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An in vitro model for long-term hepatotoxicity testing utilizing rat hepatocytes entrapped in micro-hollow fiber reactor
Biochemical Engineering Journal 34 (2007) 267–272
An in vitro model for long-term hepatotoxicity testing utilizing rat hepatocytes entrapped in micro-hollow fiber reactor Chong Shen a , Guoliang Zhang b , Qin Meng a,∗ a
Department of Chemical Engineering and Biochemical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou, Zhejiang 310027, PR China b Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, PR China Received 12 January 2006; received in revised form 7 December 2006; accepted 8 December 2006
Abstract A long-term hepatocyte model in vitro is preferable for chronic hepatotoxicity research because hepatocytes in this model of culture can preserve liver-specific functions for long period. Micro-hollow fiber reactors (MHFR), composed of polysulphone (PS) hollow fibers with a molecular weight cut-off 100 kDa, were applied to test the hepatotoxicity of acetaminophen, isoniazid and rifampicin, respectively. Monolayer culture was used as a control model for hepatocyte culture. It was found that hepatocytes within MHFR were more sensitive to toxicity of acetaminophen (0.38–1.51 g/L) than those in monolayer cultures. Furthermore, significant hepatotoxicity of isoniazid (15 mg/L) and rifampicin (10 mg/L) were detected in hepatocytes cultured in MHFR but not detected in hepatocyte monolayer, which could be due to well-preserved drug metabolizing enzymes in MHFR. These results indicate that the MHFR may be an effective model for long-term hepatotoxicity research in vitro. © 2006 Elsevier B.V. All rights reserved. Keywords: Micro-hollow fiber reactor; Hepatocyte monolayer; Rat hepatocytes
1. Introduction Monolayer cultures of primary hepatocytes were widely used for hepatotoxicity studies in vitro [1,2]. Unfortunately, hepatocytes in monolayer cultures lost their liver-specific functions such as cytochrome P 450 (CYP 450) activity within 1–3 days [3,4] due to the absence of three-dimensional organization. Because chronic hepatotoxicity in vivo was often caused over several days or weeks, short-term assays in vitro cannot reflect it well. Therefore, long-term models of primary cultured hepatocytes, where liver-specific functions can be maintained for 1–2 weeks, were suggested for investigating chronic toxicity of drugs in vitro. An ideal in vitro model of hepatocytes should recreate an in vivo-like microenvironment [5]. Hepatocytes in such in vitro model should be of three-dimensional structure instead of flat morphology in monolayer culture and possess abundant cytoplasmic organelles, the presence of bile canaliculi and gap junctions [6]. Moreover, the hepatocytes in tissue-like cultures maintained liver-specific functions higher and longer
∗
Corresponding author. Tel.: +86 571 87961091/53193; fax: +86 571 87951227. E-mail addresses: [email protected], [email protected] (Q. Meng). 1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.12.010
than monolayer cultures. Therefore, tissue-like cultures may be a suitable model for investigating chronic hepatotoxicity in vitro [7,8]. Hepatocytes in spheroids and sandwich culture are two typical long-term culture models. Hepatocyte spheroids could sustain several liver-specific functions for several weeks, such as albumin and transferring secretion, CYP 450 activity [9]. Similarly, hepatocytes cultured in sandwich of collagen expressed stable liver-specific functions over weeks [10]. However, for hepatotoxicity studies in vitro, hepatocyte spheroids and sandwich culture still have some disadvantages. First, although sandwich culture of hepatocytes performed better in maintenance of liver-specific functions than hepatocyte monolayer [11], the flattened morphology of hepatocytes [12] and gap junctions disappearance [10] resulted in significant decrease on CYP 450 in sandwich culture compared to hepatocytes in vivo. And hepatocyte spheroids are inconveniently used for toxicology studies in vitro, as a couple of days will be particularly needed to form spheroids. Moreover, hepatocyte spheroids were considered to have diffusion barriers for their tight construction [6]. In spite of other newly formulated tissue-like hepatocyte models [13,14], there is a need to further develop an effectively and conveniently handled model of hepatocyte in vitro system for hepatotoxicity studies.
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Gel entrapment culture of hepatocytes within hollow fibers was initially developed for bio-artificial liver (BAL) [15]. This configuration of BAL supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis and oxygen consumption [15]. Hence, these features make this device a favorable candidate for drug research in vitro. However, BAL, with a large volume over 200 mL and a complicated hemoperfusion circuit including pumps and oxygenators, was not applicable for drug testing. In our previous study, we minimized this bio-artificial liver to a simple micro-hollow fiber reactor (MHFR), which is composed of hollow fibers and silicone tube. In this study, we proposed to discuss the possibility of using such MHFR in hepatotoxicity studies. The monolayer culture of hepatocytes was used as a control. Acetaminophen, an analgesic and antipyretic drug, was first applied as a hepatotoxin to compare its toxicity in rat hepatocyte culture in both monolayer and MHFR. Then, isoniazid and rifampicin, anti-tuberculosis drugs, were included to highlight the long-term performance of hepatocytes in MHFR at usual therapeutic doses.
2.3. Evaluation of drug-induced hepatotoxicity After 4 h incubation of hepatocytes in either monolayer culture or MHFR, the hepatocytes were washed in warm phosphate buffer solution and refilled with culture medium containing 0.38, 0.76 and 1.51 g/L acetaminophen, while non-treated hepatocytes were incubated in the corresponding medium without acetaminophen. Furthermore, different cultures were exposed to 10 mg/L rifampicin and 15 mg/L isoniazid dissolved in Williams’ E medium in the similar way. The culture medium was refreshed every 2 days. 2.4. Cell viability assays
2. Materials and methods
MTT assay was used to evaluate hepatocyte viability. Briefly, hepatocytes were immerged within 650 L of 1.15 mg/mL MTT-phosphate buffer solution in 24-well plates, which were incubated for 3 h in the incubator at 5% CO2 , 37 ◦ C. At the end of incubation, 1.5 mL isopropanol (with 0.4 mol/L HCl) was added to each well. The trays were then shaken 1 h for extracting the indiscerptible colored products and the solution was read on a spectrophotometer with the wavelength of 570 nm [17].
2.1. Chemicals
2.5. Assays on liver-specific functions
Acetaminophen was from Hangzhou Huadong Medicine Group Co. Ltd. (Hangzhou, China). Isoniazid and rifampicin were from Zhejiang Medicine Co. Ltd. Xinchang Pharmaceutical Factory (Shaoxing, China).
Urea assays were performed using a Urea Nitrogen Kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rat albumin was determined by enzymelinked immunosorbent assay (ELISA) by rat albumin ELISA Quantitation Kit obtained from BETHYL laboratories Inc. (Montgomery, USA).
2.2. Rat hepatocyte cultures Rat hepatocytes were isolated using a collagenase perfusion as described previously [13]. Viability of isolated hepatocytes was at least 85% by trypan blue exclusion. Hepatocytes were cultured in a basal medium of Williams’ E (Gibco) complemented with 100 U/mL penicillin and 100 g/mL streptomycin, l-glutamine 2 mmol/L (Amresco) and 5% fetal calf serum (Hangzhou Sijiqing Biological Eng. Material Co. Ltd., China). For monolayer culture, freshly isolated hepatocytes were seeded at a density of 1 × 105 cells/well in collagen-coated 24well plates in 1 mL culture medium which were cultured at 37 ◦ C in a humidified incubator in an atmosphere of 5% CO2 and 95% air. For gel entrapment culture, freshly harvest hepatocytes were mixed with the collagen solution and loaded into hollow fibers by injection [16]. Briefly, hepatocytes were inoculated into a 3:1 (v/v) mixture of type I collagen (3 g/L) and four-fold concentrated Williams’E medium at pH of 7.4. Hepatocyte suspension at cell density of 1 × 106 cells/mL was loaded into the lumen of long fibers with length of 35 cm. Hollow fibers were circuitously put into 12 cm diameter dishes and maintained in a 5% CO2 incubator for collagen gelation. Ten minutes later, hollow fibers were cut into equal four pieces and loaded into a piece of silicone tube full of 5 mL prewarmed culture medium. These tubes, with 0.4 × 106 hepatocytes each, were put back into incubator for hepatocyte cultures.
2.6. Data analysis Values for the different treatments from three separate hepatocyte harvests were compared against the control using t-tests as the statistical method. A p-value less of 0.05 was determined to be significant. All values reported in the text are mean ± standard error mean. 3. Results 3.1. Acetaminophen-induced hepatotoxicity Fig. 1 illustrated the liver-specific functions in acetaminophen treated hepatocytes of monolayer culture, as marked by urea genesis and albumin synthesis. After 72 h exposure to acetaminophen (0.38, 0.76 and 1.51 g/L), accumulated albumin synthesis respectively decreased to 48%, 36%, 28% of control but cumulative urea genesis did not decrease significantly. The effects of acetaminophen on rat hepatocytes within MHFR were shown in Fig. 2. Different from Fig. 1, both urea genesis and albumin synthesis significantly decreased compared to the control culture without exposure to acetaminophen. For an example, after 72 h treatment by acetaminophen at concentrations of 0.38, 0.76 and 1.51 g/L, cumulative urea decreased to 64%, 36% and 26% of the control value, while
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Fig. 1. Effect of increasing concentrations of acetaminophen on cumulative urea and albumin in rat hepatocyte monolayer: (♦) control, () acetaminophen 0.38 g/L, () acetaminophen 0.76 g/L, () acetaminophen 1.51 g/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, *** p < 0.001 compared with controls.
Fig. 2. Effect of increasing concentrations of acetaminophen on cumulative urea, and albumin in rat hepatocytes in MHFR: (♦) control () acetaminophen 0.38 g/L, () acetaminophen 0.76 g/L, () acetaminophen 1.51 g/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
albumin production more drastically decreased to 23%, 22% and 7% of controls, respectively. Furthermore, cell viability in both cultures was determined by MTT assay, as shown in Fig. 3. The cell viability of acetaminophen treatment was expressed as a percentage of the relative controls. There were remarkable differences of acetaminophen hepatotoxicity between hepatocytes within MHFR and hepatocytes in monolayer. Hepatocytes within MHFR significantly decreased cell viability after treatment of acetaminophen at various concentrations for 24 and 72 h, respectively, while hepatocyte monolayer only showed reluctant cell
Fig. 3. Rat hepatocytes in monolayer culture and within MHFR were exposed to the different concentrations of acetaminophen. The results at each acetaminophen concentration were compared with the control () acetaminophen 0.38 g/L, ( ) acetaminophen 0.76 g/L, () acetaminophen 1.51 g/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
damage after long exposure for 72 h but no response at short exposure for 24 h. These results again confirmed the more sensitivity of hepatocytes in MHFR than in monolayer culture when exposed to acetaminophen. 3.2. Effects of rifampicin and isoniazid on rat hepatocytes within MHFR and monolayer culture Hepatocyte monolayer was treated by 10 mg/L rifampicin and 15 mg/L isoniazid for 5 days and their liver-specific functions were shown in Fig. 4. Different from acetaminophen, rifampicin and isoniazid were studied at low concentrations comparable to their plasma concentrations in vivo. Interestingly, hepatocyte monolayer after exposure to either rifampicin or isoniazid did not show obvious decrease on both albumin synthesis and urea genesis compared to control. Under phase contrast microscope, morphology of hepatocyte monolayer after exposure to rifampicin and isoniazid also did not show any difference from that in control. The effects of anti-tuberculosis drugs on rat hepatocytes within MHFR were presented in Fig. 5. In contrast with hepatocyte monolayer, hepatocytes in MHFR elicited significant hepatotoxicity following drug treatment with either 10 mg/L rifampicin or 15 mg/L isoniazid for 4 days (* p < 0.05). Moreover, at 8 days, urea levels of hepatocytes, treated by 10 mg/L rifampicin and 15 mg/L isoniazid, decreased to approximately
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Fig. 4. Effect of rifampicin and isoniazid on cumulative urea and albumin in rat hepatocyte monolayer: (♦) control, () rifampicin 10 mg/L, () isoniazid 15 mg/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05 compared with controls.
Fig. 5. Effect of rifampicin and isoniazid on cumulative urea and albumin on rat hepatocytes within MHFR: (♦) control, () rifampicin 10 mg/L, () isoniazid 15 mg/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
80% of controls. More significant decrease on albumin production was observed with a rapid drop to 50% at an exposure time of 8 days. 4. Discussion It is well known that a number of liver-specific functions, such as CYP 450 activities, decreased drastically in conventional culture of hepatocyte monolayer. Therefore, long-term cultures of hepatocytes in three-dimensional configuration were preferable for hepatotoxicity studies. Similar to spheroid culture [9] and collagen gel sandwich culture [10], hepatocytes entrapped in collagens of hollow fiber-based BAL can sustain long viability and high liver-specific functions of hepatocytes [15]. To improve the feasibility of using such culture as a toxicity model in vitro, we minimized the typical hollow fiber bioreactor into our simple MHFR. The average performance of hepatocytes in MHFR within 8 days culture was evaluated by dividing the accumulated urea or albumin production by culture time and these average values were listed in Table 1 together with
those values from monolayer culture at a culture period of 5 days. In comparison to monolayer, the hepatocytes in MHFR expressed double albumin secretion and 18% higher of urea genesis. The higher liver-specific functions expressed by hepatocytes in MHFR than monolayer culture were consistent with the results reported in hollow-fiber-based BAL [18,19]. According to our previous studies, the activity of cytochrome P450 1A2 in rat hepatocytes within MHFR was also two-fold of that on monolayer [20], corresponding well with the previous report on BAL [19]. Therefore, we can concluded that the minimization of BAL did not decrease the liver-specific functions of entrapped hepatocytes, which may be due to the extracellular matrix of collagen and the cytocompatible hollow fibers used in this kind of MHFR. The hepatocytes within MHFR showed the cuboidal shape, which dispersed into collagen gel equably and occasionally aggregated to small aggregates. The hollow fiber served like the blood vessel in vivo and the collagen gel immobilized the cells as in liver. Because of the well simulation of MHFR to microenvironment in vivo, the hepatocytes in MHFR can maintain the liver-specific functions for a long time in vitro. Hence,
Table 1 Comparison of urea and albumin secretions in hepatocyte monolayer and hepatocyte within MHFR culture Culture mode
Average urea genesis (pg/cell h)
Average albumin secretion (pg/cell h)
Hepatocyte monolayer (for 5 days culture) Hepatocytes in MHFR (for 8 days culture) Statistical assay (compared to monolayer)
17.38 ± 0.68 20.54 ± 0.22** ** p < 0.01
0.52 ± 0.07 1.02 ± 0.03** ** p < 0.01
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our primary results on performance of hepatocytes in MHFR suggest that this system could be a suitable long-term model for chronic hepatotoxicity testing. Thus, the possibility of this culture system as an in vitro hepatotoxicity model was further evaluated using tree hepatotoxic drugs in this study. Acetaminophen, a widely used analgesic and antipyretic drug, is safe at its usual therapeutic doses but shows hepatic toxicity at high doses. It is well established that N-acetyl-p-benzoquinoneimine (NAPQI), metabolized from acetaminophen by CYP 2E1, reduces cellular glutathione (GSH) storage and thus causes hepatotoxicity [21–23]. In our study, hepatocytes in MHFR with exposure to various acetaminophen presented concentration- and time-dependent hepatotoxicity according to MTT reduction and decrease on liver-specific functions, marked by urea genesis and albumin synthesis. And these toxic responses of hepatocytes in MHFR were more sensitive than those in monolayer. It is most likely that more NAPQI was accumulated by hepatocyte in gel entrapment culture in MHFR due to higher level of maintenance of CYP 2E1 than in monolayer culture. In addition, it should be mentioned that the concentrations of tested acetaminophen used here were considerably higher than plasma concentrations. For example, at a dose of 50 mg/kg (i.p.) acetaminophen for rats, peak plasma concentrations reached 26.3 mg/L [24]. In sandwich cultured rat hepatocytes, it was reported that acetaminophen at 0.15 g/L could not induce the significant hepatotoxicity [25]. While in rat hepatocyte spheroids, the detected concentration of acetaminophen at intriguing slight hepatotoxicity was about 0.15 g/L as indicated by energy metabolism assays [26], which may indicate the higher sensitivity of hepatocyte spheroids than sandwich cultured hepatocytes to acetaminophen treatment. According to our results, hepatocytes within MHFR were severe damaged by acetaminophen at a concentration of 0.38 g/L, suggested that the treatment concentration could be reduced to a concentration as low as 0.15 g/L. To interpret the chronic in vivo toxicity, isoniazid and rifampicin were used as model chronic hepatotoxins. Isoniazid and rifampicin are currently the extensively used anti-tuberculosis drugs. Clinical investigations and animal studies frequently evidenced the hepatotoxicity of isoniazid and rifampicin at therapeutic doses [27–30]. As the mechanisms of the metabolism of isoniazid and rifampicin were not very clear yet, oxidative stress was proposed as one mechanism responsible for hepatic injury induced by isoniazid and rifampicin [30]. The oxidative stress was closely associated with decrease of glutathione levels [24]. Considering that peak plasma concentrations were reported as 11.5 and 5.5 mg/L at therapeutic doses of rifampicin (600 mg, orally) and isoniazid (300 mg, orally) for tuberculosis patients [31], we selected the concentrations comparable to peak plasma concentrations for in vitro testing. According to our experimental results in Fig. 4, hepatocyte monolayer exposed to 10 mg/L rifampicin and 15 mg/L isoniazid did not show difference in both urea genesis and albumin synthesis within 5 days’ culture. In contrast, hepatocytes in MHFR cultures at treatment of either 10 mg/L rifampicin or 15 mg/L isoniazid for 4 days started to show obvious hepatotoxicity and this cellular damage was more severe with subsequent
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culture time. In addition, the detected hepatotoxic concentration of isoniazid were reported around 650 mg/L as indicated by pyruvate uptake and lactic acid release in hepatocyte spheroids [26], which may be due to the high diffusion barriers [6]. And the insensitivity of spheroid cultures to other hepatotoxins were also reported in literature [32,33], which supported the hypothesis of high diffusion barriers in hepatocyte spheroids. Because hepatotoxicity of either isoniazid or rifampicin was only detected in hepatocytes cultured in MHFR, it is indicative that hepatocytes within MHFR could be a suitable long-term hepatocyte model for drug hepatotoxicity testing. It was noticed that hepatocytes in monolayer culture showed cytotoxic responses to rifampicin and isoniazid at very high concentrations of 1.0 and 1.5 g/L, respectively. In another word, 100-fold higher concentrations of rifampicin and isoniazid were cytotoxic to hepatocytes in monolayer culture than their detected cytotoxic concentrations in MHFR or in vivo. Coincidently, tacrine was reported to cause hepatotoxicity to primary hepatocyte in monolayer culture at concentration of 0.5 mmol/L, 100–1000-fold higher than those observed in vivo at the onset of hepatotoxicity [3]. These results on less sensitivity of hepatocyte monolayer could be due to the decreased expression of CYP 450 in monolayer culture. In summary, the aim of our study was to assess the feasibility of MHFR for hepatotoxicity research in vitro. Hepatocyte culture in MHFR was used as a biology system to test drug-induced hepatotoxicity and monolayer culture, an extensively used model in hepatotoxicity studies, was used a control system. The results from MHFR cultures indicated that it should be a suitable long-term culture model in testing drug-induced hepatotoxicity, since its urea secretion and rat albumin synthesis were higher than those in monolayer cultures. Three drugs, acetaminophen, rifampicin and isoniazid, were used as hepatotoxic drugs and it was found that hepatocytes cultured in MHFR appeared much more sensitive to the effects of drugs compared to hepatocyte monolayer. It implied that hepatocyte culture in MHFR could be a suitable model for drug hepatotoxicity research in vitro than hepatocytes in monolayer. Acknowledgments This research was supported by grants (No. 20576119) from NSFC (National Natural Science Foundation of China) and the Ph.D. Programs Foundation of Ministry of Education of China (No. 20060335083). References [1] C.J. Viau, R.D. Curren, K. Wallace, Cytotoxicity of tacrine and velnacrine metabolites in cultured rat, dog and human hepatocytes, Drug Chem. Toxicol. 16 (3) (1993) 227–239. [2] T. Harauchi, M. Hirata, Effect of P-450 inducers on glutathione (GSH) depletion by bromobenzene in primary cultures of dog hepatocytes, Biol. Pharm. Bull. 17 (5) (1994) 658–661. [3] D. Lagadic-Gossmann, M. Rissel, M.A. Le Bot, A. Guillouzo, Toxic effects of tacrine on primary hepatocytes and liver epithelial cells in culture, Cell Biol. Toxicol. 14 (5) (1998) 361–373.
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[4] E.F. Brandon, C.D. Raap, I. Meijerman, J.H. Beijnen, J.H. Schellens, An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons, Toxicol. Appl. Pharmacol. 189 (3) (2003) 233–246. [5] K. Bhadriraju, C.S. Chen, Engineering cellular microenvironments to improve cell-based drug testing, Drug Discov. Today 7 (11) (2002) 612–620. [6] T.M. Walker, P.C. Rhodes, C. Westmoreland, The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures, Toxicol. In Vitro 14 (5) (2000) 475–485. [7] J.Z. Tong, P. De Lagausie, V. Furlan, T. Cresteil, O. Bernard, F. Alvarez, Long-term culture of adult rat hepatocyte spheroids, Exp. Cell Res. 200 (2) (1992) 326–332. [8] S. Beken, T. Vanhaecke, K. De Smet, M. Pauwels, A. Vercruysse, V. Rogiers, Collagen-gel cultures of rat hepatocytes: collagen-gel sandwich and immobilization cultures, Meth. Mol. Biol. 107 (1998) 303–309. [9] T.M. Walker, A.J. Woodrooffe, Cytochrome P450 activity in control and induced long-term cultures of rat hepatocyte spheroids, Toxicol. In Vitro 15 (6) (2001) 713–719. [10] F. Berthiaume, P.V. Moghe, M. Toner, M.L. Yarmush, Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration, FASEB J. 10 (13) (1996) 1471–1484. [11] D. Farkas, S.R. Tannenbaum, Characterization of chemically induced hepatotoxicity in collagen sandwiches of rat hepatocytes, Toxicol. Sci. 85 (2) (2005) 927–934. [12] J.C. Dunn, R.G. Tompkins, M.L. Yarmush, Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration, Biotechnol. Prog. 7 (3) (1991) 237–245. [13] R. Gebhardt, J.G. Hengstler, D. Muller, R. Glockner, P. Buenning, B. Laube, E. Schmelzer, M. Ullrich, D. Utesch, N. Hewitt, M. Ringel, B.R. Hilz, A. Bader, A. Langsch, T. Koose, H.J. Burger, J. Maas, F. Oesch, New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures, Drug Metab. Rev. 35 (2–3) (2003) 145– 213. [14] M. Ringel, M.A. von Mach, R. Santos, P.J. Feilen, M. Brulport, M. Hermes, A.W. Bauer, W. Schormann, B. Tanner, M.R. Schon, F. Oesch, J.G. Hengstler, Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction, Toxicology 206 (1) (2005) 153– 167. [15] T.D. Sielaff, S.L. Nyberg, M.D. Rollins, M.Y. Hu, B. Amiot, A. Lee, F.J. Wu, W.S. Hu, F.B. Cerra, Characterization of the three-compartment gelentrapment porcine hepatocyte bioartificial liver, Cell Biol. Toxicol. 13 (4–5) (1997) 357–364. [16] D.Q. Wu, G.L. Zhang, C. Shen, Q. Zhao, H. Li, Q. Meng, Evaluation of diffusion in gel entrapment cell culture within hollow fibers, World J. Gastroenterol. 11 (11) (2005) 1599–1604. [17] H.Z. Wang, C.H. Chang, C.P. Lin, M.C. Tsai, Using MTT viability assay to test the cytotoxicity of antibiotics and steroid to cultured porcine corneal endothelial cells, J. Ocul. Pharmacol. Ther. 12 (1) (1996) 35–43.
[18] S.F. Abu-Absi, G. Seth, R.A. Narayanan, K. Groehler, P. Lai, M.L. Anderson, T. Sielaff, W.S. Hu, Characterization of a hollow fiber bioartificial liver device, Artif. Organs 29 (5) (2005) 419–422. [19] S.L. Nyberg, K. Shirabe, M.V. Peshwa, T.D. Sielaff, P.L. Crotty, H.J. Mann, R.P. Remmel, W.D. Payne, W.S. Hu, F.B. Cerra, Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions, Cell Transplant. 2 (6) (1993) 441–452. [20] H.X. Qiu, G.G. Su, X. Tang, Q. Meng, Tissue-like cultures of rat hepatocytes in study of phase I and phase II drug metabolism, J. Zhejiang Univ. Med. Sci. 35 (5) (2006) 541–546. [21] C. Cheung, A.M. Yu, J.M. Ward, K.W. Krausz, T.E. Akiyama, L. Feigenbaum, F.J. Gonzalez, The cyp2e1-humanized transgenic mouse: role of cyp2e1 in acetaminophen hepatotoxicity, Drug Metab. Dispos. 33 (3) (2005) 449–457. [22] B.E. Jones, H. Liu, C.R. Lo, D.R. Koop, M.J. Czaja, Cytochrome P450 2E1 expression induces hepatocyte resistance to cell death from oxidative stress, Antioxid. Redox. Signal. 4 (5) (2002) 701–709. [23] M.L. Bajt, T.R. Knight, J.J. Lemasters, H. Jaeschke, Acetaminopheninduced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine, Toxicol. Sci. 80 (2) (2004) 343–349. [24] A. Tran, J.M. Treluyer, E. Rey, J. Barbet, G. Ferracci, P. d’Athis, J. Vincent, G. Pons, Protective effect of stiripentol on acetaminophen-induced hepatotoxicity in rat, Toxicol. Appl. Pharmacol. 170 (3) (2001) 145–152. [25] D. Farkas, S.R. Tannenbaum, Characterization of chemically induced hepatotoxicity in collagen sandwiches of rat hepatocytes, Toxicol. Sci. 85 (2) (2005) 927–934. [26] J. Xu, W.M. Purcell, Energy metabolism and biotransformation as endpoints to pre-screen hepatotoxicity using a liver spheroid model, Toxicol. Appl. Pharmacol. 216 (2) (2006) 293–302. [27] W.W. Yew, Clinically significant interactions with drugs used in the treatment of tuberculosis, Drug Saf. 25 (2) (2002) 111–133. [28] S. Karthikeyan, Isoniazid and rifampicin treatment on phospholipids and their subfractions in liver tissue of rabbits, Drug Chem. Toxicol. 28 (3) (2005) 273–280. [29] D.J. Girling, The hepatic toxicity of antituberculosis regimens containing isoniazid, rifampicin and pyrazinamide, Tubercle 59 (1) (1978) 13–32. [30] S. Attri, S.V. Rana, K. Vaiphei, C.P. Sodhi, R. Katyal, R.C. Goel, C.K. Nain, K. Singh, Isoniazid- and rifampicin-induced oxidative hepatic injury—protection by N-acetylcysteine, Hum. Exp. Toxicol. 19 (9) (2000) 517–522. [31] S. Agrawal, I. Singh, K.J. Kaur, S.R. Bhade, C.L. Kaul, R. Panchagnula, Comparative bioavailability of rifampicin, isoniazid and pyrazinamide from a four drug fixed dose combination with separate formulations at the same dose levels, Int. J. Pharm. 276 (1–2) (2004) 41–49. [32] C. Dilworth, G.A. Hamilton, E. George, J.A. Timbrell, The use of liver spheroids as an in vitro model for studying induction of the stress response as a marker of chemical toxicity, Toxicol. In Vitro 14 (2) (2000) 169–176. [33] T. Battle, T. Maguire, H. Moulsdale, A. Doyle, Progressive maturation resistance to microcystin-LR cytotoxicity in two different hepatospheroidal models, Cell Biol. Toxicol. 15 (1) (1999) 3–12.
An in vitro model for long-term hepatotoxicity testing utilizing rat hepatocytes entrapped in micro-hollow fiber reactor Chong Shen a , Guoliang Zhang b , Qin Meng a,∗ a
Department of Chemical Engineering and Biochemical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou, Zhejiang 310027, PR China b Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, PR China Received 12 January 2006; received in revised form 7 December 2006; accepted 8 December 2006
Abstract A long-term hepatocyte model in vitro is preferable for chronic hepatotoxicity research because hepatocytes in this model of culture can preserve liver-specific functions for long period. Micro-hollow fiber reactors (MHFR), composed of polysulphone (PS) hollow fibers with a molecular weight cut-off 100 kDa, were applied to test the hepatotoxicity of acetaminophen, isoniazid and rifampicin, respectively. Monolayer culture was used as a control model for hepatocyte culture. It was found that hepatocytes within MHFR were more sensitive to toxicity of acetaminophen (0.38–1.51 g/L) than those in monolayer cultures. Furthermore, significant hepatotoxicity of isoniazid (15 mg/L) and rifampicin (10 mg/L) were detected in hepatocytes cultured in MHFR but not detected in hepatocyte monolayer, which could be due to well-preserved drug metabolizing enzymes in MHFR. These results indicate that the MHFR may be an effective model for long-term hepatotoxicity research in vitro. © 2006 Elsevier B.V. All rights reserved. Keywords: Micro-hollow fiber reactor; Hepatocyte monolayer; Rat hepatocytes
1. Introduction Monolayer cultures of primary hepatocytes were widely used for hepatotoxicity studies in vitro [1,2]. Unfortunately, hepatocytes in monolayer cultures lost their liver-specific functions such as cytochrome P 450 (CYP 450) activity within 1–3 days [3,4] due to the absence of three-dimensional organization. Because chronic hepatotoxicity in vivo was often caused over several days or weeks, short-term assays in vitro cannot reflect it well. Therefore, long-term models of primary cultured hepatocytes, where liver-specific functions can be maintained for 1–2 weeks, were suggested for investigating chronic toxicity of drugs in vitro. An ideal in vitro model of hepatocytes should recreate an in vivo-like microenvironment [5]. Hepatocytes in such in vitro model should be of three-dimensional structure instead of flat morphology in monolayer culture and possess abundant cytoplasmic organelles, the presence of bile canaliculi and gap junctions [6]. Moreover, the hepatocytes in tissue-like cultures maintained liver-specific functions higher and longer
∗
Corresponding author. Tel.: +86 571 87961091/53193; fax: +86 571 87951227. E-mail addresses: [email protected], [email protected] (Q. Meng). 1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.12.010
than monolayer cultures. Therefore, tissue-like cultures may be a suitable model for investigating chronic hepatotoxicity in vitro [7,8]. Hepatocytes in spheroids and sandwich culture are two typical long-term culture models. Hepatocyte spheroids could sustain several liver-specific functions for several weeks, such as albumin and transferring secretion, CYP 450 activity [9]. Similarly, hepatocytes cultured in sandwich of collagen expressed stable liver-specific functions over weeks [10]. However, for hepatotoxicity studies in vitro, hepatocyte spheroids and sandwich culture still have some disadvantages. First, although sandwich culture of hepatocytes performed better in maintenance of liver-specific functions than hepatocyte monolayer [11], the flattened morphology of hepatocytes [12] and gap junctions disappearance [10] resulted in significant decrease on CYP 450 in sandwich culture compared to hepatocytes in vivo. And hepatocyte spheroids are inconveniently used for toxicology studies in vitro, as a couple of days will be particularly needed to form spheroids. Moreover, hepatocyte spheroids were considered to have diffusion barriers for their tight construction [6]. In spite of other newly formulated tissue-like hepatocyte models [13,14], there is a need to further develop an effectively and conveniently handled model of hepatocyte in vitro system for hepatotoxicity studies.
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Gel entrapment culture of hepatocytes within hollow fibers was initially developed for bio-artificial liver (BAL) [15]. This configuration of BAL supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis and oxygen consumption [15]. Hence, these features make this device a favorable candidate for drug research in vitro. However, BAL, with a large volume over 200 mL and a complicated hemoperfusion circuit including pumps and oxygenators, was not applicable for drug testing. In our previous study, we minimized this bio-artificial liver to a simple micro-hollow fiber reactor (MHFR), which is composed of hollow fibers and silicone tube. In this study, we proposed to discuss the possibility of using such MHFR in hepatotoxicity studies. The monolayer culture of hepatocytes was used as a control. Acetaminophen, an analgesic and antipyretic drug, was first applied as a hepatotoxin to compare its toxicity in rat hepatocyte culture in both monolayer and MHFR. Then, isoniazid and rifampicin, anti-tuberculosis drugs, were included to highlight the long-term performance of hepatocytes in MHFR at usual therapeutic doses.
2.3. Evaluation of drug-induced hepatotoxicity After 4 h incubation of hepatocytes in either monolayer culture or MHFR, the hepatocytes were washed in warm phosphate buffer solution and refilled with culture medium containing 0.38, 0.76 and 1.51 g/L acetaminophen, while non-treated hepatocytes were incubated in the corresponding medium without acetaminophen. Furthermore, different cultures were exposed to 10 mg/L rifampicin and 15 mg/L isoniazid dissolved in Williams’ E medium in the similar way. The culture medium was refreshed every 2 days. 2.4. Cell viability assays
2. Materials and methods
MTT assay was used to evaluate hepatocyte viability. Briefly, hepatocytes were immerged within 650 L of 1.15 mg/mL MTT-phosphate buffer solution in 24-well plates, which were incubated for 3 h in the incubator at 5% CO2 , 37 ◦ C. At the end of incubation, 1.5 mL isopropanol (with 0.4 mol/L HCl) was added to each well. The trays were then shaken 1 h for extracting the indiscerptible colored products and the solution was read on a spectrophotometer with the wavelength of 570 nm [17].
2.1. Chemicals
2.5. Assays on liver-specific functions
Acetaminophen was from Hangzhou Huadong Medicine Group Co. Ltd. (Hangzhou, China). Isoniazid and rifampicin were from Zhejiang Medicine Co. Ltd. Xinchang Pharmaceutical Factory (Shaoxing, China).
Urea assays were performed using a Urea Nitrogen Kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rat albumin was determined by enzymelinked immunosorbent assay (ELISA) by rat albumin ELISA Quantitation Kit obtained from BETHYL laboratories Inc. (Montgomery, USA).
2.2. Rat hepatocyte cultures Rat hepatocytes were isolated using a collagenase perfusion as described previously [13]. Viability of isolated hepatocytes was at least 85% by trypan blue exclusion. Hepatocytes were cultured in a basal medium of Williams’ E (Gibco) complemented with 100 U/mL penicillin and 100 g/mL streptomycin, l-glutamine 2 mmol/L (Amresco) and 5% fetal calf serum (Hangzhou Sijiqing Biological Eng. Material Co. Ltd., China). For monolayer culture, freshly isolated hepatocytes were seeded at a density of 1 × 105 cells/well in collagen-coated 24well plates in 1 mL culture medium which were cultured at 37 ◦ C in a humidified incubator in an atmosphere of 5% CO2 and 95% air. For gel entrapment culture, freshly harvest hepatocytes were mixed with the collagen solution and loaded into hollow fibers by injection [16]. Briefly, hepatocytes were inoculated into a 3:1 (v/v) mixture of type I collagen (3 g/L) and four-fold concentrated Williams’E medium at pH of 7.4. Hepatocyte suspension at cell density of 1 × 106 cells/mL was loaded into the lumen of long fibers with length of 35 cm. Hollow fibers were circuitously put into 12 cm diameter dishes and maintained in a 5% CO2 incubator for collagen gelation. Ten minutes later, hollow fibers were cut into equal four pieces and loaded into a piece of silicone tube full of 5 mL prewarmed culture medium. These tubes, with 0.4 × 106 hepatocytes each, were put back into incubator for hepatocyte cultures.
2.6. Data analysis Values for the different treatments from three separate hepatocyte harvests were compared against the control using t-tests as the statistical method. A p-value less of 0.05 was determined to be significant. All values reported in the text are mean ± standard error mean. 3. Results 3.1. Acetaminophen-induced hepatotoxicity Fig. 1 illustrated the liver-specific functions in acetaminophen treated hepatocytes of monolayer culture, as marked by urea genesis and albumin synthesis. After 72 h exposure to acetaminophen (0.38, 0.76 and 1.51 g/L), accumulated albumin synthesis respectively decreased to 48%, 36%, 28% of control but cumulative urea genesis did not decrease significantly. The effects of acetaminophen on rat hepatocytes within MHFR were shown in Fig. 2. Different from Fig. 1, both urea genesis and albumin synthesis significantly decreased compared to the control culture without exposure to acetaminophen. For an example, after 72 h treatment by acetaminophen at concentrations of 0.38, 0.76 and 1.51 g/L, cumulative urea decreased to 64%, 36% and 26% of the control value, while
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Fig. 1. Effect of increasing concentrations of acetaminophen on cumulative urea and albumin in rat hepatocyte monolayer: (♦) control, () acetaminophen 0.38 g/L, () acetaminophen 0.76 g/L, () acetaminophen 1.51 g/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, *** p < 0.001 compared with controls.
Fig. 2. Effect of increasing concentrations of acetaminophen on cumulative urea, and albumin in rat hepatocytes in MHFR: (♦) control () acetaminophen 0.38 g/L, () acetaminophen 0.76 g/L, () acetaminophen 1.51 g/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
albumin production more drastically decreased to 23%, 22% and 7% of controls, respectively. Furthermore, cell viability in both cultures was determined by MTT assay, as shown in Fig. 3. The cell viability of acetaminophen treatment was expressed as a percentage of the relative controls. There were remarkable differences of acetaminophen hepatotoxicity between hepatocytes within MHFR and hepatocytes in monolayer. Hepatocytes within MHFR significantly decreased cell viability after treatment of acetaminophen at various concentrations for 24 and 72 h, respectively, while hepatocyte monolayer only showed reluctant cell
Fig. 3. Rat hepatocytes in monolayer culture and within MHFR were exposed to the different concentrations of acetaminophen. The results at each acetaminophen concentration were compared with the control () acetaminophen 0.38 g/L, ( ) acetaminophen 0.76 g/L, () acetaminophen 1.51 g/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
damage after long exposure for 72 h but no response at short exposure for 24 h. These results again confirmed the more sensitivity of hepatocytes in MHFR than in monolayer culture when exposed to acetaminophen. 3.2. Effects of rifampicin and isoniazid on rat hepatocytes within MHFR and monolayer culture Hepatocyte monolayer was treated by 10 mg/L rifampicin and 15 mg/L isoniazid for 5 days and their liver-specific functions were shown in Fig. 4. Different from acetaminophen, rifampicin and isoniazid were studied at low concentrations comparable to their plasma concentrations in vivo. Interestingly, hepatocyte monolayer after exposure to either rifampicin or isoniazid did not show obvious decrease on both albumin synthesis and urea genesis compared to control. Under phase contrast microscope, morphology of hepatocyte monolayer after exposure to rifampicin and isoniazid also did not show any difference from that in control. The effects of anti-tuberculosis drugs on rat hepatocytes within MHFR were presented in Fig. 5. In contrast with hepatocyte monolayer, hepatocytes in MHFR elicited significant hepatotoxicity following drug treatment with either 10 mg/L rifampicin or 15 mg/L isoniazid for 4 days (* p < 0.05). Moreover, at 8 days, urea levels of hepatocytes, treated by 10 mg/L rifampicin and 15 mg/L isoniazid, decreased to approximately
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Fig. 4. Effect of rifampicin and isoniazid on cumulative urea and albumin in rat hepatocyte monolayer: (♦) control, () rifampicin 10 mg/L, () isoniazid 15 mg/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05 compared with controls.
Fig. 5. Effect of rifampicin and isoniazid on cumulative urea and albumin on rat hepatocytes within MHFR: (♦) control, () rifampicin 10 mg/L, () isoniazid 15 mg/L. The data were represented as the mean ± S.E. of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with controls.
80% of controls. More significant decrease on albumin production was observed with a rapid drop to 50% at an exposure time of 8 days. 4. Discussion It is well known that a number of liver-specific functions, such as CYP 450 activities, decreased drastically in conventional culture of hepatocyte monolayer. Therefore, long-term cultures of hepatocytes in three-dimensional configuration were preferable for hepatotoxicity studies. Similar to spheroid culture [9] and collagen gel sandwich culture [10], hepatocytes entrapped in collagens of hollow fiber-based BAL can sustain long viability and high liver-specific functions of hepatocytes [15]. To improve the feasibility of using such culture as a toxicity model in vitro, we minimized the typical hollow fiber bioreactor into our simple MHFR. The average performance of hepatocytes in MHFR within 8 days culture was evaluated by dividing the accumulated urea or albumin production by culture time and these average values were listed in Table 1 together with
those values from monolayer culture at a culture period of 5 days. In comparison to monolayer, the hepatocytes in MHFR expressed double albumin secretion and 18% higher of urea genesis. The higher liver-specific functions expressed by hepatocytes in MHFR than monolayer culture were consistent with the results reported in hollow-fiber-based BAL [18,19]. According to our previous studies, the activity of cytochrome P450 1A2 in rat hepatocytes within MHFR was also two-fold of that on monolayer [20], corresponding well with the previous report on BAL [19]. Therefore, we can concluded that the minimization of BAL did not decrease the liver-specific functions of entrapped hepatocytes, which may be due to the extracellular matrix of collagen and the cytocompatible hollow fibers used in this kind of MHFR. The hepatocytes within MHFR showed the cuboidal shape, which dispersed into collagen gel equably and occasionally aggregated to small aggregates. The hollow fiber served like the blood vessel in vivo and the collagen gel immobilized the cells as in liver. Because of the well simulation of MHFR to microenvironment in vivo, the hepatocytes in MHFR can maintain the liver-specific functions for a long time in vitro. Hence,
Table 1 Comparison of urea and albumin secretions in hepatocyte monolayer and hepatocyte within MHFR culture Culture mode
Average urea genesis (pg/cell h)
Average albumin secretion (pg/cell h)
Hepatocyte monolayer (for 5 days culture) Hepatocytes in MHFR (for 8 days culture) Statistical assay (compared to monolayer)
17.38 ± 0.68 20.54 ± 0.22** ** p < 0.01
0.52 ± 0.07 1.02 ± 0.03** ** p < 0.01
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our primary results on performance of hepatocytes in MHFR suggest that this system could be a suitable long-term model for chronic hepatotoxicity testing. Thus, the possibility of this culture system as an in vitro hepatotoxicity model was further evaluated using tree hepatotoxic drugs in this study. Acetaminophen, a widely used analgesic and antipyretic drug, is safe at its usual therapeutic doses but shows hepatic toxicity at high doses. It is well established that N-acetyl-p-benzoquinoneimine (NAPQI), metabolized from acetaminophen by CYP 2E1, reduces cellular glutathione (GSH) storage and thus causes hepatotoxicity [21–23]. In our study, hepatocytes in MHFR with exposure to various acetaminophen presented concentration- and time-dependent hepatotoxicity according to MTT reduction and decrease on liver-specific functions, marked by urea genesis and albumin synthesis. And these toxic responses of hepatocytes in MHFR were more sensitive than those in monolayer. It is most likely that more NAPQI was accumulated by hepatocyte in gel entrapment culture in MHFR due to higher level of maintenance of CYP 2E1 than in monolayer culture. In addition, it should be mentioned that the concentrations of tested acetaminophen used here were considerably higher than plasma concentrations. For example, at a dose of 50 mg/kg (i.p.) acetaminophen for rats, peak plasma concentrations reached 26.3 mg/L [24]. In sandwich cultured rat hepatocytes, it was reported that acetaminophen at 0.15 g/L could not induce the significant hepatotoxicity [25]. While in rat hepatocyte spheroids, the detected concentration of acetaminophen at intriguing slight hepatotoxicity was about 0.15 g/L as indicated by energy metabolism assays [26], which may indicate the higher sensitivity of hepatocyte spheroids than sandwich cultured hepatocytes to acetaminophen treatment. According to our results, hepatocytes within MHFR were severe damaged by acetaminophen at a concentration of 0.38 g/L, suggested that the treatment concentration could be reduced to a concentration as low as 0.15 g/L. To interpret the chronic in vivo toxicity, isoniazid and rifampicin were used as model chronic hepatotoxins. Isoniazid and rifampicin are currently the extensively used anti-tuberculosis drugs. Clinical investigations and animal studies frequently evidenced the hepatotoxicity of isoniazid and rifampicin at therapeutic doses [27–30]. As the mechanisms of the metabolism of isoniazid and rifampicin were not very clear yet, oxidative stress was proposed as one mechanism responsible for hepatic injury induced by isoniazid and rifampicin [30]. The oxidative stress was closely associated with decrease of glutathione levels [24]. Considering that peak plasma concentrations were reported as 11.5 and 5.5 mg/L at therapeutic doses of rifampicin (600 mg, orally) and isoniazid (300 mg, orally) for tuberculosis patients [31], we selected the concentrations comparable to peak plasma concentrations for in vitro testing. According to our experimental results in Fig. 4, hepatocyte monolayer exposed to 10 mg/L rifampicin and 15 mg/L isoniazid did not show difference in both urea genesis and albumin synthesis within 5 days’ culture. In contrast, hepatocytes in MHFR cultures at treatment of either 10 mg/L rifampicin or 15 mg/L isoniazid for 4 days started to show obvious hepatotoxicity and this cellular damage was more severe with subsequent
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culture time. In addition, the detected hepatotoxic concentration of isoniazid were reported around 650 mg/L as indicated by pyruvate uptake and lactic acid release in hepatocyte spheroids [26], which may be due to the high diffusion barriers [6]. And the insensitivity of spheroid cultures to other hepatotoxins were also reported in literature [32,33], which supported the hypothesis of high diffusion barriers in hepatocyte spheroids. Because hepatotoxicity of either isoniazid or rifampicin was only detected in hepatocytes cultured in MHFR, it is indicative that hepatocytes within MHFR could be a suitable long-term hepatocyte model for drug hepatotoxicity testing. It was noticed that hepatocytes in monolayer culture showed cytotoxic responses to rifampicin and isoniazid at very high concentrations of 1.0 and 1.5 g/L, respectively. In another word, 100-fold higher concentrations of rifampicin and isoniazid were cytotoxic to hepatocytes in monolayer culture than their detected cytotoxic concentrations in MHFR or in vivo. Coincidently, tacrine was reported to cause hepatotoxicity to primary hepatocyte in monolayer culture at concentration of 0.5 mmol/L, 100–1000-fold higher than those observed in vivo at the onset of hepatotoxicity [3]. These results on less sensitivity of hepatocyte monolayer could be due to the decreased expression of CYP 450 in monolayer culture. In summary, the aim of our study was to assess the feasibility of MHFR for hepatotoxicity research in vitro. Hepatocyte culture in MHFR was used as a biology system to test drug-induced hepatotoxicity and monolayer culture, an extensively used model in hepatotoxicity studies, was used a control system. The results from MHFR cultures indicated that it should be a suitable long-term culture model in testing drug-induced hepatotoxicity, since its urea secretion and rat albumin synthesis were higher than those in monolayer cultures. Three drugs, acetaminophen, rifampicin and isoniazid, were used as hepatotoxic drugs and it was found that hepatocytes cultured in MHFR appeared much more sensitive to the effects of drugs compared to hepatocyte monolayer. It implied that hepatocyte culture in MHFR could be a suitable model for drug hepatotoxicity research in vitro than hepatocytes in monolayer. Acknowledgments This research was supported by grants (No. 20576119) from NSFC (National Natural Science Foundation of China) and the Ph.D. Programs Foundation of Ministry of Education of China (No. 20060335083). References [1] C.J. Viau, R.D. Curren, K. Wallace, Cytotoxicity of tacrine and velnacrine metabolites in cultured rat, dog and human hepatocytes, Drug Chem. Toxicol. 16 (3) (1993) 227–239. [2] T. Harauchi, M. Hirata, Effect of P-450 inducers on glutathione (GSH) depletion by bromobenzene in primary cultures of dog hepatocytes, Biol. Pharm. Bull. 17 (5) (1994) 658–661. [3] D. Lagadic-Gossmann, M. Rissel, M.A. Le Bot, A. Guillouzo, Toxic effects of tacrine on primary hepatocytes and liver epithelial cells in culture, Cell Biol. Toxicol. 14 (5) (1998) 361–373.
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C. Shen et al. / Biochemical Engineering Journal 34 (2007) 267–272
[4] E.F. Brandon, C.D. Raap, I. Meijerman, J.H. Beijnen, J.H. Schellens, An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons, Toxicol. Appl. Pharmacol. 189 (3) (2003) 233–246. [5] K. Bhadriraju, C.S. Chen, Engineering cellular microenvironments to improve cell-based drug testing, Drug Discov. Today 7 (11) (2002) 612–620. [6] T.M. Walker, P.C. Rhodes, C. Westmoreland, The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures, Toxicol. In Vitro 14 (5) (2000) 475–485. [7] J.Z. Tong, P. De Lagausie, V. Furlan, T. Cresteil, O. Bernard, F. Alvarez, Long-term culture of adult rat hepatocyte spheroids, Exp. Cell Res. 200 (2) (1992) 326–332. [8] S. Beken, T. Vanhaecke, K. De Smet, M. Pauwels, A. Vercruysse, V. Rogiers, Collagen-gel cultures of rat hepatocytes: collagen-gel sandwich and immobilization cultures, Meth. Mol. Biol. 107 (1998) 303–309. [9] T.M. Walker, A.J. Woodrooffe, Cytochrome P450 activity in control and induced long-term cultures of rat hepatocyte spheroids, Toxicol. In Vitro 15 (6) (2001) 713–719. [10] F. Berthiaume, P.V. Moghe, M. Toner, M.L. Yarmush, Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration, FASEB J. 10 (13) (1996) 1471–1484. [11] D. Farkas, S.R. Tannenbaum, Characterization of chemically induced hepatotoxicity in collagen sandwiches of rat hepatocytes, Toxicol. Sci. 85 (2) (2005) 927–934. [12] J.C. Dunn, R.G. Tompkins, M.L. Yarmush, Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration, Biotechnol. Prog. 7 (3) (1991) 237–245. [13] R. Gebhardt, J.G. Hengstler, D. Muller, R. Glockner, P. Buenning, B. Laube, E. Schmelzer, M. Ullrich, D. Utesch, N. Hewitt, M. Ringel, B.R. Hilz, A. Bader, A. Langsch, T. Koose, H.J. Burger, J. Maas, F. Oesch, New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures, Drug Metab. Rev. 35 (2–3) (2003) 145– 213. [14] M. Ringel, M.A. von Mach, R. Santos, P.J. Feilen, M. Brulport, M. Hermes, A.W. Bauer, W. Schormann, B. Tanner, M.R. Schon, F. Oesch, J.G. Hengstler, Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction, Toxicology 206 (1) (2005) 153– 167. [15] T.D. Sielaff, S.L. Nyberg, M.D. Rollins, M.Y. Hu, B. Amiot, A. Lee, F.J. Wu, W.S. Hu, F.B. Cerra, Characterization of the three-compartment gelentrapment porcine hepatocyte bioartificial liver, Cell Biol. Toxicol. 13 (4–5) (1997) 357–364. [16] D.Q. Wu, G.L. Zhang, C. Shen, Q. Zhao, H. Li, Q. Meng, Evaluation of diffusion in gel entrapment cell culture within hollow fibers, World J. Gastroenterol. 11 (11) (2005) 1599–1604. [17] H.Z. Wang, C.H. Chang, C.P. Lin, M.C. Tsai, Using MTT viability assay to test the cytotoxicity of antibiotics and steroid to cultured porcine corneal endothelial cells, J. Ocul. Pharmacol. Ther. 12 (1) (1996) 35–43.
[18] S.F. Abu-Absi, G. Seth, R.A. Narayanan, K. Groehler, P. Lai, M.L. Anderson, T. Sielaff, W.S. Hu, Characterization of a hollow fiber bioartificial liver device, Artif. Organs 29 (5) (2005) 419–422. [19] S.L. Nyberg, K. Shirabe, M.V. Peshwa, T.D. Sielaff, P.L. Crotty, H.J. Mann, R.P. Remmel, W.D. Payne, W.S. Hu, F.B. Cerra, Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions, Cell Transplant. 2 (6) (1993) 441–452. [20] H.X. Qiu, G.G. Su, X. Tang, Q. Meng, Tissue-like cultures of rat hepatocytes in study of phase I and phase II drug metabolism, J. Zhejiang Univ. Med. Sci. 35 (5) (2006) 541–546. [21] C. Cheung, A.M. Yu, J.M. Ward, K.W. Krausz, T.E. Akiyama, L. Feigenbaum, F.J. Gonzalez, The cyp2e1-humanized transgenic mouse: role of cyp2e1 in acetaminophen hepatotoxicity, Drug Metab. Dispos. 33 (3) (2005) 449–457. [22] B.E. Jones, H. Liu, C.R. Lo, D.R. Koop, M.J. Czaja, Cytochrome P450 2E1 expression induces hepatocyte resistance to cell death from oxidative stress, Antioxid. Redox. Signal. 4 (5) (2002) 701–709. [23] M.L. Bajt, T.R. Knight, J.J. Lemasters, H. Jaeschke, Acetaminopheninduced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine, Toxicol. Sci. 80 (2) (2004) 343–349. [24] A. Tran, J.M. Treluyer, E. Rey, J. Barbet, G. Ferracci, P. d’Athis, J. Vincent, G. Pons, Protective effect of stiripentol on acetaminophen-induced hepatotoxicity in rat, Toxicol. Appl. Pharmacol. 170 (3) (2001) 145–152. [25] D. Farkas, S.R. Tannenbaum, Characterization of chemically induced hepatotoxicity in collagen sandwiches of rat hepatocytes, Toxicol. Sci. 85 (2) (2005) 927–934. [26] J. Xu, W.M. Purcell, Energy metabolism and biotransformation as endpoints to pre-screen hepatotoxicity using a liver spheroid model, Toxicol. Appl. Pharmacol. 216 (2) (2006) 293–302. [27] W.W. Yew, Clinically significant interactions with drugs used in the treatment of tuberculosis, Drug Saf. 25 (2) (2002) 111–133. [28] S. Karthikeyan, Isoniazid and rifampicin treatment on phospholipids and their subfractions in liver tissue of rabbits, Drug Chem. Toxicol. 28 (3) (2005) 273–280. [29] D.J. Girling, The hepatic toxicity of antituberculosis regimens containing isoniazid, rifampicin and pyrazinamide, Tubercle 59 (1) (1978) 13–32. [30] S. Attri, S.V. Rana, K. Vaiphei, C.P. Sodhi, R. Katyal, R.C. Goel, C.K. Nain, K. Singh, Isoniazid- and rifampicin-induced oxidative hepatic injury—protection by N-acetylcysteine, Hum. Exp. Toxicol. 19 (9) (2000) 517–522. [31] S. Agrawal, I. Singh, K.J. Kaur, S.R. Bhade, C.L. Kaul, R. Panchagnula, Comparative bioavailability of rifampicin, isoniazid and pyrazinamide from a four drug fixed dose combination with separate formulations at the same dose levels, Int. J. Pharm. 276 (1–2) (2004) 41–49. [32] C. Dilworth, G.A. Hamilton, E. George, J.A. Timbrell, The use of liver spheroids as an in vitro model for studying induction of the stress response as a marker of chemical toxicity, Toxicol. In Vitro 14 (2) (2000) 169–176. [33] T. Battle, T. Maguire, H. Moulsdale, A. Doyle, Progressive maturation resistance to microcystin-LR cytotoxicity in two different hepatospheroidal models, Cell Biol. Toxicol. 15 (1) (1999) 3–12.