Sensitivities of gel entrapped hepatocytes in hollow fibers to hepatotoxic drug

Toxicology Letters 166 (2006) 19–26

Sensitivities of gel entrapped hepatocytes in hollow fibers to hepatotoxic drug Qin Meng a,∗ , Guoliang Zhang a,b , Chong Shen a , Hongxia Qiu a b

a College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027, China Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Zhejiang 310032, China

Received 17 January 2006; received in revised form 12 May 2006; accepted 12 May 2006 Available online 17 May 2006

Abstract The aim of this study was to determine the feasibility of detecting hepatotoxicity using gel entrapped hepatocytes in simple hollow fibers. Four typical hepatotoxic drugs were tested for hepatotoxicity in gel entrapped hepatocyte as opposed to hepatocyte monolayer, a hepatocyte system extensively used for hepatotoxicity studies in vitro. Hepatotoxicity or cell damage was assessed by the methyl tetrazolium (MTT) assay, liver-specific functions and the intracellular glutathione (GSH) content. After exposure to acetaminophen, significant cell damage of gel entrapped hepatocytes was detected at 48 h while hepatocyte monolayer was not so sensitive except for albumin synthesis and this difference between two hepatocyte systems was similar on hepatotoxic response to antituberculosis drugs including rifampicin and isoniazid. At low concentrations of either rifampicin or isoniazid, timedependent hepatotoxicity was only evidenced in gel entrapped hepatocytes after treatment and no cell damage occurred in hepatocyte monolayer at an incubation time as long as 96 h. Interestingly, hepatotoxicities of acetaminophen, isoniazid and rifampicin are all reportedly relevant to drug metabolisms of cytochrome P450. For sodium salicylate whose hepatotoxicity is unassociated to P450 activities, more significant reductions on cell viability and albumin synthesis at 5 mM than those at 1 mM apparently illustrated the concentration-dependent hepatotoxicities of gel entrapped hepatocytes as well as hepatocyte monolayer. It is highly suggested that gel entrapped hepatocyte are more sensitive in evaluation of hepatotoxicities than hepatocyte monolayer if this hepatotoxicity is related to drug metabolism. Thus, gel entrapment culture of hepatocytes with simple hollow fibers could be recommended for hepatotoxicity studies in vitro. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Gel entrapment culture; Hepatocyte; Hollow fiber; Hepatotoxicity; Drug metabolism

1. Introduction The drug discovery and development process for demonstration of efficacy and safety in humans is long and often protracted by unanticipated safety problems. In vitro models can be efficient and cost-effective tools for investigating specific mechanisms. As the most fre-

∗

Corresponding author. Fax: +86 571 87951227. E-mail address: [email protected] (Q. Meng).

quently encountered organ toxicity is liver, hepatocyte is extensively used as a cell model for in vitro hepatotoxicity studies. One principle reason for using hepatocytes in hepatotoxicity studies as opposed to other cultured cell types (i.e., hepatoma cell lines) is their capacity for drug metabolism. Hepatocytes are traditionally seeded on collagen-coated surface to form hepatocyte monolayer for in vitro hepatotoxicity studies (Acosta et al., 1985). Although hepatocyte monolayer as a model for investigative toxicology has moved well beyond the experimental stage and incorporated as key components in the drug

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discovery and development process (Lagadic-Gossmann et al., 1998), hepatocytes in monolayer culture might generate misleading results. Major problems have been encountered with hepatocyte monolayers because of their loss of liver-specific functions together with phase I and phase II drug-metabolizing enzyme activities (Baker et al., 2001). Cytochrome P450 activities significantly decreased during the first 24–48 h in monolayer culture due to the lack of a three-dimensional organization in vivo (Hu et al., 1997; Guillouzo, 1998). Gel entrapment culture of hepatocytes, employed in a bioartificial liver configured by hollow fiber cartridges, has been greatly regarded for its clear advantages of three-dimensional structures and close cell–cell contacts in contrast to hepatocyte monolayers (Nyberg et al., 1993; Wu et al., 1996). Nevertheless, little has been reported on the application of gel entrapment cultures in hepatotoxicity studies. This could be highly due to the large size of commercial hollow fiber cartridges. Further, the inconvenient operation of such big hollow fiber cartridges and an enormous requirement of hepatocytes as well as tested compound most possibly inhibit its application in hepatotoxicity studies. In this study, we discussed the feasibility of using a couple of simple hollow fibers for hepatotoxicity research. Several drugs with known hepatotoxic activities were employed as model drugs and hepatocyte monolayer was selected as a control culture system. Cell viability detected by the MTT reduction was marked as an endpoint for mitochondrial activity while urea genesis and albumin synthesis were assayed as liver-specific functions. In addition, the GSH content was usually lowered in hepatocytes following exposure to hepatotoxic drugs as a result of oxidative-induced depletion of glutathione. Thus, intracellular GSH content was also determined as an endpoint for cell damage. 2. Materials and methods 2.1. Chemicals Acetaminophen and sodium salicylate were from Hangzhou Huadong Medicine Group Co. Ltd. (Hangzhou, China). Isoniazid and rifampicin were from Zhejiang Medicine Co. Ltd. Xinchang Pharmaceutical Factory (Shaoxing, China). 2.2. Hepatocyte culture and drug treatment Hepatocyte harvest and culture medium compositions were previously reported (Meng et al., 2004). Hepatocytes were isolated from female Sprague-Dawley rats, using a collagenase perfusion as described previously (Wu et al., 2005). 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 l-glutamine 2 mmol/l (Amresco), 100 U/ml penicillin and 100 ␮g/ml streptomycin, dexamethasone 392 ng/ml, insulin 0.2 U/ml, glucagon 4 ng/ml 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. Rat-tail collagen prepared in our laboratory was used in this study (Wu et al., 2005). For gel entrapment culture, immediately after cell harvest, soluble collagen from rat-tail was first mixed with four-fold concentrated cold Williams’ E medium at a final concentration range of 3.0 mg/ml and at pH of 7.4. Freshly harvested hepatocytes were dispersed in that mixture at a density of 1 × 106 cells/ml. The cell mixture was then loaded into the lumen of hollow fibers with a length of 35 cm. After gelation for 10 min in a 5% CO2 incubator, each hollow fiber was cut into equal four pieces and put into a 60-mm dish full of 5 ml prewarmed culture medium. This dish with total hepatocytes of 0.4 × 106 in hollow fibers was put back into incubator for hepatocyte cultures. Polysulfone hollow fibers with MWCO of 100 kDa (Yuandong Pharmaceutical Machinery Corporation, Shanghai, China) were configured with an outer diameter of 1 mm, an inner diameter of 0.7 mm and membrane porosity of about 90%. At 4 h of rat hepatocyte culture in either monolayer or hollow fibers, medium was discarded. After washed by warm phosphate buffer solution, the cultures were refilled with Williams’ E medium containing tested drug and then incubated with medium change every 2 days. 2.3. Viability assays A sampled hollow fiber loaded with a known number of hepatocytes was first cut into small pieces with lengths of about 1 cm and then the absorbance on MTT was detected to evaluate hepatocyte viability as same as cell monolayer (Fotakis and Timbrell, 2006). In either monolayer culture or gel entrapment culture, control cultures without exposure to drug were considered to be 100% viable and the viability resulting from the drug-treated cells was displayed as percent viability compared to controls. 2.4. Assays on liver-specific functions Samples for analysis of urea secretion and albumin production were taken at regular intervals subsequent to mild mixing of the medium. Urea assays were performed using a Urea Nitrogen Kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rat albumin concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The rat albumin ELISA quantitation kit was obtained from BETHYL laboratories Inc. (Montgomery, USA). In either

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monolayer culture or gel entrapment culture, the liver-specific functions in control cultures without exposure to drug were accumulatively calculated and considered to be 100% so that the liver-specific functions of the drug-treated hepatocytes are presented as percentage of control values.

Table 1 The intracellular glutathione content, urea and albumin synthesis of hepatocytes cultured in monolayer and collagen gel entrapment following incubation for 48 h without any drug treatment; data presented as mean ± S.E. for three separate experiments Culture time

GSH (␮mol/106 cells)

Urea (mg/106 cells)

48 h Monolayer Gel entrapment

4.30 ± 0.29 5.67 ± 0.32

1.12 ± 0.10 1.85 ± 0.14

41.1 ± 0.50 79.3 ± 19.3

96 h Monolayer Gel entrapment

3.40 ± 0.38 5.21 ± 0.36

1.80 ± 0.11 2.81 ± 0.12

58.2 ± 12.0 101.4 ± 18.2

2.5. Measurement of glutathione Gel entrapped hepatocyte noodle was extruded from hollow fiber by injection with a syringer of 5 ml and then subjected to glutathione assay following the same procedure as GSH assays in hepatocyte monolayer (Griffith, 1980). Assays were performed in 96-well microplates. After addition of reagents, the change in absorbance at 412 nm was monitored, using a microplate reader. The GSH content of control hepatocytes without treatment by any drugs were considered to be 100% and the GSH content resulting from the drug-treated cells is presented as percentage of the control values. 2.6. Statistical analyses All values reported in the text are presented as mean ± standard error. Comparisons between multiple groups were performed with one-way ANOVA or results for two different treatments were compared using t-test for statistical comparisons. A p-value < 0.05 was determined to be significant.

3. Results The normal hepatocyte cultures in monolayer and gel entrapment were compared based on morphology and performance on liver-specific functions without any drug treatment. Morphologies of hepatocyte monolayer and gel entrapped hepatocyte, extruded from hollow fiber by injection with a syringer after culture for 48 h, were detected by phase contrast light microscopy (Fig. 1). From this figure, it can be seen that the hepatocytes in monolayer culture attached on collagen-coated plates in the form of much flattened configuration while hepatocytes in gel entrapment culture were immobilized into cylindrical shaped collagen gels with three-dimensional

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Albumin (␮g/106 cells)

organization. Except for the difference on morphology, the liver-specific functions and GSH content were different in two culture systems according to the data shown in Table 1. The intracellular GSH content decreased in both culture systems but more significantly decreased in monolayer culture, especially at the culture time of 96 h. At each culture time, accumulative albumin production is almost twice for gel entrapped hepatocytes than for hepatocyte monolayer while urea productions in gel entrapment were higher than in monolayer culture. More liver-specific functions and intracellular GSH content were noticed in gel entrapped hepatocytes than in hepatocyte monolayer. Four hepatotoxic drugs were subsequently tested here to evaluate the sensitivities of gel entrapped hepatocytes for hepatotoxicity study in comparison to hepatocyte monolayer. Hepatotoxicities were assessed by the MTT reduction, liver-specific functions and the intracellular GSH content after hepatocytes were incubated with hepatotoxic drug for a period of time. Acetaminophen is a widely used analgesic and antipyretic drug. Isoniazid and rifampicin are extensively used to treat tuberculosis which is caused by a bacterial infection. As both isoniazid (15 mg/l) and rifampicin (10 mg/l) were investigated

Fig. 1. Morphology of hepatocyte monolayer and gel entrapped hepatocyte after culture for 48 h without drug treatment. (a) Hepatocyte monolayers attached on the collagen-coated plates. (b and c) Hepatocytes were immobilized into the cylindrical shaped collagen gels within hollow fibers. For investigation under a phase contrast light microscope, collagen gels were extruded from hollow fiber by injection with a syringer of 5 ml.

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at a low concentration comparable to peak blood concentrations at usual clinical doses, an exposure to these drugs for totally 96 h was included in this experiment to evaluate the feasibility of gel entrapped hepatocytes

Fig. 2. Comparison of cell viability, intracellular GSH content, urea and protein productions of hepatocytes in gel entrapment culture and monolayer culture after exposure to acetaminophen (10 mM) for 48 h; data presented as percentage of control and given as mean ± S.E. for three separate experiments (* p < 0.05; ** p < 0.01 compared to controls).

for long-term hepatotoxicity study. Also, concentrationdependent hepatotoxicity was particularly evaluated at different concentrations of sodium salicylate (1 and 5 mM). Fig. 2 illustrates the different hepatotoxic performs of hepatocyte monolayers or gel entrapped hepatocytes after incubation with 2.5 mM acetaminophen for 48 h. Due to acetaminophen treatment, hepatocyte monolayers maintained relative cellular levels over 80% for

Fig. 3. Comparison of cell viability, intracellular GSH content, urea and protein productions of hepatocytes in gel entrapment culture and monolayer culture after exposure to isoniazid (15 mg/l) for 96 h; data presented as percentage of control and given as mean ± S.E. for three separate experiments (* p < 0.05 compared to controls).

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cell viability and urea genesis and intracellular GSH content and expressed a relative level of about 50% for albumin synthesis. By exposure to acetaminophen at the same concentration, gel entrapped hepatocytes possessed relative cellular levels below 45% for cell viability, albumin synthesis and intracellular GSH content and this value was around 62% for urea genesis. Hence, gel entrapped hepatocytes were more sensitive to acetaminophen-induced hepatotoxicity than hepatocyte monolayers. Fig. 3 depicts the hepatotoxic responses of hepatocytes in both monolayer culture and gel entrapment culture when incubated with isoniazid (15 mg/l) for 96 h. At 48 h of incubation with isoniazid, both hepatocyte monolayer and gel entrapped hepatocyte did not show significant cell damage (data were not shown). After exposure to isoniazid for 96 h, hepatocyte monolayer still presented no decreases on cell viability, intracellular glutathione content and liver-specific functions. In contrast, gel entrapped hepatocytes showed a significant reduction on the intracellular GSH level and albumin synthesis after subjected to isoniazid for 96 h. Both of them (GSH content and albumin synthesis) fell down to a relative level of around 44% and even urea genesis and MTT reduction showed an apparent reduction.

In generally, urea genesis is not a sensitive marker for hepatotoxicity. Fig. 4 represents the liver-specific functions of both hepatocyte monolayer and gel entrapped hepatocytes following treatment by rifampicin (10 mg/l) for 96 h. Similar to isoniazid, in both cultures of hepatocytes exposed to rifampicin for 48 h, no hepatotoxic responses were evidenced according to their liver-specific activities (data were not shown). But at 96 h exposure to rifampicin, gel entrapped hepatocytes maintained about 70% of urea genesis and 60% of albumin synthesis relative to the control culture without drug treatment whereas no significant decrease on liver-specific functions occurred in hepatocyte monolayer. The cell viability and intracellular GSH content were also detected as 70% and 72% for gel entrapped hepatocytes when exposure to rifampicin for 96 h, in comparison to almost no reduction for hepatocyte monolayers (data were not listed in Fig. 3). Fig. 5 illustrates the results on hepatotoxic responses of hepatocytes treated by sodium salicylate (1 and 5 mM) for 48 h. Although concentration-dependent hepatotoxicities of acetaminophen and antituberculosis drugs were

Fig. 4. Comparison of urea and protein productions of hepatocytes in gel entrapment culture and monolayer culture after exposure to rifampicin (10 mg/l) for 96 h; data presented as percentage of control and given as mean ± S.E. for three separate experiments (* p < 0.05 compared to controls).

Fig. 5. Comparison of hepatotoxic responses of gel entrapped hepatocytes and hepatocyte monolayer after exposure to sodium salicylate at two different concentrations (1 and 5 mM) for 48 h; data presented as percentage of control and given as mean ± S.E. for three separate experiments (* p < 0.05, ** p < 0.01 compared to controls).

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all noticed in both cultures of hepatocytes (data were not shown here), sodium salicylate were particularly used in this paper to illustrate the concentration-dependent hepatotoxicity. After exposure to sodium salicylate at a low concentration of 1 mM, the hepatotoxicities were almost equivalent for hepatocytes in two culture systems but at a higher concentration of 5 mM, hepatocyte monolayers were a little more sensitive to sodium salicylate than gel entrapped hepatocytes. Hence, hepatocytes in both cultures showed concentration-dependent hepatotoxicity by treatment with sodium salicylate. 4. Discussion The specific goal of this paper was to determine the feasibility of hepatotoxicity studies employing gel entrapped hepatocytes by exposure to four typical hepatotoxic drugs. In this regard, traditional monolayer hepatocytes were used as a control system. Both acetaminophen (Lewerenz et al., 2003; Villeneuve and Pichette, 2004) and isoniazid (Yue et al., 2004) have been widely studied on their hepatotoxicities which are relevantly associated with reactive metabolites of cytochrome P450 2E1 and GSH depletion (Kim et al., 1997; Villeneuve and Pichette, 2004). The early investigations about acetaminophen identified the critical role of CYP2E1 (Cheung et al., 2005) in toxicity and the function of intracellular glutathione (Jaeschke et al., 2003) in detoxifying of the acetaminophen metabolite, N-acetylp-benzoquinoneimine. This metabolite biotransformed by cytochrome P450 is a highly reactive compound that acts as an oxidizing agent that converts reduced glutathione (GSH) to oxidized glutathione (GSSG). Usually, the metabolites or reactive oxidant species are detoxified by GSH, however, at higher concentrations of hepatotoxic drugs, this detoxification system becomes overwhelmed and the subsequent depletion of GSH renders the hepatocyte vulnerable to further attack by the reactive metabolites leading to inhibition of cell functions and ultimate cell death (Allameh, 2002). Similar to acetaminophen, isoniazid undergoes cell damage in the liver resulting in the production of active radicals due to the enzyme activity of P450 2E1 (Tasduq et al., 2005; Yue et al., 2004). Clinical investigations and animal studies frequently evidenced the hepatotoxicity of isoniazid at therapeutic doses (Attri et al., 2000). Considering the well-established information about hepatotoxicities of both acetaminophen and isoniazid, this paper first detected their hepatotoxicities in order to examine the applicability of using gel entrapped hepatocytes for hepatotoxicity studies. After gel entrapped hep-

atocytes were respectively exposed to acetaminophen for 48 h and isoniazid for 96 h, significant GSH depletion and albumin synthesis inhibition were evidenced followed by slight decreases on the urea production while significant MTT reduction occurred in acetaminophen treatment but not in isoniazid treatment which was operated at a rather low concentration (Figs. 1 and 2). The fact that urea genesis of hepatocytes was less sensitive to hepatotoxic drugs than albumin synthesis could be due to the robustness of urea synthesis enzymes after cellular damage occurs (Meng, 2003). It seems that the cellular GSH content as well as albumin synthesis decrease either simultaneously with cell viability in acetaminophen-treated hepatocytes or earlier than cell viability in isoniazid-treated hepaotyctes. This phenomenon can be explained by the fact that the depletion of intracellular GSH content could impair the cell’s defense against toxic compounds and thus result in decrease on cell functions and even cell death (Neuman et al., 1999, 2001). Opposite to gel entrapped hepatocytes, hepatocyte monolayers possessed a little decrease on cell viability, GSH content and urea production when treated by acetaminophen for 48 h and showed almost no reduction on cell activities when treated by isoniazid for 96 h (Figs. 1 and 2). In another word, based on the data presented in Figs. 1 and 2, exposure to these hepatotoxic drugs caused more dramatic cell damage in gel entrapped hepatocytes than in hepatocyte monolayers. Hence, entrapped hepatocytes were more sensitive to hepatotoxic drugs of acetaminophen and isoniazid than hepatocyte monolayers. The time-dependent hepatotoxicities were also investigated using antituberculosis drugs, isoniazid and rifampicin, by detecting the cellular damage at 48 and 96 h, respectively. In contrast to no significant cellular damage of gel entrapped hepatocytes at 48 h after exposure to isoniazid (15 mg/l), cellular damages detected by albumin synthesis and intracellular GSH content were obviously noticed at 96 h while hepatocyte monolayer did not show any cell damage during the whole 96 h of isoniazid treatment. Similarly, the fact that rifampicin (10 mg/l) treatment caused more significant reductions on albumin synthesis after an exposure of 96 h than that of 48 h indicated again the time-dependent hepatotoxicity of gel entrapped hepatocytes but this time-dependent hepatotoxicity was not observed in hepatocyte monolayer within a low drug concentration range because of its insensitivity to rifampicin hepatotoxicity. The further investigations on concentrationdependent hepatotoxicities were illustrated by treating hepatocyes with sodium salicylate at different concentrations (1 and 5 mM) and it was found that both

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hepatocyte monolayers and gel entrapped hepatocytes responded to the toxicities of sodium salicylate at a concentration-dependent manner. MTT reduction and albumin synthesis at 5 mM than those at 1 mM apparently illustrated the concentration-dependent hepatotoxicities of gel entrapped hepatocytes as well as hepatocyte monolayers. It should be mentioned here that gel entrapped hepatocytes showed the similar concentration-dependent hepatotoxicity of drugs including acetaminophen, isoniazid and rifampicin (data were not shown here). Nevertheless, hepatocyte monolayers did not show concentration-dependent responses due to their quite low sensitivities at the same concentration range of the drugs mentioned above. Overall, gel entrapped hepatocytes were sensitive to hepatotoxic drugs of acetaminophen and antituberculosis drugs of isoniazid and rifampicin than monolayer hepatocytes. This sensitivity of gel entrapped hepatocytes in hepatotoxicity studies could be associated with their higher enzymatic activities of drug metabolism sustained in gel entrapped hepatocytes, as cytochrome P450 were reported to be well preserved in a bioartificial liver (Wu et al., 1996). In our laboratory, drug metabolism assay using 4-nitrophenol as a substrate marker for P450 2E1, a major enzyme involved in hepatotoxicities of acetaminophen and isoniacid, indicated that gel entrapped hepatocyte sustained this enzyme activity for at least 4 days and its activity was more than twice of those in hepatocyte monolayer within 2 days, a period of which metabolites of CYP 2E1 in hepatocyte monolayer can be detectable by HPLC. Hence, metabolic assays confirmed that gel entrapped hepatocytes expressed higher cytochrome P450 enzymatic activities than hepatocyte monolayer. As rifampicin was commonly investigated at co-administration with other antituberculosis drugs for hepatotoxicity studies, its individual mechanism of hepatotoxicity is unclear so far. But this cell damage resulted from the formation of reactive oxygen species (Skakun and Tabachuk, 1991), similar to both acetaminophen and isoniazid. We premise that rifampicin-induced hepatotoxicity might result from the enzyme activities of cytochrome P450 due to its higher sensitivity in gel entrapment culture. On the contrary, sodium salicylate induced hepatotoxicities by the way of inhibition of mitochondria, not associated with drug metabolism of cytochrome P450 (Trost and Lemasters, 1997), which might cause no significant difference on hepatotoxic responses between gel entrapped hepatocyte and hepatocyte monolayer. In vitro hepatotoxicity studies using hepatocyte monolayer can be used to predict human hepatotoxicity and for the general screening of drugs. Nevertheless,

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it has been previously reported that the detected hepatotoxic concentration of tacrine by hepatocyte monolayer was as high as 100–1000-fold of the concentration observed in vivo at the onset of hepatotoxicity (Lagadic-Gossmann et al., 1998). As shown in this paper, the insensitivities of hepatocyte monolayer to isoniazid, rifampicin and acetaminophen were also noticed at a low concentration equivalent or approaching to a detected hepatotoxic blood concentration in clinicals. In contrast, gel entrapped hepatocytes were more sensitive to the hepatotoxic drugs like acetaminophen, isoniazid and rifampicin. Significant cell damage of gel entrapped hepatocytes treated by drug, whose hapatotoxicity is related to drug metabolism, could be evident at both an early time and a low concentration. In conclusion, gel entrapped hepatocyte are more sensitive than hepatocyte monolayer to the treatment by acetaminophen, isoniazid and rifampicin. A higher cytochrome P450 enzymatic activity sustained by gel entrapment culture of hepatocyte is suggested to be the main contributor for the sensitivities of gel entrapped hepatocytes in hepatotoxicity study. Thus, gel entrapment culture of hepatocytes with simple hollow fibers could be recommended for hepatotoxicity studies in vitro. Acknowledgments This research was supported by National Natural Science Foundation of China (NSFC, Nos. 90209053 and 20576119) and Zhejiang Scientific Project (No. 2003C31042), China. References Acosta, D., Scorensen, E.M.B., Anuforo, D.C., Mitchell, D.B., Ramos, K., Santone, K.S., Simth, M.A., 1985. An in vitro approach to the study of target organ toxicity of drugs and chemicals. In Vitro Cell Dev. Biol. 21, 495–504. Allameh, N.A., 2002. Acetaminophen–glutathione conjugate formation in a coupled cytochrome P-450-glutathione S-transferase assay system mediated by subcellular preparations from adult and weanling rat tissues. Toxicol. in Vitro 16, 637–641. Attri, S., Rana, S.V., Vaiphei, K., Sodhi, C.P., Katyal, R., Goel, R.C., Nain, C.K., Singh, K., 2000. Isoniazid- and rifampicin-induced oxidative hepatic injury—protection by N-acetylcysteine. Human Exp. Toxicol. 19, 517–522. Baker, T.K., Carfagna, M.A., Gao, H., Dow, E.R., Li, Q., Searfoss, G.H., Ryan, T.P., 2001. Temporal gene expression analysis of monolayer cultured rat hepatocytes. Chem. Res. Toxicol. 14, 1218–1231. Cheung, C., Yu, A.M., Ward, J.M., Krausz, K.W., Akiyama, T.E., Feigenbaum, L., Gonzalez, F.J., 2005. The CYP2E1-humanized transgenic mouse: role of CYP2E1 in acetaminophen hepatotoxicity. Drug Metab. Dispos. 33, 449–457.

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