw = 2.5 × 105) (18). This means that the molecular weight of fraction is controlled by PLL
FIG. 3. Time course increase in resorufin concentrations after addition of culture medium containing 35 μM ER and cofactors to the culture medium containing (a and c) 5.0 × 105 beads L− 1 S9-encapsulated gel microbeads or (b) 5% S9. (c) S9-encapsulated gel microbeads were removed from the culture medium after 30 min. All plots in the figure were the mean± SD from at least three independent experiments.
Cytotoxicity of S9-encapsulated gel microbeads As mentioned above, S9 was stably encapsulated in the alginate microbeads. However, it was not clear whether S9-derived microsomal lipid peroxides leaked out of the gel microbeads. Therefore, we determined the cytotoxicity of the S9-encapsulated gel microbeads to NIH3T3 cells incubated in the culture medium containing cofactors. Expecting that cell growth might be inhibited if cells were exposed to toxic lipid peroxidase, cytotoxicity was assessed by measuring cell growth. The cell growth ratio was defined as 100×R/R0, where R and R0 were the numbers of cells in the culture medium at day 7 after an initial 4-hour incubation respectively with and without gel microbeads. We first examined the cytotoxicity of the normal gel microbeads. The surface of the NIH3T3 cells layer in the culture dish was directly and almost entirely covered with 4.4× 102 beads cm− 2 of normal gel microbeads. The cells were incubated for 4 h, followed by further incubation for 7 days without microbeads. Unfortunately, the cell growth ratio on day 7 was nearly 0%. In contrast, the cell growth ratio was nearly 100% when NIH3T3 cells were only exposed to cofactors for 4 h. These results are likely due to the fact that the normal gel microbeads caused physical cell damage by direct contact and/or the cells preferentially adhered to the surface of the normal gel microbeads and removed with the microbeads. In addition, a layer of gel microbeads covering the NIH3T3 cells might interfere with diffusion of O2 into the cells. To avoid these problems, a culture insert filled with 4.4×102 beads cm− 2 of normal gel microbeads was set in the culture dish and the cells were incubated. As a result, the cell growth ratio was nearly 100%. Subsequently, we examined the cytotoxicity of S9 and S9-encapsulated gel microbeads using the culture insert-based incubation system as illustrated in Fig. 1. First, we confirmed the cytotoxicity of S9. NIH3T3 cells were incubated in the S9-dispersed culture medium containing cofactors for 4 h (Fig. 1A), followed by incubation in the culture medium without S9 and cofactors for 7 days. This exposure system corresponds to the conventional test system. As expected, the cell growth ratio on day 7 decreased as S9 concentration increased (Fig. 4, plot a). These results indicate that microsomal lipid peroxides generated by CYPs-based metabolism caused damage to NIH3T3 cells. For comparison, NIH3T3 cells were also incubated in culture medium containing cofactors for 4 h after the culture insert filled with S9 suspension was set in the dish (Fig. 1B), followed by further incubation in culture medium without cofactors for 7 days after removal of the culture insert. As in the case of S9-dispersed culture medium, the cell growth ratio decreased as S9 concentration increased (Fig. 4, plot b). This result indicates that the membrane of the culture insert could not prevent microsomal lipid peroxides from diffusing to and affecting the cells. Thus, the cytotoxicity of S9-derived substances is non-negligible in cytotoxicity tests for reactive metabolites. Next, we evaluated the cytotoxicity of the S9-encapsulated gel microbeads (Fig. 1C). Here, we employed the culture inserts with the membrane surface densely covered with 4.4 × 102 or 8.8 × 102 beads cm− 2 of S9-encapsulated gel microbeads, resulting in S9 concentrations in the 4.1 mL culture medium roughly equivalent to 5 and 10%, respectively. Interestingly, the cell growth ratio was not affected by the number of the S9-encapsulated gel microbeads (Fig. 4, plot c). This result might indicate that the fine polymer network described above prevented
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FIG. 4. Changes in the cell growth ratio of NIH3T3 cells grown in normal culture medium for 7 days after a 4 h incubation in the PE-membrane culture insert-based incubation system. Each plot a, b, and c in this figure corresponds to the schematic illustration A, B, and C in Fig. 1, respectively. All plots in the figure were the mean ± SD from at least three independent experiments.
the leakage of microsomal lipid peroxides from the gel microbeads. Furthermore, it has been shown that the cytotoxicity of S9 is reduced if a certain amount of Ca2+ is added to the culture medium (9). There is a possibility that Ca2+ involved in the formation of the alginate gel microbeads contributes to reduce the cytotoxicity of microsomal lipid peroxides. In any case, the S9-induced cytotoxicity was certainly negligible thanks to encapsulation of S9 in the gel microbeads coated with PLL. Application to metabolic activation tests As S9 cytotoxicity could be neglected by encapsulation of S9, we evaluated the cytotoxicity of the anti-cancer drug CPA (Mw=279) using the S9-encapsulated gel microbeads. It is known that CPA is metabolized to 4-hydroxy-CPA in the liver mainly by CYP 2B6, resulting in cell apoptosis due to DNA alkylation (26). In the present test system (Fig. 1C), CPA is metabolized by CYP 2B6 in S9-encapsulated gel microbeads. Then, 4-hydroxy-CPA diffuses out of the gel microbeads, through the PE membrane, and into NIH3T3 cells, resulting in cell apoptosis. The CPA cytotoxicity was determined on the
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basis of the cell growth ratio on day 7 of cell culture after CPA exposure for 4 h. NIH3T3 cells were also exposed to CPA in the culture medium containing 0% and 5% S9. Dose–response curves were shown in Fig. 5. As can be seen, the dose–response curve in the presence of the S9-encapsulated gel microbeads (plot a) shifted towards lower CPA concentrations compared with that in the absence of S9 (plot b) and towards higher CPA concentrations compared with that in the presence of 5% S9 (plot c). Based in Fig. 5, each median effective concentration (EC50) was estimated at 7.0 × 10− 3 M for no S9, 5.8 × 10− 5 M for S9-encapsulated gel microbeads, and 1.2 × 10− 5 M for 5% S9, respectively. These differences may be explained as follows: in the absence of S9, decrease in the cell growth ratio was attributed to the toxicity of CPA itself. The toxicity mechanism is reckoned to be a disruption of the phospholipid membrane due to permeation and/or accumulation of CPA. In the presence of S9-encapsulated gel microbeads, decrease in the cell growth ratio is mainly attributed to the toxicity of the CPA metabolite by S9-encapsulated gel microbeads, which causes DNA alkylation. In the case of 5% S9, decrease in the cell growth ratio might be attributed to the combined toxicities of CPA- and microsomal lipid-derived metabolites. Although the detailed toxicity mechanism is unclear, use of the S9-encapsulated gel microbeads might prevent the microsomal lipid-derived metabolite toxicity. Thus, the evaluation of reactive metabolites without the S9-induced cytotoxicity is now possible by using S9-encapsulated gel microbeads. Under the present condition, it is necessary to miniaturize the S9encapsulated gel microbeads, because diffusion of chemicals, such as xenobiotis and their metabolites, in and out of the present gel microbeads was limited due to large size as shown in Fig. 3. For example, inkjet techniques (27), which can prepare gel microbeads with diameters in the micrometer range, could enable the large scale production of smaller S9-encapsulated gel microbeads in a short time. In addition, the metabolic kinetic analysis for various model substances is needed. The miniaturized S9-encapsulated gel microbeads would permit direct comparison of net cytotoxicity of reactive metabolites for various types of target cells. ACKNOWLEDGEMENT This work was supported by the Bio Electromechanical Autonomous Nano Systems (BEANS) Project from NEDO, Japan. References
FIG. 5. Dose–response curves of NIH3T3 cells incubated in culture medium for 7 days after 4 h exposure to CPA in the presence of (a) 4.4 × 102 S9-encapsulated gel microbeads per cm2, (b) no S9, and (c) 5% S9. All plots in the figure were the mean ± SD of nine wells from three independent experiments.
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Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads Naoko Yamamoto,1,2 Kikuo Komori,1,⁎ Kevin Montagne,1,3 Hitoshi Matsui,2 Hidenari Nakayama,1 Shoji Takeuchi,1,2 and Yasuyuki Sakai1,2 Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan, 1 Life BEANS Center BEANS Project, Komaba, Meguro-ku, Tokyo 153-8505, Japan, 2 and LIMMS/CNRS-IIS, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan 3 Received 20 August 2010; accepted 1 December 2010 Available online 15 January 2011
We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cytotoxicity test; Rat liver homogenate microsome; Gel microbeads; Encapsulation; Reactive metabolite]
A rapid and simple way to evaluate the toxicity of xenobiotics, such as drugs, pesticides, and food additives, has been increasingly sought for the early stages of their development. During xenobiotic metabolism, a xenobiotic introduced into the human body is often converted into a reactive metabolite, mainly by detoxification enzymes produced in the liver, resulting in expression and/or enhancement of toxicity. Reactive metabolites are widely recognized as mutagens and carcinogens based on their covalent binding with DNA and proteins (1,2). For instance, it is generally known that aflatoxin B1, which is a natural carcinogen, is itself lowly toxic, while its reactive metabolites, aflatoxin B1-8,9-epoxide, exhibit severe toxicity to the liver (3,4). Therefore, toxicities of not only xenobiotics but also their metabolites should be determined. In drug metabolism and detoxification pathways, cytochrome P450 (CYP) enzymes are generally involved in the formation of reactive metabolites and can be isolated in microsomes of human and animal cells. The S9 microsomal fraction of rat liver homogenate is widely used in in vitro bioassays to evaluate the toxicity of reactive metabolites (5–7). Since S9 contains enzymes of the CYP superfamily, phase I metabolism in the liver is partially replicable in the presence of cofactors, such as NADP+, which is used as an electron donor. Therefore, comprehensive evaluation of CYP-metabolized xenobiotic toxicity may become possible. However, it is difficult to determine net cytotoxicity of the xenobiotic metabolites, because lipids that make up ⁎ Corresponding author. Tel./fax: + 81 3 5452 6349. E-mail address: [email protected] (K. Komori).
S9 microsomes are known to be metabolized by CYPs, resulting in the formation of toxic microsomal lipid peroxides (8,9). Therefore, due to the different sensitivity of target cells to S9 and its metabolites, the concentration of S9 is adjusted in cytotoxicity tests in order to minimize cell damage. To overcome this problem, encapsulation of S9 in hydrogel microbeads may be an effective way of preventing leakage of the microsomal lipid peroxides. It has been reported that hydrogel microbeads are extensively employed as supporting materials for biocatalysts (10), proteins (11,12), and cells (13,14). In particular, there has been considerable research on gel microbeads of alginic acid, which is a well known as polysaccharide, thanks to its possible handling in mild conditions and high biocompatibility (15,16). Moreover, coating the beads with poly-L-lysine (PLL) can control diffusion of chemicals due to the polyion complex-based polymer network (17,18). This means that high-molecular-weight chemicals larger than the polymer network pores can be retained in the gel microbeads, whereas low-molecular-weight chemicals can move in and out of the gel microbeads. Therefore, it is expected that metabolic activity of CYPs in S9 will be retained and leakage of microsomal lipid peroxides prevented by encapsulation of S9 in alginate gel microbeads coated with PLL. Such S9-encapsulated gel microbeads may lead to the formation of artificial liver cells. In this work, using a coaxial two-fluid nozzle, we fabricated non toxic S9-encapsulated alginate gel microbeads, the surfaces of which were coated with PLL. Moreover, we established a simple in vitro cytotoxicity test using an incubation system based on a membrane
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culture insert, on which the S9-encapsulated gel microbeads were seeded. Here, we used a mouse fibroblast NIH3T3 cells as a cell model and the anti-cancer drug cyclophosphamide (CPA) as an example of a reactive metabolite precursor, as CPA is known as an indirect mutagen. Cytotoxicity was determined from the ratio between cell growth after exposure to the xenobiotic and that observed in a standard culture system. The present test might readily permit direct comparison of the cytotoxicity of various S9-generated xenobiotic metabolites under the same concentration of S9, regardless of the type of cell. MATERIALS AND METHODS Preparation of gel microbeads Using a coaxial two-fluid nozzle, 2.5 wt.% alginate (Wako Pure Chemical Industries, Ltd., Japan, Mw = 64 000) aqueous solution with 0 or 50% S9 of rat liver homogenate suspension (Kikkoman, Japan), containing 14 μM CYPs, was discharged from the inner nozzle (0.27 mm in internal diameter (ID)) into 5 wt.% CaCl2 aqueous solution stirred at 700 rpm under running N2 gas (2.0 L min− 1) from the outer nozzle (1.3 mm in ID). Microbeads thus obtained were suspended in 1.5 wt.% PLL (Sigma-Aldrich, USA, Mw = 24 000) aqueous solution, followed by suspension in D-MEM/F12 (Invitrogen, USA) containing 10% fetal bovine serum (FBS; Gemini Bio-Products, USA). Consequently, we obtained normal and S9encapsulated gel microbeads, the surfaces of which were electrostatically coated with PLL and proteins. Determination of metabolic activity The metabolic activity of CYP 1A1/2 in S9 was determined on the basis of fluorescence intensity of resorufin (λex = 530 nm and λem = 585 nm), which is enzymatically converted from ethoxyresorufin (ER, SigmaAldrich) (19–21). 5.0× 105 S9-encapsulated gel microbeads L− 1 and 20 μM dicumarol (Sigma-Aldrich) were added to D-MEM/F12-based culture medium containing 10% FBS, 25 mM hydroxyethylpiperadine-N′ 2-ethanesulfonic acid (Dojindo Laboratories, Japan), 100 units mL− 1 penicillin (Wako), 100 μg mL− 1 streptomycin (Wako), and 0.25 μg mL− 1 amphotericin B (Sigma-Aldrich). Then, the culture medium containing S9-encapsulated gel microbeads was gently stirred at 37°C, followed by the addition of culture medium containing 35 μM ER and cofactors, such as 0.83 mM D-glucose-6-phosphate disodium salt hydrate (Sigma-Aldrich), 0.67 mM β-NADP+ (Oriental Yeast Co., Ltd, Japan), and 0.83 mM MgCl2 (final concentrations). A spectrofluorophotometer RF-5300PC (Shimadzu, Japan) was used for spectrofluorometric measurements. Cytotoxicity tests To evaluate the cytotoxicity of S9-encapsulated gel microbeads, we used a culture insert-based incubation system as illustrated in Fig. 1. A polyester (PE) membrane culture insert (4.67 cm2 with 0.4 μm pores) filled with S9 or S9-encapsulated gel microbeads in culture mediums was set in the accompanying culture dish, on which NIH3T3 cells were incubated for 24 h after cell seeding at an initial density of 2.0 × 104 cells cm− 2. NIH3T3 cells were incubated in the culture medium (4.1 mL in total) containing cofactors for 4 h in a shaking incubator (5% CO2, 37°C), followed by further incubation in the culture medium without cofactors for 7 days in a stationary incubator (5% CO2, 37°C) after removal of the culture insert. As a control experiment, NIH3T3 cells were incubated in the culture medium containing S9 and cofactors for 4 h, followed by incubation in the culture medium. Based on 4′,6-diamino-2-phenylindole (DAPI) fluorometry (22), the number of cells on day 7 of cell culture was measured. The number of cells obtained in a standard culture conditions was also measured as a reference. To evaluate the cytotoxicity of CPA, the measurement system was essentially the same as the one described above. The PE membrane culture insert filled with 4.4 × 102 S9-encapsulated gel microbeads per cm2 and culture medium was set in the accompanying dish with NIH3T3 cells. NIH3T3 cells were incubated in the culture medium (4.1 mL in total) containing cofactors and CPA for 4 h in the shaking incubator, followed by further incubation in the culture medium without cofactors and CPA for
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7 days in the stationary incubator after removal of the culture insert. The number of cells on day 7 of cell culture was measured on the basis of DAPI fluorometry.
RESULTS AND DISCUSSION Shape of gel microbeads It is known that spherical alginate microbeads are formed when the microbeads are prepared at 1.2 wt.% or higher alginate concentrations (9). The normal and S9-encapsulated gel microbeads prepared for this study and observed under a microscope were spherical (Fig. 2), consistent with the fact that a 2.5 wt.% alginate aqueous solution was used. In addition, the size of normal and S9encapsulated gel microbeads was found to be 480 ± 20 μm and 720 ± 30 μm in diameter, respectively. This difference may be explained as follows: normal microbeads are densely packed with alginate polymers thanks to coordinate bonds between carboxyl groups via Ca2+. Regarding S9-encapsulated microbeads, since the surface of rat liver microsomes is known to be negatively charged at neutral pH (23), microbeads swelled likely due to electrostatic repulsion between parts of carboxyl groups in the gel microbeads and the surfaces of S9 microsomes. Note that reversible expansion/contraction of a coordination bond-based hydrogel is known to be controllable by electrostatic interaction based on dissociation/formation of the coordination bond (24). Biochemical characteristics of S9-encapsulated gel microbeads To determine the metabolic capacity of the S9-encapsulated gel microbeads, we measured the activity of CYP 1A1/2 contained in S9 as an example of the CYPs superfamily by measuring the fluorescence intensity of resorufin, the metabolite of ER by CYP1A1/2 activity (19–21). The plot a in Fig. 3 shows that resorufin production in the culture medium containing 5.0×105 beads L− 1 of S9-encapsulated gel microbeads, 35 μM ER, and cofactors increased for up to 2 h and then leveled off. As a control experiment, plot b in Fig. 3 shows resorufin production in the culture medium containing 5% S9, 35 μM ER, and cofactors. Note that the S9 concentration in the culture medium containing 5% S9 was roughly equal to that containing 5.0×105 beads L− 1 of S9-encapsulated gel microbeads. As can be seen in plot a and b of Fig. 3, the final amount of resorufin produced by 5.0×105 beads L− 1 of S9-encapsulated gel microbeads was actually equal to that by 5% S9. However, the initial rate of resorufin production in the former was slower than in the latter. Based on plot a and b in Fig. 3, each initial rate was determined to be 15 and 19 nM min− 1, respectively. This difference is likely due to the lower diffusion rates of ER and resorufin in the gel microbeads. In addition, we examined leakage of S9 from gel microbeads. After S9-encapsulated gel microbeads were removed from the culture medium containing 35 μM ER and cofactors after 30 min, resorufin production was further measured. If resorufin production increased after removal of the S9-encapsulated gel microbeads from the culture medium, S9 might have leaked out of the gel microbeads. However, resorufin production did not increase after 60 min (Fig. 3, plot c). This
FIG. 1. Schematic illustration of the PE membrane culture insert-based incubation system for the metabolic activation test. (A) S9 is present in both culture insert and culture dish. (B) S9 is present only in the culture insert. (C) S9-encapsulated gel microbeads are present only in the culture insert.
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J. BIOSCI. BIOENG., coating. Correspondingly, S9 (ca. 0.1 μm in diameter or larger) (8) was apparently larger than PLL (Mw = 2.5 × 104) employed in the present work, resulting in stably encapsulation in the gel microbeads. In contrast, the molecular weights of cofactors, ER, and resorufin are smaller than 750, so that they might move in or out of the microbeads regardless of surface modification by PLL. In the present investigation, we focused on the activity of only CYP 1A1/2 in S9. In contrast, it is well-known that S9 contains several CYP enzymes, such as CYP 2B6, CYP 2C19, CYP 2D6, CYP 2E1, and CYP 3A4/ 5 (25). Therefore, S9-encapsulated gel microbeads prepared here might be expected to exhibit the metabolic capacity based on not only CYP 1A1/2 but also other CYP enzymes.
FIG. 2. Microphotographs of (A) normal and (B) S9-encapsulated gel microbeads. Scale bar indicates 500 μm.
result suggests that S9 was retained in the gel microbeads thanks to the formation of fine polymer networks based on a polyion complex between the alginate gel microbead surface and PLL. It has been reported that leakage of hemoglobin (Mw = 6.8 × 104) does not occur from alginate gel microbeads coated with PLL (Mw = (1.7 or 2.5) × 104), whereas chymotrypsin (Mw = 2.5 × 104) can enter the alginate gel microbeads coated with PLL (Mw = 2.5 × 105) (18). This means that the molecular weight of fraction is controlled by PLL
FIG. 3. Time course increase in resorufin concentrations after addition of culture medium containing 35 μM ER and cofactors to the culture medium containing (a and c) 5.0 × 105 beads L− 1 S9-encapsulated gel microbeads or (b) 5% S9. (c) S9-encapsulated gel microbeads were removed from the culture medium after 30 min. All plots in the figure were the mean± SD from at least three independent experiments.
Cytotoxicity of S9-encapsulated gel microbeads As mentioned above, S9 was stably encapsulated in the alginate microbeads. However, it was not clear whether S9-derived microsomal lipid peroxides leaked out of the gel microbeads. Therefore, we determined the cytotoxicity of the S9-encapsulated gel microbeads to NIH3T3 cells incubated in the culture medium containing cofactors. Expecting that cell growth might be inhibited if cells were exposed to toxic lipid peroxidase, cytotoxicity was assessed by measuring cell growth. The cell growth ratio was defined as 100×R/R0, where R and R0 were the numbers of cells in the culture medium at day 7 after an initial 4-hour incubation respectively with and without gel microbeads. We first examined the cytotoxicity of the normal gel microbeads. The surface of the NIH3T3 cells layer in the culture dish was directly and almost entirely covered with 4.4× 102 beads cm− 2 of normal gel microbeads. The cells were incubated for 4 h, followed by further incubation for 7 days without microbeads. Unfortunately, the cell growth ratio on day 7 was nearly 0%. In contrast, the cell growth ratio was nearly 100% when NIH3T3 cells were only exposed to cofactors for 4 h. These results are likely due to the fact that the normal gel microbeads caused physical cell damage by direct contact and/or the cells preferentially adhered to the surface of the normal gel microbeads and removed with the microbeads. In addition, a layer of gel microbeads covering the NIH3T3 cells might interfere with diffusion of O2 into the cells. To avoid these problems, a culture insert filled with 4.4×102 beads cm− 2 of normal gel microbeads was set in the culture dish and the cells were incubated. As a result, the cell growth ratio was nearly 100%. Subsequently, we examined the cytotoxicity of S9 and S9-encapsulated gel microbeads using the culture insert-based incubation system as illustrated in Fig. 1. First, we confirmed the cytotoxicity of S9. NIH3T3 cells were incubated in the S9-dispersed culture medium containing cofactors for 4 h (Fig. 1A), followed by incubation in the culture medium without S9 and cofactors for 7 days. This exposure system corresponds to the conventional test system. As expected, the cell growth ratio on day 7 decreased as S9 concentration increased (Fig. 4, plot a). These results indicate that microsomal lipid peroxides generated by CYPs-based metabolism caused damage to NIH3T3 cells. For comparison, NIH3T3 cells were also incubated in culture medium containing cofactors for 4 h after the culture insert filled with S9 suspension was set in the dish (Fig. 1B), followed by further incubation in culture medium without cofactors for 7 days after removal of the culture insert. As in the case of S9-dispersed culture medium, the cell growth ratio decreased as S9 concentration increased (Fig. 4, plot b). This result indicates that the membrane of the culture insert could not prevent microsomal lipid peroxides from diffusing to and affecting the cells. Thus, the cytotoxicity of S9-derived substances is non-negligible in cytotoxicity tests for reactive metabolites. Next, we evaluated the cytotoxicity of the S9-encapsulated gel microbeads (Fig. 1C). Here, we employed the culture inserts with the membrane surface densely covered with 4.4 × 102 or 8.8 × 102 beads cm− 2 of S9-encapsulated gel microbeads, resulting in S9 concentrations in the 4.1 mL culture medium roughly equivalent to 5 and 10%, respectively. Interestingly, the cell growth ratio was not affected by the number of the S9-encapsulated gel microbeads (Fig. 4, plot c). This result might indicate that the fine polymer network described above prevented
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FIG. 4. Changes in the cell growth ratio of NIH3T3 cells grown in normal culture medium for 7 days after a 4 h incubation in the PE-membrane culture insert-based incubation system. Each plot a, b, and c in this figure corresponds to the schematic illustration A, B, and C in Fig. 1, respectively. All plots in the figure were the mean ± SD from at least three independent experiments.
the leakage of microsomal lipid peroxides from the gel microbeads. Furthermore, it has been shown that the cytotoxicity of S9 is reduced if a certain amount of Ca2+ is added to the culture medium (9). There is a possibility that Ca2+ involved in the formation of the alginate gel microbeads contributes to reduce the cytotoxicity of microsomal lipid peroxides. In any case, the S9-induced cytotoxicity was certainly negligible thanks to encapsulation of S9 in the gel microbeads coated with PLL. Application to metabolic activation tests As S9 cytotoxicity could be neglected by encapsulation of S9, we evaluated the cytotoxicity of the anti-cancer drug CPA (Mw=279) using the S9-encapsulated gel microbeads. It is known that CPA is metabolized to 4-hydroxy-CPA in the liver mainly by CYP 2B6, resulting in cell apoptosis due to DNA alkylation (26). In the present test system (Fig. 1C), CPA is metabolized by CYP 2B6 in S9-encapsulated gel microbeads. Then, 4-hydroxy-CPA diffuses out of the gel microbeads, through the PE membrane, and into NIH3T3 cells, resulting in cell apoptosis. The CPA cytotoxicity was determined on the
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basis of the cell growth ratio on day 7 of cell culture after CPA exposure for 4 h. NIH3T3 cells were also exposed to CPA in the culture medium containing 0% and 5% S9. Dose–response curves were shown in Fig. 5. As can be seen, the dose–response curve in the presence of the S9-encapsulated gel microbeads (plot a) shifted towards lower CPA concentrations compared with that in the absence of S9 (plot b) and towards higher CPA concentrations compared with that in the presence of 5% S9 (plot c). Based in Fig. 5, each median effective concentration (EC50) was estimated at 7.0 × 10− 3 M for no S9, 5.8 × 10− 5 M for S9-encapsulated gel microbeads, and 1.2 × 10− 5 M for 5% S9, respectively. These differences may be explained as follows: in the absence of S9, decrease in the cell growth ratio was attributed to the toxicity of CPA itself. The toxicity mechanism is reckoned to be a disruption of the phospholipid membrane due to permeation and/or accumulation of CPA. In the presence of S9-encapsulated gel microbeads, decrease in the cell growth ratio is mainly attributed to the toxicity of the CPA metabolite by S9-encapsulated gel microbeads, which causes DNA alkylation. In the case of 5% S9, decrease in the cell growth ratio might be attributed to the combined toxicities of CPA- and microsomal lipid-derived metabolites. Although the detailed toxicity mechanism is unclear, use of the S9-encapsulated gel microbeads might prevent the microsomal lipid-derived metabolite toxicity. Thus, the evaluation of reactive metabolites without the S9-induced cytotoxicity is now possible by using S9-encapsulated gel microbeads. Under the present condition, it is necessary to miniaturize the S9encapsulated gel microbeads, because diffusion of chemicals, such as xenobiotis and their metabolites, in and out of the present gel microbeads was limited due to large size as shown in Fig. 3. For example, inkjet techniques (27), which can prepare gel microbeads with diameters in the micrometer range, could enable the large scale production of smaller S9-encapsulated gel microbeads in a short time. In addition, the metabolic kinetic analysis for various model substances is needed. The miniaturized S9-encapsulated gel microbeads would permit direct comparison of net cytotoxicity of reactive metabolites for various types of target cells. ACKNOWLEDGEMENT This work was supported by the Bio Electromechanical Autonomous Nano Systems (BEANS) Project from NEDO, Japan. References
FIG. 5. Dose–response curves of NIH3T3 cells incubated in culture medium for 7 days after 4 h exposure to CPA in the presence of (a) 4.4 × 102 S9-encapsulated gel microbeads per cm2, (b) no S9, and (c) 5% S9. All plots in the figure were the mean ± SD of nine wells from three independent experiments.
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